Structured Review

Cytoskeleton Inc actin cytoskeleton
Actin <t>cytoskeleton</t> organization at the two poles of a migrating T cell . Schematic representations of the ultrastructure of the actin cytoskeleton networks at the leading and trailing edges of the migrating T cell shown in Figure 1 . At the leading edge, the T cell that migrates on a 2D surface emits a protrusion that alternates between a lamellipodium and a pseudopodium. It contains a very dynamical and highly branched actin meshwork. At the trailing edge, the T cell uropod is made of a network of parallel actin bundles that can slide along each other to generate contractile forces.
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1) Product Images from "T Lymphocyte Migration: An Action Movie Starring the Actin and Associated Actors"

Article Title: T Lymphocyte Migration: An Action Movie Starring the Actin and Associated Actors

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2015.00586

Actin cytoskeleton organization at the two poles of a migrating T cell . Schematic representations of the ultrastructure of the actin cytoskeleton networks at the leading and trailing edges of the migrating T cell shown in Figure 1 . At the leading edge, the T cell that migrates on a 2D surface emits a protrusion that alternates between a lamellipodium and a pseudopodium. It contains a very dynamical and highly branched actin meshwork. At the trailing edge, the T cell uropod is made of a network of parallel actin bundles that can slide along each other to generate contractile forces.
Figure Legend Snippet: Actin cytoskeleton organization at the two poles of a migrating T cell . Schematic representations of the ultrastructure of the actin cytoskeleton networks at the leading and trailing edges of the migrating T cell shown in Figure 1 . At the leading edge, the T cell that migrates on a 2D surface emits a protrusion that alternates between a lamellipodium and a pseudopodium. It contains a very dynamical and highly branched actin meshwork. At the trailing edge, the T cell uropod is made of a network of parallel actin bundles that can slide along each other to generate contractile forces.

Techniques Used:

The different facets of actin cytoskeleton remodeling in migrating T cells . Represented at the center of the scheme (green zone) are the dominant receptors in the control of T cell motility. They include chemokine receptors, the TCR and integrins such as LFA-1, each being interconnected with the actin cytoskeleton with specific sets of signaling molecules. Ligand-mediated triggering of these receptors leads to the activation of the RhoGTPases Rac, Rho, and Cdc42 via GEFs and GAPs (purple zone). Such activation is highly controlled in time and space to orchestrate the assembly of distinct actin networks. Rac activates the WAVE complex, leading to Arp2/3-mediated actin polymerization at the leading edge to form a branched actin network. Cdc42 also favors membrane extension by activating the Arp2/3 complex via WASP. In addition to its major role at the uropod, Rho plays a dual role at the leading edge by promoting actin filament elongation via the formin mDia and by favoring membrane retraction via myosin activation. The left side of the scheme depicts the role of ERM proteins as anchors of the actin cytoskeleton in the plasma membrane (orange zone). The right side of the scheme illustrates the role of BAR-domain proteins as molecular links to guarantee local coordination of membrane curvature and actin polymerization (yellow zone). The actin meshwork is represented as blue filaments that are intertwined with the different signaling areas.
Figure Legend Snippet: The different facets of actin cytoskeleton remodeling in migrating T cells . Represented at the center of the scheme (green zone) are the dominant receptors in the control of T cell motility. They include chemokine receptors, the TCR and integrins such as LFA-1, each being interconnected with the actin cytoskeleton with specific sets of signaling molecules. Ligand-mediated triggering of these receptors leads to the activation of the RhoGTPases Rac, Rho, and Cdc42 via GEFs and GAPs (purple zone). Such activation is highly controlled in time and space to orchestrate the assembly of distinct actin networks. Rac activates the WAVE complex, leading to Arp2/3-mediated actin polymerization at the leading edge to form a branched actin network. Cdc42 also favors membrane extension by activating the Arp2/3 complex via WASP. In addition to its major role at the uropod, Rho plays a dual role at the leading edge by promoting actin filament elongation via the formin mDia and by favoring membrane retraction via myosin activation. The left side of the scheme depicts the role of ERM proteins as anchors of the actin cytoskeleton in the plasma membrane (orange zone). The right side of the scheme illustrates the role of BAR-domain proteins as molecular links to guarantee local coordination of membrane curvature and actin polymerization (yellow zone). The actin meshwork is represented as blue filaments that are intertwined with the different signaling areas.

Techniques Used: Activation Assay

Actin cytoskeleton underlies T cell morphological changes during directional migration . (A) Snapshots of a movie showing a primary CD8 + human T cell expressing Dendra2-LifeAct moving along a CXCL12 gradient created in a collagen IV-coated Ibidi μ-Slide Chemotaxis 2D . The cell extends dynamic protrusions and moves toward the source of CXCL12 (top). See Movie S1 in Supplementary Material. The organization of the actin cytoskeleton in T lymphocytes can also be appreciated in previous reports on the dynamics of actin during scanning of target cells ( 40 ) and polarization in response to CXCL12 ( 41 ). (B) Velocity of the cell shown in (A) , calculated for successive 12 s intervals on the basis of the tracking of the cell. (C) Morphology of the cell shown in (A) , calculated as Aspect Ratio (length/width of a fitted ellipsis) for each frame.
Figure Legend Snippet: Actin cytoskeleton underlies T cell morphological changes during directional migration . (A) Snapshots of a movie showing a primary CD8 + human T cell expressing Dendra2-LifeAct moving along a CXCL12 gradient created in a collagen IV-coated Ibidi μ-Slide Chemotaxis 2D . The cell extends dynamic protrusions and moves toward the source of CXCL12 (top). See Movie S1 in Supplementary Material. The organization of the actin cytoskeleton in T lymphocytes can also be appreciated in previous reports on the dynamics of actin during scanning of target cells ( 40 ) and polarization in response to CXCL12 ( 41 ). (B) Velocity of the cell shown in (A) , calculated for successive 12 s intervals on the basis of the tracking of the cell. (C) Morphology of the cell shown in (A) , calculated as Aspect Ratio (length/width of a fitted ellipsis) for each frame.

Techniques Used: Migration, Expressing, Chemotaxis Assay

Migratory challenges faced by T cells during their journey through the organism . (A) The crossing of the endothelium barrier follows steps of T cell tethering and rolling on the luminal surface of the endothelial cells. The combination of chemokines and adhesion molecules triggers firm adhesion to the endothelial surface via the emission of filopodia-like protrusions. Depending on their activation state, T cells then either use the transcellular route by emitting invadopodia-like protrusions that go across the entire endothelial cell body, or the paracellular by squeezing through the junction between two adjacent endothelial cells. (B) Following the crossing of the endothelial barrier and underlying basal membrane, T cells undergo interstitial migration through the tissue they have entered in. Using an amoeboid mode of motility, they crawl and squeeze along and through extracellular matrix (ECM) fibers of various nature. In the lymph node cortex, T cells preferentially migrate along a network of fibroblastic reticular cells decorated with chemokines. (C) During the scanning of antigen-presenting cells (APC), T cells make multiple encounters of various duration and quality. Some contacts may last only a few minutes in the form of an immature immunological synapse. Upon recognition of an APC bearing specific antigens, the T cell stops migrating to assemble a long-lasting immunological synapse. In addition to controlling the interaction between the T cell and the APC, the dynamical actin cytoskeleton serves as a physical platform for numerous signaling events that take place at the immunological synapse to activate the T cell. After a few hours, the activated T cell detaches from its APC partner and regains its motility behavior.
Figure Legend Snippet: Migratory challenges faced by T cells during their journey through the organism . (A) The crossing of the endothelium barrier follows steps of T cell tethering and rolling on the luminal surface of the endothelial cells. The combination of chemokines and adhesion molecules triggers firm adhesion to the endothelial surface via the emission of filopodia-like protrusions. Depending on their activation state, T cells then either use the transcellular route by emitting invadopodia-like protrusions that go across the entire endothelial cell body, or the paracellular by squeezing through the junction between two adjacent endothelial cells. (B) Following the crossing of the endothelial barrier and underlying basal membrane, T cells undergo interstitial migration through the tissue they have entered in. Using an amoeboid mode of motility, they crawl and squeeze along and through extracellular matrix (ECM) fibers of various nature. In the lymph node cortex, T cells preferentially migrate along a network of fibroblastic reticular cells decorated with chemokines. (C) During the scanning of antigen-presenting cells (APC), T cells make multiple encounters of various duration and quality. Some contacts may last only a few minutes in the form of an immature immunological synapse. Upon recognition of an APC bearing specific antigens, the T cell stops migrating to assemble a long-lasting immunological synapse. In addition to controlling the interaction between the T cell and the APC, the dynamical actin cytoskeleton serves as a physical platform for numerous signaling events that take place at the immunological synapse to activate the T cell. After a few hours, the activated T cell detaches from its APC partner and regains its motility behavior.

Techniques Used: Activation Assay, Migration

Actin cytoskeleton architecture . (A) Actin cytoskeleton dynamics rely in part on the tightly controlled cycle of polymerization and depolymerization, also known as treadmilling. ATP-bound actin is added to the fast growing barbed end of filaments via the combined action of profilin, which prevents self-nucleation of actin monomers and actin-nucleating proteins such as the formin FMLN1 or WASP-family proteins, both of which are under the control of RhoGTPases. Depolymerization is promoted by cofilin, which stimulates dissociation of ADP-bound actin at the pointed end of filaments. The rate of cofilin-mediated depolymerization can be controlled by Rho via Rock and LimK. (B) In addition to be elongated by formins, actin filaments can build networks in multiple ways. Actin bundles or cables with parallel or anti-parallel orientation of actin filaments are assembled by cross-linking proteins such as fimbrin. Actin filaments can also be cross-linked in a non-parallel fashion via filamin to create a gelled network. Branched networks are promoted by the Arp2/3 complex that initiates nucleation of branched filaments on the side of existing ones. This activity is driven by WASP-family proteins and stabilized by HS-1. An additional important regulation of actin cytoskeleton networks is mediated by capping proteins such as gelsolin, which bind the plus end of actin filaments to prevent monomer exchange. (C) Actin filaments not only generate forces while they elongate. They also generate the cell contractile forces via the intercalation of the molecular motor myosin between parallel actin filaments, which results in filament sliding. Such process is regulated by the control of the myosin light chain phosphatase and kinase activities, as well as by the degree of actin cross-linking via α-actinin.
Figure Legend Snippet: Actin cytoskeleton architecture . (A) Actin cytoskeleton dynamics rely in part on the tightly controlled cycle of polymerization and depolymerization, also known as treadmilling. ATP-bound actin is added to the fast growing barbed end of filaments via the combined action of profilin, which prevents self-nucleation of actin monomers and actin-nucleating proteins such as the formin FMLN1 or WASP-family proteins, both of which are under the control of RhoGTPases. Depolymerization is promoted by cofilin, which stimulates dissociation of ADP-bound actin at the pointed end of filaments. The rate of cofilin-mediated depolymerization can be controlled by Rho via Rock and LimK. (B) In addition to be elongated by formins, actin filaments can build networks in multiple ways. Actin bundles or cables with parallel or anti-parallel orientation of actin filaments are assembled by cross-linking proteins such as fimbrin. Actin filaments can also be cross-linked in a non-parallel fashion via filamin to create a gelled network. Branched networks are promoted by the Arp2/3 complex that initiates nucleation of branched filaments on the side of existing ones. This activity is driven by WASP-family proteins and stabilized by HS-1. An additional important regulation of actin cytoskeleton networks is mediated by capping proteins such as gelsolin, which bind the plus end of actin filaments to prevent monomer exchange. (C) Actin filaments not only generate forces while they elongate. They also generate the cell contractile forces via the intercalation of the molecular motor myosin between parallel actin filaments, which results in filament sliding. Such process is regulated by the control of the myosin light chain phosphatase and kinase activities, as well as by the degree of actin cross-linking via α-actinin.

Techniques Used: Activity Assay

2) Product Images from "Regulation of the Postsynaptic Compartment of Excitatory Synapses by the Actin Cytoskeleton in Health and Its Disruption in Disease"

Article Title: Regulation of the Postsynaptic Compartment of Excitatory Synapses by the Actin Cytoskeleton in Health and Its Disruption in Disease

Journal: Neural Plasticity

doi: 10.1155/2016/2371970

Schematic representation of the cytoskeleton-dependent trafficking of neurotransmitter receptors. Depicted are the key structures of the synapse and associated cytoskeletal molecules. Numbers indicate the following steps: (1) Myosin V traffics vesicles with receptors from the soma to the distal dendritic sites via microtubules (MT) [ 116 ]. MT plus ends are indicated by green circles. (2) Once within spines, myosin V transports receptors to plasma membrane via actin filaments [ 117 ]. (3) Anchoring of receptors in the PSD relies on myosin II contractile force on actin cytoskeleton in combination with constant actin treadmilling/turnover [ 48 ]. Lateral diffusion of receptors to and from the PSD to presynaptic regions can occur. (4) Receptor internalization involves myosin VI activity. Myosin VI transports internalized receptors to endosomal organelles, facilitating recycling of receptors back to the membrane or to degradation pathways [ 95 ]. (5) Receptors can also travel between the PSD and peripheral sites [ 118 , 119 ].
Figure Legend Snippet: Schematic representation of the cytoskeleton-dependent trafficking of neurotransmitter receptors. Depicted are the key structures of the synapse and associated cytoskeletal molecules. Numbers indicate the following steps: (1) Myosin V traffics vesicles with receptors from the soma to the distal dendritic sites via microtubules (MT) [ 116 ]. MT plus ends are indicated by green circles. (2) Once within spines, myosin V transports receptors to plasma membrane via actin filaments [ 117 ]. (3) Anchoring of receptors in the PSD relies on myosin II contractile force on actin cytoskeleton in combination with constant actin treadmilling/turnover [ 48 ]. Lateral diffusion of receptors to and from the PSD to presynaptic regions can occur. (4) Receptor internalization involves myosin VI activity. Myosin VI transports internalized receptors to endosomal organelles, facilitating recycling of receptors back to the membrane or to degradation pathways [ 95 ]. (5) Receptors can also travel between the PSD and peripheral sites [ 118 , 119 ].

Techniques Used: Diffusion-based Assay, Activity Assay

Amyloid- β disrupts the actin cytoskeleton and receptor trafficking through multiple pathways. There are many conflicting pathways through which A β is proposed to alter the actin cytoskeleton. These may involve both up- and downregulation of cofilin activity. Activation of RhoA by A β [ 204 ] antagonistically inhibits Rac1 [ 206 ], both leading to increased cofilin activity. In contrast, A β can cause decrease in cofilin activity via activation of Cdc42 [ 211 ]. Both active and inactive cofilin are thought to be required for the formation of cofilin rods, which lead to impairment of intracellular transport [ 215 ]. Alternatively or in addition to this, altered expression and/or processing of actin filament stabilizing proteins [ 217 , 218 ] may impact the trafficking of neurotransmitter receptors.
Figure Legend Snippet: Amyloid- β disrupts the actin cytoskeleton and receptor trafficking through multiple pathways. There are many conflicting pathways through which A β is proposed to alter the actin cytoskeleton. These may involve both up- and downregulation of cofilin activity. Activation of RhoA by A β [ 204 ] antagonistically inhibits Rac1 [ 206 ], both leading to increased cofilin activity. In contrast, A β can cause decrease in cofilin activity via activation of Cdc42 [ 211 ]. Both active and inactive cofilin are thought to be required for the formation of cofilin rods, which lead to impairment of intracellular transport [ 215 ]. Alternatively or in addition to this, altered expression and/or processing of actin filament stabilizing proteins [ 217 , 218 ] may impact the trafficking of neurotransmitter receptors.

Techniques Used: Activity Assay, Activation Assay, Expressing

3) Product Images from "The Actin Cytoskeleton Is Involved in Glial Cell Line-Derived Neurotrophic Factor (GDNF)-Induced Ret Translocation into Lipid Rafts in Dopaminergic Neuronal Cells"

Article Title: The Actin Cytoskeleton Is Involved in Glial Cell Line-Derived Neurotrophic Factor (GDNF)-Induced Ret Translocation into Lipid Rafts in Dopaminergic Neuronal Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18091922

The concentration-dependent effects of Lat B and Jas on the actin cytoskeleton. ( A ) Untreated MN9D cells were stained with phalloidin at the given time points; ( B ) MN9D cells were stimulated with Lat B at the indicated concentrations and time points; ( C ) MN9D cells were stimulated with Jas at the indicated concentrations and time points. (Scale bar = 5 μm).
Figure Legend Snippet: The concentration-dependent effects of Lat B and Jas on the actin cytoskeleton. ( A ) Untreated MN9D cells were stained with phalloidin at the given time points; ( B ) MN9D cells were stimulated with Lat B at the indicated concentrations and time points; ( C ) MN9D cells were stimulated with Jas at the indicated concentrations and time points. (Scale bar = 5 μm).

Techniques Used: Concentration Assay, Staining

4) Product Images from "All-Round Manipulation of the Actin Cytoskeleton by HIV"

Article Title: All-Round Manipulation of the Actin Cytoskeleton by HIV

Journal: Viruses

doi: 10.3390/v10020063

Actin-related changes in cellular behavior induced by HIV in relevant cell types. Coordinated manipulation of actin regulators results in global and cell-type specific changes in cellular morphology and motility that contribute to viral spread, impairment of immune function and HIV comorbidities. Many of these changes can be mapped to specific HIV accessory proteins, which act as master regulators of the cytoskeleton. Nef and Tat are expressed in HIV-infected cells but are also present extracellularly in serum and, thus, can affect both infected and uninfected cells. ( a ) Nef; leads to Rac1 activation in a wide range of cell types. In T-cells, this is associated with inactivation of Cofilin and severe cytoskeletal disorganization, which impairs cell migration and immunological synapse formation. In myeloid cells, Nef enhances formation of several membrane protrusions which promote cell motility and contacts with uninfected cells; ( b ) Tat modulates the expression of numerous genes involved in actin regulation. In T-cells, Tat interferes with chemotaxis and F-actin remodeling, whereas in monocytes it increases cell motility, chemotaxis and phagocytosis. Tat also induces expression of adhesion molecules and promotes leukocyte binding to the endothelium. Upward arrows represent enhancement of biological processes or increases in number of structures, whereas downward arrows represent impairment of processes. * = Effects also induced by HIV envelope glycoprotein (Env).
Figure Legend Snippet: Actin-related changes in cellular behavior induced by HIV in relevant cell types. Coordinated manipulation of actin regulators results in global and cell-type specific changes in cellular morphology and motility that contribute to viral spread, impairment of immune function and HIV comorbidities. Many of these changes can be mapped to specific HIV accessory proteins, which act as master regulators of the cytoskeleton. Nef and Tat are expressed in HIV-infected cells but are also present extracellularly in serum and, thus, can affect both infected and uninfected cells. ( a ) Nef; leads to Rac1 activation in a wide range of cell types. In T-cells, this is associated with inactivation of Cofilin and severe cytoskeletal disorganization, which impairs cell migration and immunological synapse formation. In myeloid cells, Nef enhances formation of several membrane protrusions which promote cell motility and contacts with uninfected cells; ( b ) Tat modulates the expression of numerous genes involved in actin regulation. In T-cells, Tat interferes with chemotaxis and F-actin remodeling, whereas in monocytes it increases cell motility, chemotaxis and phagocytosis. Tat also induces expression of adhesion molecules and promotes leukocyte binding to the endothelium. Upward arrows represent enhancement of biological processes or increases in number of structures, whereas downward arrows represent impairment of processes. * = Effects also induced by HIV envelope glycoprotein (Env).

Techniques Used: Activated Clotting Time Assay, Infection, Activation Assay, Migration, Expressing, Chemotaxis Assay, Binding Assay

Manipulation of the actin cytoskeleton by human immunodeficiency virus (HIV). ( a ) Actin regulators subjected to modulation by HIV. Mechanistically diverse strategies enable the virus to alter cellular cytoskeletal functions. Manipulation of host factors can be either direct, when mediated by physical interaction with viral proteins, or indirect, when requiring upstream cellular factors. Exploitation mechanisms increase native protein activity by upregulation of gene expression, or indirect activation within a cellular pathway. Neutralization of host factors is achieved by downregulation of gene expression or protein inactivation. Hijacking alters the functional outcome of host protein activity, either by overriding regulatory mechanisms (i.e., direct protein activation), changing protein subcellular localization, and/or modifying protein interaction partners. Note that some host factors can be manipulated by multiple strategies at diverse stages of the viral life cycle, as well as differentially in infected and uninfected cells. Examples of actin regulators corresponding to each strategy are provided, however this is not a complete list; ( b ) Functional consequences of actin-dependent changes induced by HIV. Normal immunological functions are compromised upon HIV infection, partly due to actin-remodeling changes orchestrated by viral proteins. CD4+ lymphocytes display severe impairment of chemotaxis and immunological synapse formation. Myeloid cells display aberrant enhancement of actin dependent structures, which alters cell motility and tissue distribution. Concurrent changes in actin remodeling in both cell types also promote viral spread via actin-dependent cell-cell contacts and support infection by inbound cell-free virus.
Figure Legend Snippet: Manipulation of the actin cytoskeleton by human immunodeficiency virus (HIV). ( a ) Actin regulators subjected to modulation by HIV. Mechanistically diverse strategies enable the virus to alter cellular cytoskeletal functions. Manipulation of host factors can be either direct, when mediated by physical interaction with viral proteins, or indirect, when requiring upstream cellular factors. Exploitation mechanisms increase native protein activity by upregulation of gene expression, or indirect activation within a cellular pathway. Neutralization of host factors is achieved by downregulation of gene expression or protein inactivation. Hijacking alters the functional outcome of host protein activity, either by overriding regulatory mechanisms (i.e., direct protein activation), changing protein subcellular localization, and/or modifying protein interaction partners. Note that some host factors can be manipulated by multiple strategies at diverse stages of the viral life cycle, as well as differentially in infected and uninfected cells. Examples of actin regulators corresponding to each strategy are provided, however this is not a complete list; ( b ) Functional consequences of actin-dependent changes induced by HIV. Normal immunological functions are compromised upon HIV infection, partly due to actin-remodeling changes orchestrated by viral proteins. CD4+ lymphocytes display severe impairment of chemotaxis and immunological synapse formation. Myeloid cells display aberrant enhancement of actin dependent structures, which alters cell motility and tissue distribution. Concurrent changes in actin remodeling in both cell types also promote viral spread via actin-dependent cell-cell contacts and support infection by inbound cell-free virus.

Techniques Used: Activity Assay, Expressing, Activation Assay, Neutralization, Functional Assay, Infection, Chemotaxis Assay

Functional protein interaction network of actin regulators targeted by HIV manipulation. Subversion of the actin cytoskeleton occurs throughout all layers of the actin cytoskeleton, including surface proteins, their linkers to actin filaments, diverse signaling nodes, their effectors, and proteins that directly mediate actin remodeling. Color coding of host factors indicates the main viral protein involved in their deregulation; Nef = green, Gag = yellow, Tat = pink, Env = blue, presently undefined = grey. So far, only proteins shown in bold have been experimentally confirmed to physically interact with the indicated viral proteins (i.e., direct manipulation). Network data was obtained from the STRING database [ 93 ] using the following settings: network edge meaning = evidence, active sources = databases, minimum confidence score = 0.9, number of interactors = query only (proteins covered in this review). The network nodes were rearranged using Cytoscape software [ 94 ], to represent functional groups within the cytoskeleton.
Figure Legend Snippet: Functional protein interaction network of actin regulators targeted by HIV manipulation. Subversion of the actin cytoskeleton occurs throughout all layers of the actin cytoskeleton, including surface proteins, their linkers to actin filaments, diverse signaling nodes, their effectors, and proteins that directly mediate actin remodeling. Color coding of host factors indicates the main viral protein involved in their deregulation; Nef = green, Gag = yellow, Tat = pink, Env = blue, presently undefined = grey. So far, only proteins shown in bold have been experimentally confirmed to physically interact with the indicated viral proteins (i.e., direct manipulation). Network data was obtained from the STRING database [ 93 ] using the following settings: network edge meaning = evidence, active sources = databases, minimum confidence score = 0.9, number of interactors = query only (proteins covered in this review). The network nodes were rearranged using Cytoscape software [ 94 ], to represent functional groups within the cytoskeleton.

Techniques Used: Functional Assay, Software

5) Product Images from "Role of the Cytoskeleton in Formation and Maintenance of Angiogenic Sprouts"

Article Title: Role of the Cytoskeleton in Formation and Maintenance of Angiogenic Sprouts

Journal: Journal of Vascular Research

doi: 10.1159/000324751

Schematic illustration illustrating key steps in angiogenesis, along with molecules that transduce signals to the cytoskeleton to induce sprouting angiogenesis. a Quiescent endothelium exhibiting intact basement membrane (red), mural cell (yellow) and
Figure Legend Snippet: Schematic illustration illustrating key steps in angiogenesis, along with molecules that transduce signals to the cytoskeleton to induce sprouting angiogenesis. a Quiescent endothelium exhibiting intact basement membrane (red), mural cell (yellow) and

Techniques Used:

6) Product Images from "Early Events in Actin Cytoskeleton Dynamics and E-Cadherin-Mediated Cell-Cell Adhesion during Epithelial-Mesenchymal Transition"

Article Title: Early Events in Actin Cytoskeleton Dynamics and E-Cadherin-Mediated Cell-Cell Adhesion during Epithelial-Mesenchymal Transition

Journal: Cells

doi: 10.3390/cells9030578

Reorganization of the actin cytoskeleton in IAR-20 cells treated with EGF. ( a ) Disruption of the circumferential actin bundle (arrow) and appearance of pseudopodia at the cell-cell-boundaries (arrowheads) after the addition of EGF in IAR-20 cells stably expressing F-tractin-tdTomato. Selected frames from Supplementary Video S3 . ( b ) Immunostaining of p34 and β-actin. In control cells, the Arp2/3 complex resides at cell-cell boundaries. In EGF-treated cells, Arp2/3 is enriched in lamellipodia close to cell-cell boundaries (arrowheads) where the disintegrated circumferential bundle is also seen. Dashed rectangles indicate the cell-cell boundaries enlarged in boxed regions with corresponding fluorescence intensity profiles. Arrows indicate p34 fluorescence intensity peak corresponding to the pseudopodia edge. ( c ) Immunostaining of p34 and E-cadherin ( Figure S1 ) and corresponding fluorescence intensity profiles. In control cells, p34 colocalizes with E-cadherin. In EGF-treated cells, a second, smaller peak of p34 intensity appears which corresponds to the extending edges of the pseudopodia (arrows).
Figure Legend Snippet: Reorganization of the actin cytoskeleton in IAR-20 cells treated with EGF. ( a ) Disruption of the circumferential actin bundle (arrow) and appearance of pseudopodia at the cell-cell-boundaries (arrowheads) after the addition of EGF in IAR-20 cells stably expressing F-tractin-tdTomato. Selected frames from Supplementary Video S3 . ( b ) Immunostaining of p34 and β-actin. In control cells, the Arp2/3 complex resides at cell-cell boundaries. In EGF-treated cells, Arp2/3 is enriched in lamellipodia close to cell-cell boundaries (arrowheads) where the disintegrated circumferential bundle is also seen. Dashed rectangles indicate the cell-cell boundaries enlarged in boxed regions with corresponding fluorescence intensity profiles. Arrows indicate p34 fluorescence intensity peak corresponding to the pseudopodia edge. ( c ) Immunostaining of p34 and E-cadherin ( Figure S1 ) and corresponding fluorescence intensity profiles. In control cells, p34 colocalizes with E-cadherin. In EGF-treated cells, a second, smaller peak of p34 intensity appears which corresponds to the extending edges of the pseudopodia (arrows).

Techniques Used: Stable Transfection, Expressing, Immunostaining, Fluorescence

Rearrangement of E-cadherin-based AJs in parallel with reorganization of the actin cytoskeleton in IAR-20 cells treated with EGF. ( a ) Immunostaining of E-cadherin and β-actin. Top—red and green channels merged. The cell-cell boundaries enlarged below are indicated with arrows. Disorganization of AJs begins 5 min after addition of EGF, simultaneously with dissolution of the circumferential actin bundle and formation of lamellipodia. Beginning from 10–15 min after the addition of EGF, punctate AJs were connected with straight actin fibers appear. ( b ) Fluorescence intensity profiles of F-actin and β-catenin in the zones of cell-cell contacts. N = 30 for every graph.
Figure Legend Snippet: Rearrangement of E-cadherin-based AJs in parallel with reorganization of the actin cytoskeleton in IAR-20 cells treated with EGF. ( a ) Immunostaining of E-cadherin and β-actin. Top—red and green channels merged. The cell-cell boundaries enlarged below are indicated with arrows. Disorganization of AJs begins 5 min after addition of EGF, simultaneously with dissolution of the circumferential actin bundle and formation of lamellipodia. Beginning from 10–15 min after the addition of EGF, punctate AJs were connected with straight actin fibers appear. ( b ) Fluorescence intensity profiles of F-actin and β-catenin in the zones of cell-cell contacts. N = 30 for every graph.

Techniques Used: Immunostaining, Fluorescence

Schematic representation of the actin cytoskeleton reorganization and rearrangement of E-cadherin-based AJs during EGF-induced EMT (created by I.Y. Zhitnyak).
Figure Legend Snippet: Schematic representation of the actin cytoskeleton reorganization and rearrangement of E-cadherin-based AJs during EGF-induced EMT (created by I.Y. Zhitnyak).

Techniques Used:

IAR-20 epithelial cells undergoing epidermal growth factor (EGF)-induced epithelial-mesenchymal transition (EMT). ( a ) In sparse culture, control IAR-20 epithelial cells form islands. DIC-microscopy. ( b ) In IAR-20 cells, the actin cytoskeleton is organized into the marginal actin bundle (asterisk) and circumferential actin bundles (arrow). ( c ) E-cadherin-based AJs (arrowhead) in an IAR-20 monolayer exhibit linear organization and colocalize with circumferential actin bundles (arrow). ( d ) Scattering of IAR-20 epithelial cells in response to EGF (50 ng/mL). In the control (45 min and 1 min before treatment with EGF), cells are joined into an island with stable cell-cell contacts. Addition of EGF leads to stimulation of protrusive activity at the free cell edges (cell 1), disruption of cell-cell contacts (asterisks), and initiation of cell migration. The migratory cells can form new transient contacts with neighboring cells (arrowheads). Both individual (cell 1) and collective (cells 2, 3, and 4) migration can be observed. Selected frames from Supplementary Video S1 . ( e ) The centroid trajectories of cells migrating for 6 h. ( f ) Western blot showing the expression levels of E-cadherin in IAR-20 cells treated with EGF. β-actin was used as loading control. Densitometry results are averaged across three independent experiments. Data are presented as mean ± SEM, * p
Figure Legend Snippet: IAR-20 epithelial cells undergoing epidermal growth factor (EGF)-induced epithelial-mesenchymal transition (EMT). ( a ) In sparse culture, control IAR-20 epithelial cells form islands. DIC-microscopy. ( b ) In IAR-20 cells, the actin cytoskeleton is organized into the marginal actin bundle (asterisk) and circumferential actin bundles (arrow). ( c ) E-cadherin-based AJs (arrowhead) in an IAR-20 monolayer exhibit linear organization and colocalize with circumferential actin bundles (arrow). ( d ) Scattering of IAR-20 epithelial cells in response to EGF (50 ng/mL). In the control (45 min and 1 min before treatment with EGF), cells are joined into an island with stable cell-cell contacts. Addition of EGF leads to stimulation of protrusive activity at the free cell edges (cell 1), disruption of cell-cell contacts (asterisks), and initiation of cell migration. The migratory cells can form new transient contacts with neighboring cells (arrowheads). Both individual (cell 1) and collective (cells 2, 3, and 4) migration can be observed. Selected frames from Supplementary Video S1 . ( e ) The centroid trajectories of cells migrating for 6 h. ( f ) Western blot showing the expression levels of E-cadherin in IAR-20 cells treated with EGF. β-actin was used as loading control. Densitometry results are averaged across three independent experiments. Data are presented as mean ± SEM, * p

Techniques Used: Microscopy, Stable Transfection, Activity Assay, Migration, Western Blot, Expressing

7) Product Images from "Na+/H+ exchangers are required for development and function of vertebrate mucociliary epithelia"

Article Title: Na+/H+ exchangers are required for development and function of vertebrate mucociliary epithelia

Journal: Cells, tissues, organs

doi: 10.1159/000492973

NHE1-3 loss-of-function affects neural tube closure as well as subapical actin formation and basal body positioning in MCCs ( A ) slc9a1. 2 or 3 were knocked down unilaterally (visualized by co-injection of RFP encoding mRNA; individual knockdown at 4pmol, combined knockdown at 1.33pmol of each MO) and neural tube closure was analyzed at stage 19-20. In contrast to control-injected embryos and the non-injected control side of individual embryos, slc9a ). Representative examples are shown in anterior-dorsal view and the outline of the injected neural folds is marked by dashed blue line. ( B ) Embryos were injected with clamp-rfp (red) mRNAs and indicated MOs (same set of embryos as depiced in A). Immunofluorescence staining against acetylated-α-tubulin (Ac.-α-Tub., blue) revealed cilia and phalloidin stained the actin cytoskeleton (green). Loss of NHE function induced cilia defects as well as loss of subapical actin links between basal bodies in all treatments. Lateral projections further revealed severe basal body apical transport defects in slc9a1 and 2 morphants, but only mild or no defects after combined knockdown or after slc9a3 MO injections. Scale bars indicate magnification.
Figure Legend Snippet: NHE1-3 loss-of-function affects neural tube closure as well as subapical actin formation and basal body positioning in MCCs ( A ) slc9a1. 2 or 3 were knocked down unilaterally (visualized by co-injection of RFP encoding mRNA; individual knockdown at 4pmol, combined knockdown at 1.33pmol of each MO) and neural tube closure was analyzed at stage 19-20. In contrast to control-injected embryos and the non-injected control side of individual embryos, slc9a ). Representative examples are shown in anterior-dorsal view and the outline of the injected neural folds is marked by dashed blue line. ( B ) Embryos were injected with clamp-rfp (red) mRNAs and indicated MOs (same set of embryos as depiced in A). Immunofluorescence staining against acetylated-α-tubulin (Ac.-α-Tub., blue) revealed cilia and phalloidin stained the actin cytoskeleton (green). Loss of NHE function induced cilia defects as well as loss of subapical actin links between basal bodies in all treatments. Lateral projections further revealed severe basal body apical transport defects in slc9a1 and 2 morphants, but only mild or no defects after combined knockdown or after slc9a3 MO injections. Scale bars indicate magnification.

Techniques Used: Injection, Immunofluorescence, Staining

8) Product Images from "Order and Disorder: The Role of Extracellular Matrix in Epithelial Cancer"

Article Title: Order and Disorder: The Role of Extracellular Matrix in Epithelial Cancer

Journal: Cancer investigation

doi:

Architecture of the basement membrane and cell surface connections. Basement membrane is composed of interconnected networks of collagen IV, laminin, nidogen/entactin, and proteoglycans such as perlecan. These connect to the cell through cell surface receptors such as integrins and dystroglycan. These, in turn, connect to the actin cytoskeleton through cytoplasmic adapter protein complexes.
Figure Legend Snippet: Architecture of the basement membrane and cell surface connections. Basement membrane is composed of interconnected networks of collagen IV, laminin, nidogen/entactin, and proteoglycans such as perlecan. These connect to the cell through cell surface receptors such as integrins and dystroglycan. These, in turn, connect to the actin cytoskeleton through cytoplasmic adapter protein complexes.

Techniques Used:

9) Product Images from "Cytoskeletal Rearrangements in Synovial Fibroblasts as a Novel Pathophysiological Determinant of Modeled Rheumatoid Arthritis"

Article Title: Cytoskeletal Rearrangements in Synovial Fibroblasts as a Novel Pathophysiological Determinant of Modeled Rheumatoid Arthritis

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.0010048

Mouse Arthritic SFs have a Rearranged Cytoskeleton and Increased ECM Adhesion (A) Immunofluoresence of arthritic (RA) or normal (WT) primary and immortalized synovial fibroblasts (pSFs and iSFs respectively) for F-actin. (B) Adhesion assays of arthritic (RA) or normal (WT) primary and immortalized SFs on purified ECM components as described in Materials and Methods. Error bars indicate standard deviation of triplicate samples from their mean value. (C) Transmission electron microscopy (TEM, magnification 5,000×) of SFs from ankle joints isolated from WT and arthritic mice. Arrowheads indicate dilation r-ER, while arrows point to swollen mitochondria with distorted cristae. N, nuclei.
Figure Legend Snippet: Mouse Arthritic SFs have a Rearranged Cytoskeleton and Increased ECM Adhesion (A) Immunofluoresence of arthritic (RA) or normal (WT) primary and immortalized synovial fibroblasts (pSFs and iSFs respectively) for F-actin. (B) Adhesion assays of arthritic (RA) or normal (WT) primary and immortalized SFs on purified ECM components as described in Materials and Methods. Error bars indicate standard deviation of triplicate samples from their mean value. (C) Transmission electron microscopy (TEM, magnification 5,000×) of SFs from ankle joints isolated from WT and arthritic mice. Arrowheads indicate dilation r-ER, while arrows point to swollen mitochondria with distorted cristae. N, nuclei.

Techniques Used: Purification, Standard Deviation, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Isolation, Mouse Assay

10) Product Images from "Opposing Activities of LIT-1/NLK and DAF-6/Patched-Related Direct Sensory Compartment Morphogenesis in C. elegans"

Article Title: Opposing Activities of LIT-1/NLK and DAF-6/Patched-Related Direct Sensory Compartment Morphogenesis in C. elegans

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1001121

The actin cytoskeleton is involved in amphid sensory compartment morphogenesis. (A) Growth assay (left) and quantitative β-galactosidase enzymatic activity assay (right) demonstrating the interaction between LexA fused to the LIT-1 carboxy-terminal domain and GAD fused to fragments of ACT-4 or WSP-1. Error bars, standard deviation. f, fragment. −WL, medium without Tryptophan and Leucine. –WLH, medium without Tryptophan, Leucine, and Histidine. 3AT, 3-amino-1,2,4-triazole. A.U., arbitrary units. (B) Amphid channel localization of GFP::ACT-4 (transgene nsEx2876 ). Anterior is to the left. Scale bar, 10 µm. (C, D) fEM (see Experimental Procedures) of a cross-section through the amphid channel (blue trace) just below the socket-sheath junction (C) or 2 µm posterior (D). White puncta indicate mEos::ACT-4 localization. Transgene used nsEx2970 . Asterisks, cilia. Scale bars, 1 µm. (E–G) Co-localization of GFP::WSP-1 and mCherry::LIT-1 at the amphid sensory compartment (transgene nsEx3245 ). The T02B11.3 amphid sheath promoter [32] was used to drive all constructs. Anterior is to the left. Scale bars, 10 µm. (H) The carboxy-terminal domain of LIT-1 co-immunoprecipitates with WSP-1. Drosophila S2 cells were transfected with HA::eGFP::LIT-1Ct and with or without MYC::WSP-1. Cell lysates were immunoprecipitated using anti-MYC-conjugated agarose beads and analyzed by anti-HA immunoblot. (I) Dye filling in animals of the indicated genotypes ( n ≥90). The alleles used are: daf-6 ( n1543 ), lit-1 ( ns132 ), wsp-1 ( gm324 ). daf-6 is marked with unc-3 ( e151 ) in all strains. unc-3 ( e151 ) does not affect dye filling (unpublished data). Error bars, SEM. See also Table S2 .
Figure Legend Snippet: The actin cytoskeleton is involved in amphid sensory compartment morphogenesis. (A) Growth assay (left) and quantitative β-galactosidase enzymatic activity assay (right) demonstrating the interaction between LexA fused to the LIT-1 carboxy-terminal domain and GAD fused to fragments of ACT-4 or WSP-1. Error bars, standard deviation. f, fragment. −WL, medium without Tryptophan and Leucine. –WLH, medium without Tryptophan, Leucine, and Histidine. 3AT, 3-amino-1,2,4-triazole. A.U., arbitrary units. (B) Amphid channel localization of GFP::ACT-4 (transgene nsEx2876 ). Anterior is to the left. Scale bar, 10 µm. (C, D) fEM (see Experimental Procedures) of a cross-section through the amphid channel (blue trace) just below the socket-sheath junction (C) or 2 µm posterior (D). White puncta indicate mEos::ACT-4 localization. Transgene used nsEx2970 . Asterisks, cilia. Scale bars, 1 µm. (E–G) Co-localization of GFP::WSP-1 and mCherry::LIT-1 at the amphid sensory compartment (transgene nsEx3245 ). The T02B11.3 amphid sheath promoter [32] was used to drive all constructs. Anterior is to the left. Scale bars, 10 µm. (H) The carboxy-terminal domain of LIT-1 co-immunoprecipitates with WSP-1. Drosophila S2 cells were transfected with HA::eGFP::LIT-1Ct and with or without MYC::WSP-1. Cell lysates were immunoprecipitated using anti-MYC-conjugated agarose beads and analyzed by anti-HA immunoblot. (I) Dye filling in animals of the indicated genotypes ( n ≥90). The alleles used are: daf-6 ( n1543 ), lit-1 ( ns132 ), wsp-1 ( gm324 ). daf-6 is marked with unc-3 ( e151 ) in all strains. unc-3 ( e151 ) does not affect dye filling (unpublished data). Error bars, SEM. See also Table S2 .

Techniques Used: Growth Assay, Enzyme Activity Assay, Activated Clotting Time Assay, Standard Deviation, Construct, Transfection, Immunoprecipitation

11) Product Images from "Hypoxia alters the recruitment of tropomyosins into the actin stress fibres of neuroblastoma cells"

Article Title: Hypoxia alters the recruitment of tropomyosins into the actin stress fibres of neuroblastoma cells

Journal: BMC Cancer

doi: 10.1186/s12885-015-1741-8

Hypoxia leads to a more ordered actin cytoskeleton. SH-EP cells were grown in normoxia (20 % O 2 ) or hypoxia (1 % O 2 ) for 72 – 96 h. Coverslips were fixed with 4 % PFA and stained with TRITC-phalloidin and DAPI. Single z-plane images were obtained by confocal microscopy. Linear feature detection software was used to quantitate actin cytoskeletal organization. a - d Representative linear detection after 72 h ± hypoxia. Hypoxia visibly increases the number of actin filament bundles known as stress fibres ( b vs. d ). Scale bar = 50 μm, or 5 μm in inset. e Hypoxia increases mean actin filament bundle width at 72 h. f Hypoxia increases total actin filament length per cell number ( AFLC ) and ( g ) per cell area ( AFLA ). Data is mean ± SEM ( n = 3). 650+ cells analysed per timepoint. ** P
Figure Legend Snippet: Hypoxia leads to a more ordered actin cytoskeleton. SH-EP cells were grown in normoxia (20 % O 2 ) or hypoxia (1 % O 2 ) for 72 – 96 h. Coverslips were fixed with 4 % PFA and stained with TRITC-phalloidin and DAPI. Single z-plane images were obtained by confocal microscopy. Linear feature detection software was used to quantitate actin cytoskeletal organization. a - d Representative linear detection after 72 h ± hypoxia. Hypoxia visibly increases the number of actin filament bundles known as stress fibres ( b vs. d ). Scale bar = 50 μm, or 5 μm in inset. e Hypoxia increases mean actin filament bundle width at 72 h. f Hypoxia increases total actin filament length per cell number ( AFLC ) and ( g ) per cell area ( AFLA ). Data is mean ± SEM ( n = 3). 650+ cells analysed per timepoint. ** P

Techniques Used: Staining, Confocal Microscopy, Software

12) Product Images from "Mechanisms of Epithelial Cell-Cell Adhesion and Cell Compaction Revealed by High-resolution Tracking of E-Cadherin- Green Fluorescent Protein "

Article Title: Mechanisms of Epithelial Cell-Cell Adhesion and Cell Compaction Revealed by High-resolution Tracking of E-Cadherin- Green Fluorescent Protein

Journal: The Journal of Cell Biology

doi:

Redistribution of EcadGFP during cell–cell contact occurs in two stages and correlates with reorganization of the actin cytoskeleton. Three 0.3-μm z-sections were collected from EcadGFP-expressing cells every 4.2 min at 0.5 micron/pixel at 6 sites. ( A ) Combined stacks from two sites are shown ( Contact 1 and Contact 2 ). The age of each contact is displayed in min; the zero time point defines when a stable cell–cell contact had formed. Bar, 12 μm. The arrow points to a circumferential pattern of EcadGFP observed in single cells. ( B and C ) Immunofluorescence of the same cells stained, after formaldehyde fixation, with rhodamine phalloidin (actin; B ) and mAb 3G8/CY5 (E-cadherin; C ). ( D ) EcadGFP organization shown in C is plotted as a function of fluorescence intensity (y axis, arbitrary fluorescence units) vs. position in the contact (x axis, μm). The dashed gray lines running between C and D approximately register the edges of the contact in the image and graph. ( E and F ) Triton X-100 extracted wild-type MDCK cells (i.e., without EcadGFP) that had formed a contact for
Figure Legend Snippet: Redistribution of EcadGFP during cell–cell contact occurs in two stages and correlates with reorganization of the actin cytoskeleton. Three 0.3-μm z-sections were collected from EcadGFP-expressing cells every 4.2 min at 0.5 micron/pixel at 6 sites. ( A ) Combined stacks from two sites are shown ( Contact 1 and Contact 2 ). The age of each contact is displayed in min; the zero time point defines when a stable cell–cell contact had formed. Bar, 12 μm. The arrow points to a circumferential pattern of EcadGFP observed in single cells. ( B and C ) Immunofluorescence of the same cells stained, after formaldehyde fixation, with rhodamine phalloidin (actin; B ) and mAb 3G8/CY5 (E-cadherin; C ). ( D ) EcadGFP organization shown in C is plotted as a function of fluorescence intensity (y axis, arbitrary fluorescence units) vs. position in the contact (x axis, μm). The dashed gray lines running between C and D approximately register the edges of the contact in the image and graph. ( E and F ) Triton X-100 extracted wild-type MDCK cells (i.e., without EcadGFP) that had formed a contact for

Techniques Used: Expressing, Stable Transfection, Immunofluorescence, Staining, Fluorescence

A three-stage model for cell–cell adhesion and colony formation. Stage I: multiple E-cadherin puncta form along the developing contact and loosly hold contacting cells together. A circumferential actin cable ( thick red line ) surrounds isolated cells. As cells adhere, E-cadherin clusters into puncta within the cell–cell contact interface ( blue circle ) and rapidly associates with thin actin bundles and filaments ( thin red lines ). As the contact lengthens, puncta continue to develop along the length of the contact at a constant average density during the first 2 h. Stage II: E-cadherin plaques develop at the edges of the contact which compact and strengthen cell–cell interactions. Stabilization of actin filaments by E-cadherin puncta within the cell–cell contact results in gradual dissolution of the circumferential actin cable behind the developing contact and insertion of the circumferential actin cables into the cell–cell contact accompanied by additional clustering of E-cadherin puncta into E-cadherin plaques ( green ovals ). Between cell plaques, E-cadherin is more diffusely distributed ( green line ) and associates with actin filaments oriented along the axis of the cell–cell contact ( red line ). Stage III: E-cadherin plaques cinch together to form multicellular vertices, further condensing cell colonies. In multicellular colonies, contractility within the circumferential actin cable brings E-cadherin plaques from adjacent cells together. Dynamics within the cytoskeleton result in continual rearrangement.
Figure Legend Snippet: A three-stage model for cell–cell adhesion and colony formation. Stage I: multiple E-cadherin puncta form along the developing contact and loosly hold contacting cells together. A circumferential actin cable ( thick red line ) surrounds isolated cells. As cells adhere, E-cadherin clusters into puncta within the cell–cell contact interface ( blue circle ) and rapidly associates with thin actin bundles and filaments ( thin red lines ). As the contact lengthens, puncta continue to develop along the length of the contact at a constant average density during the first 2 h. Stage II: E-cadherin plaques develop at the edges of the contact which compact and strengthen cell–cell interactions. Stabilization of actin filaments by E-cadherin puncta within the cell–cell contact results in gradual dissolution of the circumferential actin cable behind the developing contact and insertion of the circumferential actin cables into the cell–cell contact accompanied by additional clustering of E-cadherin puncta into E-cadherin plaques ( green ovals ). Between cell plaques, E-cadherin is more diffusely distributed ( green line ) and associates with actin filaments oriented along the axis of the cell–cell contact ( red line ). Stage III: E-cadherin plaques cinch together to form multicellular vertices, further condensing cell colonies. In multicellular colonies, contractility within the circumferential actin cable brings E-cadherin plaques from adjacent cells together. Dynamics within the cytoskeleton result in continual rearrangement.

Techniques Used: Isolation

13) Product Images from "Mechanisms of Epithelial Cell-Cell Adhesion and Cell Compaction Revealed by High-resolution Tracking of E-Cadherin- Green Fluorescent Protein "

Article Title: Mechanisms of Epithelial Cell-Cell Adhesion and Cell Compaction Revealed by High-resolution Tracking of E-Cadherin- Green Fluorescent Protein

Journal: The Journal of Cell Biology

doi:

Redistribution of EcadGFP during cell–cell contact occurs in two stages and correlates with reorganization of the actin cytoskeleton. Three 0.3-μm z-sections were collected from EcadGFP-expressing cells every 4.2 min at 0.5 micron/pixel at 6 sites. ( A ) Combined stacks from two sites are shown ( Contact 1 and Contact 2 ). The age of each contact is displayed in min; the zero time point defines when a stable cell–cell contact had formed. Bar, 12 μm. The arrow points to a circumferential pattern of EcadGFP observed in single cells. ( B and C ) Immunofluorescence of the same cells stained, after formaldehyde fixation, with rhodamine phalloidin (actin; B ) and mAb 3G8/CY5 (E-cadherin; C ). ( D ) EcadGFP organization shown in C is plotted as a function of fluorescence intensity (y axis, arbitrary fluorescence units) vs. position in the contact (x axis, μm). The dashed gray lines running between C and D approximately register the edges of the contact in the image and graph. ( E and F ) Triton X-100 extracted wild-type MDCK cells (i.e., without EcadGFP) that had formed a contact for
Figure Legend Snippet: Redistribution of EcadGFP during cell–cell contact occurs in two stages and correlates with reorganization of the actin cytoskeleton. Three 0.3-μm z-sections were collected from EcadGFP-expressing cells every 4.2 min at 0.5 micron/pixel at 6 sites. ( A ) Combined stacks from two sites are shown ( Contact 1 and Contact 2 ). The age of each contact is displayed in min; the zero time point defines when a stable cell–cell contact had formed. Bar, 12 μm. The arrow points to a circumferential pattern of EcadGFP observed in single cells. ( B and C ) Immunofluorescence of the same cells stained, after formaldehyde fixation, with rhodamine phalloidin (actin; B ) and mAb 3G8/CY5 (E-cadherin; C ). ( D ) EcadGFP organization shown in C is plotted as a function of fluorescence intensity (y axis, arbitrary fluorescence units) vs. position in the contact (x axis, μm). The dashed gray lines running between C and D approximately register the edges of the contact in the image and graph. ( E and F ) Triton X-100 extracted wild-type MDCK cells (i.e., without EcadGFP) that had formed a contact for

Techniques Used: Expressing, Stable Transfection, Immunofluorescence, Staining, Fluorescence

A three-stage model for cell–cell adhesion and colony formation. Stage I: multiple E-cadherin puncta form along the developing contact and loosly hold contacting cells together. A circumferential actin cable ( thick red line ) surrounds isolated cells. As cells adhere, E-cadherin clusters into puncta within the cell–cell contact interface ( blue circle ) and rapidly associates with thin actin bundles and filaments ( thin red lines ). As the contact lengthens, puncta continue to develop along the length of the contact at a constant average density during the first 2 h. Stage II: E-cadherin plaques develop at the edges of the contact which compact and strengthen cell–cell interactions. Stabilization of actin filaments by E-cadherin puncta within the cell–cell contact results in gradual dissolution of the circumferential actin cable behind the developing contact and insertion of the circumferential actin cables into the cell–cell contact accompanied by additional clustering of E-cadherin puncta into E-cadherin plaques ( green ovals ). Between cell plaques, E-cadherin is more diffusely distributed ( green line ) and associates with actin filaments oriented along the axis of the cell–cell contact ( red line ). Stage III: E-cadherin plaques cinch together to form multicellular vertices, further condensing cell colonies. In multicellular colonies, contractility within the circumferential actin cable brings E-cadherin plaques from adjacent cells together. Dynamics within the cytoskeleton result in continual rearrangement.
Figure Legend Snippet: A three-stage model for cell–cell adhesion and colony formation. Stage I: multiple E-cadherin puncta form along the developing contact and loosly hold contacting cells together. A circumferential actin cable ( thick red line ) surrounds isolated cells. As cells adhere, E-cadherin clusters into puncta within the cell–cell contact interface ( blue circle ) and rapidly associates with thin actin bundles and filaments ( thin red lines ). As the contact lengthens, puncta continue to develop along the length of the contact at a constant average density during the first 2 h. Stage II: E-cadherin plaques develop at the edges of the contact which compact and strengthen cell–cell interactions. Stabilization of actin filaments by E-cadherin puncta within the cell–cell contact results in gradual dissolution of the circumferential actin cable behind the developing contact and insertion of the circumferential actin cables into the cell–cell contact accompanied by additional clustering of E-cadherin puncta into E-cadherin plaques ( green ovals ). Between cell plaques, E-cadherin is more diffusely distributed ( green line ) and associates with actin filaments oriented along the axis of the cell–cell contact ( red line ). Stage III: E-cadherin plaques cinch together to form multicellular vertices, further condensing cell colonies. In multicellular colonies, contractility within the circumferential actin cable brings E-cadherin plaques from adjacent cells together. Dynamics within the cytoskeleton result in continual rearrangement.

Techniques Used: Isolation

14) Product Images from "A Role for the Actin Cytoskeleton of Saccharomyces cerevisiae in Bipolar Bud-Site Selection"

Article Title: A Role for the Actin Cytoskeleton of Saccharomyces cerevisiae in Bipolar Bud-Site Selection

Journal: The Journal of Cell Biology

doi:

Pseudo–wild-type actin mutants stained with rhodamine-phalloidin to visualize the actin cytoskeleton. ( a ) DDY440, wild-type cells; ( b ) DDY976, act1-115 mutant cells; ( c ) DDY977, act1-116 mutant cells; and ( d ) DDY978, act1-117 mutant cells. act1-116 and act1117 mutants show a bipolar budding defect (with act1-116 showing the more pronounced defect), while act1-115 mutants do not show a bipolar budding defect. The same exposure and printing times were used for each panel. Bar, 5 μm.
Figure Legend Snippet: Pseudo–wild-type actin mutants stained with rhodamine-phalloidin to visualize the actin cytoskeleton. ( a ) DDY440, wild-type cells; ( b ) DDY976, act1-115 mutant cells; ( c ) DDY977, act1-116 mutant cells; and ( d ) DDY978, act1-117 mutant cells. act1-116 and act1117 mutants show a bipolar budding defect (with act1-116 showing the more pronounced defect), while act1-115 mutants do not show a bipolar budding defect. The same exposure and printing times were used for each panel. Bar, 5 μm.

Techniques Used: Staining, Mutagenesis

Models for the placement of the spatial cues for the bipolar budding pattern. The placement of bipolar budding cues (represented by the shaded areas), starting from a daughter cell. (Models 1 and 2 are adapted from Chant and Pringle [1995] and Zahner et al. [1996]). The left pole represents the proximal pole with the birth scar being represented by a thin curved line. ( A ) Wild-type diploid cells. For model 1, the spatial budding cue is concentrated at the presumptive bud site at the beginning of the cell cycle. As the bud emerges, the cue is partitioned between the growing tip and the mother–daughter neck. For model 2, the cue is first positioned at the bud tip by the cell surface growth apparatus as the bud emerges and grows. When the growth apparatus is reoriented toward the mother–daughter neck for septation late in the cell cycle, the cue is placed at the neck region and is partitioned between the mother and daughter at cytokinesis. These models do not attempt to explain why daughter cells have an extremely strong bias toward forming their first bud at the distal pole. ( B ) Based on the two models in A several possible explanations exist for the bipolar budding defect in diploid mother cells with an altered actin cytoskeleton. In all of the models, the bipolar budding cue is initially placed correctly at the distal pole, and to a lesser degree at the proximal pole, in daughter cells. For model 1a, the cue might become properly partitioned, but there is a defect in retention of the cue at the region where septation ocurred. For model 1b, the cue might not get properly partitioned as the bud emerges. For model 2, the actin cytoskeleton might function properly in cytokinesis, but might be defective in placement and/or maintenance of the cue at the septal region.
Figure Legend Snippet: Models for the placement of the spatial cues for the bipolar budding pattern. The placement of bipolar budding cues (represented by the shaded areas), starting from a daughter cell. (Models 1 and 2 are adapted from Chant and Pringle [1995] and Zahner et al. [1996]). The left pole represents the proximal pole with the birth scar being represented by a thin curved line. ( A ) Wild-type diploid cells. For model 1, the spatial budding cue is concentrated at the presumptive bud site at the beginning of the cell cycle. As the bud emerges, the cue is partitioned between the growing tip and the mother–daughter neck. For model 2, the cue is first positioned at the bud tip by the cell surface growth apparatus as the bud emerges and grows. When the growth apparatus is reoriented toward the mother–daughter neck for septation late in the cell cycle, the cue is placed at the neck region and is partitioned between the mother and daughter at cytokinesis. These models do not attempt to explain why daughter cells have an extremely strong bias toward forming their first bud at the distal pole. ( B ) Based on the two models in A several possible explanations exist for the bipolar budding defect in diploid mother cells with an altered actin cytoskeleton. In all of the models, the bipolar budding cue is initially placed correctly at the distal pole, and to a lesser degree at the proximal pole, in daughter cells. For model 1a, the cue might become properly partitioned, but there is a defect in retention of the cue at the region where septation ocurred. For model 1b, the cue might not get properly partitioned as the bud emerges. For model 2, the actin cytoskeleton might function properly in cytokinesis, but might be defective in placement and/or maintenance of the cue at the septal region.

Techniques Used:

15) Product Images from "The Membrane Skeleton Controls Diffusion Dynamics and Signaling through the B Cell Receptor"

Article Title: The Membrane Skeleton Controls Diffusion Dynamics and Signaling through the B Cell Receptor

Journal: Immunity

doi: 10.1016/j.immuni.2009.12.005

The Membrane Cytoskeleton Linker Protein Ezrin Regulates BCR Diffusion in Resting B Cells (A) Dual-color TIRFM to visualize the distribution of Lifeact-mRFPruby (red) together with Ezrin-GFP (cyan) in A20 B cells on fibronectin-coated coverslips. (B) Selected pseudocolor TIRFM images of magnified view of Ezrin-GFP from (A) indicated by white square. Threshold outline (white) shows rapidly modified ezrin “holes.” (C–H) Dual-view TIRFM to simultaneously visualize ezrin-GFP (cyan) and track single molecules of IgM (BCR) in A20 B cells on fibronectin-coated coverslips. (C) Selected TIRFM images from 200 frames/10 s time sequence at the indicated times during tracking of BCR (red). The images in the right panels are magnified time sequence images of the left panel (white square) with an example of a 2D trajectory indicated in yellow, with the diffusing particle outlined with a white circle. (D) Magnified image showing 2D trajectories of BCR inside (red) and outside (yellow) ezrin-rich regions (grayscale). Magnified trajectories of IgM (E) inside (red) and (F) outside (yellow) ezrin-rich regions demarcated by white lines. (G) Diffusion coefficients of single molecules of BCR inside (circles) and outside (triangles) ezrin-rich areas with the median indicated by red bar. (H) Relative frequencies of single molecules of BCR inside (circles) and outside (triangles) actin-rich areas with diffusion coefficients in the indicated bins. ∗∗∗ p
Figure Legend Snippet: The Membrane Cytoskeleton Linker Protein Ezrin Regulates BCR Diffusion in Resting B Cells (A) Dual-color TIRFM to visualize the distribution of Lifeact-mRFPruby (red) together with Ezrin-GFP (cyan) in A20 B cells on fibronectin-coated coverslips. (B) Selected pseudocolor TIRFM images of magnified view of Ezrin-GFP from (A) indicated by white square. Threshold outline (white) shows rapidly modified ezrin “holes.” (C–H) Dual-view TIRFM to simultaneously visualize ezrin-GFP (cyan) and track single molecules of IgM (BCR) in A20 B cells on fibronectin-coated coverslips. (C) Selected TIRFM images from 200 frames/10 s time sequence at the indicated times during tracking of BCR (red). The images in the right panels are magnified time sequence images of the left panel (white square) with an example of a 2D trajectory indicated in yellow, with the diffusing particle outlined with a white circle. (D) Magnified image showing 2D trajectories of BCR inside (red) and outside (yellow) ezrin-rich regions (grayscale). Magnified trajectories of IgM (E) inside (red) and (F) outside (yellow) ezrin-rich regions demarcated by white lines. (G) Diffusion coefficients of single molecules of BCR inside (circles) and outside (triangles) ezrin-rich areas with the median indicated by red bar. (H) Relative frequencies of single molecules of BCR inside (circles) and outside (triangles) actin-rich areas with diffusion coefficients in the indicated bins. ∗∗∗ p

Techniques Used: Diffusion-based Assay, Modification, Sequencing

Alteration of the Actin Cytoskeleton Is Sufficient to Induce BCR Signaling (A–H) Alteration of the actin cytoskeleton induces intracellular signaling. (A–E) Ratiometric intracellular Ca 2+ flux in primary naive B cells upon addition (indicated by black arrow) of vehicle control (DMSO) (A), 5 μg/ml anti-IgM F(‘ab) 2 (B), 0.5 μM LatA (C), 10 μM CytoD (D), or 1 μM JP (E) measured by flow cytometry. Mean indicated by red line. (F) Primary naive B cells were treated with 0.5 μM LatA (+) or vehicle control (DMSO) (–) at 37°C for the indicated time. Cells were lysed and analyzed by SDS-PAGE followed by immunoblotting with anti-phospho-p44 and 42 MAPK (Erk1 and 2), anti-phospho-Akt, or anti-p44 and 42 MAPK. (G) Quantification of the fold increase in pERK and pAkt upon LatA treatment. (H) Primary naive B cells were treated or not (gray shaded) with 0.5 μM LatA (red line), 10 μM Cyto D (blue line), 1 μM JP (green line), or 5 μg/ml anti-IgM F(‘ab) 2 (black dotted line) for 5 min and then cultured for 24 hr. Cells were stained for the activation marker CD86 and analyzed by flow cytometry. (I–L) Signaling induced by alteration of actin is predominantly mediated via the BCR. Ratiometric intracellular Ca 2+ flux in primary wild-type (WT), PLCγ2-deficient, and Vav1 and 2 double-deficient B cells treated with 0.5 μM Lat A (I) or 200 ng/ml SDF-1 (J) measured by flow cytometry. (K) Wild-type DT40 and various signaling-deficient cells including Lyn −/− (Lyn-KO), Blnk −/− (BLNK-KO), Btk −/− (Btk-KO), Plcg2 −/− (PLCγ2-KO), Itpr1 −/− Itpr2 −/− Itpr3 −/− (IP3R-KO), Vav3 −/− (Vav3-KO), and Pik3ca −/− (PI3K-KO) were treated with 0.5 μM LatA or vehicle control (DMSO) for 5 min at 37°C. Cells were lysed and analyzed by SDS-PAGE followed by immunoblotting with anti-phospho-p44 and 42 MAPK (Erk1 and 2) and anti-p44 and 42 MAPK. (L) Quantification of the induction of pERK upon LatA treatment shown in (K).
Figure Legend Snippet: Alteration of the Actin Cytoskeleton Is Sufficient to Induce BCR Signaling (A–H) Alteration of the actin cytoskeleton induces intracellular signaling. (A–E) Ratiometric intracellular Ca 2+ flux in primary naive B cells upon addition (indicated by black arrow) of vehicle control (DMSO) (A), 5 μg/ml anti-IgM F(‘ab) 2 (B), 0.5 μM LatA (C), 10 μM CytoD (D), or 1 μM JP (E) measured by flow cytometry. Mean indicated by red line. (F) Primary naive B cells were treated with 0.5 μM LatA (+) or vehicle control (DMSO) (–) at 37°C for the indicated time. Cells were lysed and analyzed by SDS-PAGE followed by immunoblotting with anti-phospho-p44 and 42 MAPK (Erk1 and 2), anti-phospho-Akt, or anti-p44 and 42 MAPK. (G) Quantification of the fold increase in pERK and pAkt upon LatA treatment. (H) Primary naive B cells were treated or not (gray shaded) with 0.5 μM LatA (red line), 10 μM Cyto D (blue line), 1 μM JP (green line), or 5 μg/ml anti-IgM F(‘ab) 2 (black dotted line) for 5 min and then cultured for 24 hr. Cells were stained for the activation marker CD86 and analyzed by flow cytometry. (I–L) Signaling induced by alteration of actin is predominantly mediated via the BCR. Ratiometric intracellular Ca 2+ flux in primary wild-type (WT), PLCγ2-deficient, and Vav1 and 2 double-deficient B cells treated with 0.5 μM Lat A (I) or 200 ng/ml SDF-1 (J) measured by flow cytometry. (K) Wild-type DT40 and various signaling-deficient cells including Lyn −/− (Lyn-KO), Blnk −/− (BLNK-KO), Btk −/− (Btk-KO), Plcg2 −/− (PLCγ2-KO), Itpr1 −/− Itpr2 −/− Itpr3 −/− (IP3R-KO), Vav3 −/− (Vav3-KO), and Pik3ca −/− (PI3K-KO) were treated with 0.5 μM LatA or vehicle control (DMSO) for 5 min at 37°C. Cells were lysed and analyzed by SDS-PAGE followed by immunoblotting with anti-phospho-p44 and 42 MAPK (Erk1 and 2) and anti-p44 and 42 MAPK. (L) Quantification of the induction of pERK upon LatA treatment shown in (K).

Techniques Used: Flow Cytometry, Cytometry, SDS Page, Cell Culture, Staining, Activation Assay, Marker

BCR Diffusion Is Decreased in B Cells Deficient in PLCγ2 and Vav1/2 (A and B) Signaling-deficient B cells have altered actin cytoskeleton. (A) DT40 WT or Syk-deficient B cells were allowed to settle on fibronectin-coated coverslips and cell morphology and adhesion were examined by differential interference contrast (DIC) and interference reflection microscopy (IRM). (B) DT40 WT and Syk-deficient B cells on fibronectin-coated coverslips were fixed and F-actin was stained with phalloidin and visualized by TIRFM. Scale bars represent 2 μm. (C and D) Signaling-deficient B cells have reduced IgM diffusion. Single-molecule tracking of IgM in primary naive B6 (black circle), PLCγ2-deficient (green square), and Vav1 and 2 double-deficient (blue triangle). Diffusion coefficients (C) and distribution histogram (D) with diffusion coefficients in the indicated bins. Median indicated by red line. ∗∗∗ p
Figure Legend Snippet: BCR Diffusion Is Decreased in B Cells Deficient in PLCγ2 and Vav1/2 (A and B) Signaling-deficient B cells have altered actin cytoskeleton. (A) DT40 WT or Syk-deficient B cells were allowed to settle on fibronectin-coated coverslips and cell morphology and adhesion were examined by differential interference contrast (DIC) and interference reflection microscopy (IRM). (B) DT40 WT and Syk-deficient B cells on fibronectin-coated coverslips were fixed and F-actin was stained with phalloidin and visualized by TIRFM. Scale bars represent 2 μm. (C and D) Signaling-deficient B cells have reduced IgM diffusion. Single-molecule tracking of IgM in primary naive B6 (black circle), PLCγ2-deficient (green square), and Vav1 and 2 double-deficient (blue triangle). Diffusion coefficients (C) and distribution histogram (D) with diffusion coefficients in the indicated bins. Median indicated by red line. ∗∗∗ p

Techniques Used: Diffusion-based Assay, Microscopy, Staining

The Actin Cytoskeleton Defines BCR Diffusion Dynamics in Resting B Cells Dual-view TIRFM to simultaneously visualize Lifeact-GFP (green) and track single molecules of IgM (A–F) or IgM-H2 (G–J) in A20 B cells on fibronectin-coated coverslips. (A and G) Selected TIRFM images from 200 frame/10 s time sequence at the indicated times during tracking of IgM or IgM-H2 (red). The images in the right panels are magnified time sequences of the left panel (white square) with an example of a 2D trajectory of IgM (A) or IgM-H2 (G) indicated in yellow, with the diffusing particle outlined with a white circle. (B and H) Magnified image showing trajectories of IgM (B) or IgM-H2 (H) inside (red) and outside (yellow) actin-rich regions (grayscale). (C and D) Trajectories of IgM inside (C) and outside (D) actin-rich regions, demarcated by white lines. (E and I) Diffusion coefficients of single molecules of IgM or IgM-H2 inside (circles) and outside (triangles) actin-rich areas with the median indicated by red bar. (F and J) Relative frequencies of single molecules of IgM (F) or IgM-H2 (J) inside (circles) and outside (triangles) actin-rich areas with diffusion coefficients in the indicated bins. Scale bar represents 2 μm. ∗∗∗ p
Figure Legend Snippet: The Actin Cytoskeleton Defines BCR Diffusion Dynamics in Resting B Cells Dual-view TIRFM to simultaneously visualize Lifeact-GFP (green) and track single molecules of IgM (A–F) or IgM-H2 (G–J) in A20 B cells on fibronectin-coated coverslips. (A and G) Selected TIRFM images from 200 frame/10 s time sequence at the indicated times during tracking of IgM or IgM-H2 (red). The images in the right panels are magnified time sequences of the left panel (white square) with an example of a 2D trajectory of IgM (A) or IgM-H2 (G) indicated in yellow, with the diffusing particle outlined with a white circle. (B and H) Magnified image showing trajectories of IgM (B) or IgM-H2 (H) inside (red) and outside (yellow) actin-rich regions (grayscale). (C and D) Trajectories of IgM inside (C) and outside (D) actin-rich regions, demarcated by white lines. (E and I) Diffusion coefficients of single molecules of IgM or IgM-H2 inside (circles) and outside (triangles) actin-rich areas with the median indicated by red bar. (F and J) Relative frequencies of single molecules of IgM (F) or IgM-H2 (J) inside (circles) and outside (triangles) actin-rich areas with diffusion coefficients in the indicated bins. Scale bar represents 2 μm. ∗∗∗ p

Techniques Used: Diffusion-based Assay, Sequencing

Signaling Induced by Disruption of Actin Correlates with BCR Diffusion (A–E) Pharmacological agents of actin disruption increase BCR diffusion. (A) Lifeact-GFP-expressing cells were visualized by TIRFM before (top) and 5 min after treatment (bottom) with 0.5 μM LatA, 10 μM CytoD, or 2 μM JP. (B and C) Single-molecule tracking of IgM in primary naive B cells on MHC II-coated coverslips upon treatment with 0.5 μM LatA (red), 10 μM CytoD (blue), 2 μM JP (green), or vehicle control (DMSO; black). Diffusion coefficients with the median indicated in red (B) and distribution histogram (C) with diffusion coefficients in the indicated bins. (D and E) Single-molecule tracking of IgD in primary naive B cells treated with 0.5 μM LatA (red squares) or vehicle control (black circles). Diffusion coefficients with the median indicated in red (D) and distribution histogram (E) with diffusion coefficients in the indicated bins. (F and G) Genetic alteration of membrane-cytoskeleton link modifies BCR diffusion. Diffusion coefficients of single molecules of BCR in A20 B cells expressing wild-type ezrin-GFP (circles) and ezrin-310-GFP (squares) (F) or DT40 B cells expressing wild-type ezrin-GFP (circles) and ezrin-TD-GFP (squares) (G) with the median indicated in red. (H–K) Increasing concentrations of CytoD increase intracellular calcium flux and the mobile fraction of the BCR. (H) Ratiometric intracellular Ca 2+ flux in primary naive B cells treated with 0.5–10 μM CytoD measured by flow cytometry. (I and J) Diffusion coefficients (I) and distribution histogram (J) of single molecules of IgM in primary naive B cells upon treatment with increasing concentrations of CytoD. (K) The integrated area under the calcium curve in (H) was plotted against the mobile fraction (all bins except lowest) of the BCR from (J). ∗∗∗ p
Figure Legend Snippet: Signaling Induced by Disruption of Actin Correlates with BCR Diffusion (A–E) Pharmacological agents of actin disruption increase BCR diffusion. (A) Lifeact-GFP-expressing cells were visualized by TIRFM before (top) and 5 min after treatment (bottom) with 0.5 μM LatA, 10 μM CytoD, or 2 μM JP. (B and C) Single-molecule tracking of IgM in primary naive B cells on MHC II-coated coverslips upon treatment with 0.5 μM LatA (red), 10 μM CytoD (blue), 2 μM JP (green), or vehicle control (DMSO; black). Diffusion coefficients with the median indicated in red (B) and distribution histogram (C) with diffusion coefficients in the indicated bins. (D and E) Single-molecule tracking of IgD in primary naive B cells treated with 0.5 μM LatA (red squares) or vehicle control (black circles). Diffusion coefficients with the median indicated in red (D) and distribution histogram (E) with diffusion coefficients in the indicated bins. (F and G) Genetic alteration of membrane-cytoskeleton link modifies BCR diffusion. Diffusion coefficients of single molecules of BCR in A20 B cells expressing wild-type ezrin-GFP (circles) and ezrin-310-GFP (squares) (F) or DT40 B cells expressing wild-type ezrin-GFP (circles) and ezrin-TD-GFP (squares) (G) with the median indicated in red. (H–K) Increasing concentrations of CytoD increase intracellular calcium flux and the mobile fraction of the BCR. (H) Ratiometric intracellular Ca 2+ flux in primary naive B cells treated with 0.5–10 μM CytoD measured by flow cytometry. (I and J) Diffusion coefficients (I) and distribution histogram (J) of single molecules of IgM in primary naive B cells upon treatment with increasing concentrations of CytoD. (K) The integrated area under the calcium curve in (H) was plotted against the mobile fraction (all bins except lowest) of the BCR from (J). ∗∗∗ p

Techniques Used: Diffusion-based Assay, Expressing, Flow Cytometry, Cytometry

16) Product Images from "Patterns of gene expression associated with recovery and injury in heat-stressed rats"

Article Title: Patterns of gene expression associated with recovery and injury in heat-stressed rats

Journal: BMC Genomics

doi: 10.1186/1471-2164-15-1058

Aggregation propensities for modulated pathways. Proteins and genes with concordant differential expression in heat-injured animals at 48 hours were mapped to subcellular localization (see Figure 6 ). Up-regulated proteins/gene pairs mapped to the KEGG pathways Ribosome, Antigen Processing and Presentation, Regulation of Actin Cytoskeleton, Complement and Coagulation Cascade, and Pentose Phosphate Pathway. Down-regulated protein/gene pairs mapped to Hypertrophic/Dilated Cardiomyopathy and Cardiac Muscle Contraction, Fatty Acid Metabolism, and Oxidative Phosphorylation. Gene/protein pairs are mapped to target subcellular organelles. For simplicity in presentation, only protein designations are shown.
Figure Legend Snippet: Aggregation propensities for modulated pathways. Proteins and genes with concordant differential expression in heat-injured animals at 48 hours were mapped to subcellular localization (see Figure 6 ). Up-regulated proteins/gene pairs mapped to the KEGG pathways Ribosome, Antigen Processing and Presentation, Regulation of Actin Cytoskeleton, Complement and Coagulation Cascade, and Pentose Phosphate Pathway. Down-regulated protein/gene pairs mapped to Hypertrophic/Dilated Cardiomyopathy and Cardiac Muscle Contraction, Fatty Acid Metabolism, and Oxidative Phosphorylation. Gene/protein pairs are mapped to target subcellular organelles. For simplicity in presentation, only protein designations are shown.

Techniques Used: Expressing, Coagulation

17) Product Images from "Subcellular Control Over Focal Adhesion Anisotropy, Independent of Cell Morphology, Dictates Stem Cell Fate"

Article Title: Subcellular Control Over Focal Adhesion Anisotropy, Independent of Cell Morphology, Dictates Stem Cell Fate

Journal: ACS nano

doi: 10.1021/acsnano.9b03937

Fluorescence micrographs of the focal adhesions and actin cytoskeleton within cells on patterns.
Figure Legend Snippet: Fluorescence micrographs of the focal adhesions and actin cytoskeleton within cells on patterns.

Techniques Used: Fluorescence

18) Product Images from "Subcellular Control Over Focal Adhesion Anisotropy, Independent of Cell Morphology, Dictates Stem Cell Fate"

Article Title: Subcellular Control Over Focal Adhesion Anisotropy, Independent of Cell Morphology, Dictates Stem Cell Fate

Journal: ACS nano

doi: 10.1021/acsnano.9b03937

Fluorescence micrographs of the focal adhesions and actin cytoskeleton within cells on patterns.
Figure Legend Snippet: Fluorescence micrographs of the focal adhesions and actin cytoskeleton within cells on patterns.

Techniques Used: Fluorescence

19) Product Images from "An automated quantitative analysis of cell, nucleus and focal adhesion morphology"

Article Title: An automated quantitative analysis of cell, nucleus and focal adhesion morphology

Journal: PLoS ONE

doi: 10.1371/journal.pone.0195201

Quantitative analysis of the morphological features of a single cell, nucleus and individual focal adhesions (FAs) obtained under different experimental conditions, e.g. cell type, pharmacological, and substrate manipulation. A: Representative immunofluorescence images of the actin cytoskeleton (green), nucleus (blue), and FAs (magenta) for a HVSC (control) and a MEF (cell type) on a substrate homogeneously coated with fibronectin, HVSC in the presence of 10 μM of Y-27632 (drugs), and HVSC on 5x5 μm lines of fibronectin (red, anisotropy). The detected outlines are shown in green, blue, and magenta respectively. Scale bar: 50 μm. B: Analyzed morphological features of a single cell, and corresponding nucleus and FAs for the control, cell type (MEF), drug (Y-27632) and environment (anisotropy) situation. C: Boxplot of the orientation of the FAs comparing cells cultured on isotropic and anisotropic substrates. The box and whisker plot indicate the median (black line in the box), 25th percentile (bottom line of the box), 75th percentile (top line of the box), and 5th and 95th percentiles (whiskers). Next to this, the orientation of the cell and nucleus are represented by the green and blue lines, respectively. 90° represents the direction of the lines.
Figure Legend Snippet: Quantitative analysis of the morphological features of a single cell, nucleus and individual focal adhesions (FAs) obtained under different experimental conditions, e.g. cell type, pharmacological, and substrate manipulation. A: Representative immunofluorescence images of the actin cytoskeleton (green), nucleus (blue), and FAs (magenta) for a HVSC (control) and a MEF (cell type) on a substrate homogeneously coated with fibronectin, HVSC in the presence of 10 μM of Y-27632 (drugs), and HVSC on 5x5 μm lines of fibronectin (red, anisotropy). The detected outlines are shown in green, blue, and magenta respectively. Scale bar: 50 μm. B: Analyzed morphological features of a single cell, and corresponding nucleus and FAs for the control, cell type (MEF), drug (Y-27632) and environment (anisotropy) situation. C: Boxplot of the orientation of the FAs comparing cells cultured on isotropic and anisotropic substrates. The box and whisker plot indicate the median (black line in the box), 25th percentile (bottom line of the box), 75th percentile (top line of the box), and 5th and 95th percentiles (whiskers). Next to this, the orientation of the cell and nucleus are represented by the green and blue lines, respectively. 90° represents the direction of the lines.

Techniques Used: Immunofluorescence, Cell Culture, Whisker Assay

Overview of the steps of the automated image analysis pipeline. Representative immunofluorescent image of Human Vena Saphena Cells (HVSCs), (A) stained for the actin cytoskeleton (green), nucleus (blue), and focal adhesions (magenta). To automatically detect and analyze cells (B), nuclei (C) and focal adhesions (D), corresponding grey-scale images were processed using the automated image analysis pipeline. Scale bars: 50 μm.
Figure Legend Snippet: Overview of the steps of the automated image analysis pipeline. Representative immunofluorescent image of Human Vena Saphena Cells (HVSCs), (A) stained for the actin cytoskeleton (green), nucleus (blue), and focal adhesions (magenta). To automatically detect and analyze cells (B), nuclei (C) and focal adhesions (D), corresponding grey-scale images were processed using the automated image analysis pipeline. Scale bars: 50 μm.

Techniques Used: Staining

The effect of the Rho-associated kinase (ROCK) inhibitor Y-27632 on the morphological features of focal adhesions (FAs) in Human Vena Saphena Cells (HVSCs). A: Representative immunofluorescence images of the actin cytoskeleton (green), nucleus (blue), FAs (magenta) and zoom-in images of FAs of HVSCs treated with different doses (0-20 μM) of ROCK inhibitor or DMSO (control). Scale bar: 50 μm. Quantitative analysis of FA area (B), FA aspect ratio (C), and fraction of FAs with a defined length (D) reveals that Y-27632 affects FA morphology. At least 20 cells were analyzed per each condition and the results are expressed as the mean ± standard error of the mean (SEM). To assess differences between the different concentrations of ROCK inhibitor on the morphological features of the FAs, the One-Way ANOVA with a Bonferroni post-hoc test was used. ***: p
Figure Legend Snippet: The effect of the Rho-associated kinase (ROCK) inhibitor Y-27632 on the morphological features of focal adhesions (FAs) in Human Vena Saphena Cells (HVSCs). A: Representative immunofluorescence images of the actin cytoskeleton (green), nucleus (blue), FAs (magenta) and zoom-in images of FAs of HVSCs treated with different doses (0-20 μM) of ROCK inhibitor or DMSO (control). Scale bar: 50 μm. Quantitative analysis of FA area (B), FA aspect ratio (C), and fraction of FAs with a defined length (D) reveals that Y-27632 affects FA morphology. At least 20 cells were analyzed per each condition and the results are expressed as the mean ± standard error of the mean (SEM). To assess differences between the different concentrations of ROCK inhibitor on the morphological features of the FAs, the One-Way ANOVA with a Bonferroni post-hoc test was used. ***: p

Techniques Used: Immunofluorescence

Detection of a single cell, nucleus and focal adhesions (FAs). Representative grey-scale images of the actin cytoskeleton, nucleus, and FAs of HVSCs on a substrate homogeneously coated with fibronectin. The detected outlines are shown in green, blue, and magenta, respectively, and the orange rectangles marked areas show zoom-in images of the cell, nucleus and FAs. The white arrows indicate some small actin-rich membrane protrusions that were not detected.
Figure Legend Snippet: Detection of a single cell, nucleus and focal adhesions (FAs). Representative grey-scale images of the actin cytoskeleton, nucleus, and FAs of HVSCs on a substrate homogeneously coated with fibronectin. The detected outlines are shown in green, blue, and magenta, respectively, and the orange rectangles marked areas show zoom-in images of the cell, nucleus and FAs. The white arrows indicate some small actin-rich membrane protrusions that were not detected.

Techniques Used:

20) Product Images from "Phorbol Ester-dependent Phosphorylation Regulates the Association of p57/Coronin-1 with the Actin Cytoskeleton *"

Article Title: Phorbol Ester-dependent Phosphorylation Regulates the Association of p57/Coronin-1 with the Actin Cytoskeleton *

Journal:

doi: 10.1074/jbc.M709990200

Association of EGFP-p57FL, but not EGFP-p57LZ, with the actin cytoskeleton. A , schematic diagrams of the structures of EGFP-p57FL (an EGFP fusion protein with the full-length of p57/coronin-1) and EGFP-p57LZ (an EGFP fusion protein with the C-terminal
Figure Legend Snippet: Association of EGFP-p57FL, but not EGFP-p57LZ, with the actin cytoskeleton. A , schematic diagrams of the structures of EGFP-p57FL (an EGFP fusion protein with the full-length of p57/coronin-1) and EGFP-p57LZ (an EGFP fusion protein with the C-terminal

Techniques Used:

Effects of PMA and chelerythrine on the intracellular distribution of p57/coronin-1. A , HL60 cells were treated with PMA (150 n m ), chelerythrine ( Che , 30 μ m ), or chelerythrine plus PMA at 37 °C for 30 min and lysed with cytoskeleton
Figure Legend Snippet: Effects of PMA and chelerythrine on the intracellular distribution of p57/coronin-1. A , HL60 cells were treated with PMA (150 n m ), chelerythrine ( Che , 30 μ m ), or chelerythrine plus PMA at 37 °C for 30 min and lysed with cytoskeleton

Techniques Used:

Association of p57/coronin-1 with the actin cytoskeleton in HL60 cells. A ), and the lysate was separated into the detergent-soluble supernatant ( S ) and
Figure Legend Snippet: Association of p57/coronin-1 with the actin cytoskeleton in HL60 cells. A ), and the lysate was separated into the detergent-soluble supernatant ( S ) and

Techniques Used:

Analysis of p57/coronin-1 associated with the actin cytoskeleton by two-dimensional gel electrophoresis. PMA-treated HL60 cells were separated into cytosol ( Supernatant ) and cytoskeleton ( Precipitate ) fractions by extraction with cytoskeleton isolation
Figure Legend Snippet: Analysis of p57/coronin-1 associated with the actin cytoskeleton by two-dimensional gel electrophoresis. PMA-treated HL60 cells were separated into cytosol ( Supernatant ) and cytoskeleton ( Precipitate ) fractions by extraction with cytoskeleton isolation

Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Isolation

21) Product Images from "Glucocorticoid Receptor-mediated Expression of Caldesmon Regulates Cell Migration via the Reorganization of the Actin Cytoskeleton *Glucocorticoid Receptor-mediated Expression of Caldesmon Regulates Cell Migration via the Reorganization of the Actin Cytoskeleton * S⃞"

Article Title: Glucocorticoid Receptor-mediated Expression of Caldesmon Regulates Cell Migration via the Reorganization of the Actin Cytoskeleton *Glucocorticoid Receptor-mediated Expression of Caldesmon Regulates Cell Migration via the Reorganization of the Actin Cytoskeleton * S⃞

Journal:

doi: 10.1074/jbc.M801606200

Effects of CaD depletion on the actin cytoskeleton and cell migration. A , depletion of CaD expression using siRNA. A549 cells were transfected with CaD siRNAs or control siRNA, and incubated with or without 1 μ m DEX for 48 h. The expression
Figure Legend Snippet: Effects of CaD depletion on the actin cytoskeleton and cell migration. A , depletion of CaD expression using siRNA. A549 cells were transfected with CaD siRNAs or control siRNA, and incubated with or without 1 μ m DEX for 48 h. The expression

Techniques Used: Migration, Expressing, Transfection, Incubation

Effect of forced expression of GFP-CaD on the actin cytoskeleton. A , forced expression of GFP-CaD. The expression levels of GFP-CaD, control GFP, and endogenous CaD in A549 transfectants were analyzed by Western blotting with anti-CaD or anti-GFP antibody.
Figure Legend Snippet: Effect of forced expression of GFP-CaD on the actin cytoskeleton. A , forced expression of GFP-CaD. The expression levels of GFP-CaD, control GFP, and endogenous CaD in A549 transfectants were analyzed by Western blotting with anti-CaD or anti-GFP antibody.

Techniques Used: Expressing, Western Blot

Effects of steroid hormones on cell migration and the actin cytoskeleton in A549 cells. A , effects of steroid hormones on cell migration. A549 cells were cultured with the indicated steroid hormones for 48 h and treated with mitomycin C for 2 h preceding
Figure Legend Snippet: Effects of steroid hormones on cell migration and the actin cytoskeleton in A549 cells. A , effects of steroid hormones on cell migration. A549 cells were cultured with the indicated steroid hormones for 48 h and treated with mitomycin C for 2 h preceding

Techniques Used: Migration, Cell Culture

22) Product Images from "Phorbol Ester-dependent Phosphorylation Regulates the Association of p57/Coronin-1 with the Actin Cytoskeleton *"

Article Title: Phorbol Ester-dependent Phosphorylation Regulates the Association of p57/Coronin-1 with the Actin Cytoskeleton *

Journal:

doi: 10.1074/jbc.M709990200

Association of EGFP-p57FL, but not EGFP-p57LZ, with the actin cytoskeleton. A , schematic diagrams of the structures of EGFP-p57FL (an EGFP fusion protein with the full-length of p57/coronin-1) and EGFP-p57LZ (an EGFP fusion protein with the C-terminal
Figure Legend Snippet: Association of EGFP-p57FL, but not EGFP-p57LZ, with the actin cytoskeleton. A , schematic diagrams of the structures of EGFP-p57FL (an EGFP fusion protein with the full-length of p57/coronin-1) and EGFP-p57LZ (an EGFP fusion protein with the C-terminal

Techniques Used:

Effects of PMA and chelerythrine on the intracellular distribution of p57/coronin-1. A , HL60 cells were treated with PMA (150 n m ), chelerythrine ( Che , 30 μ m ), or chelerythrine plus PMA at 37 °C for 30 min and lysed with cytoskeleton
Figure Legend Snippet: Effects of PMA and chelerythrine on the intracellular distribution of p57/coronin-1. A , HL60 cells were treated with PMA (150 n m ), chelerythrine ( Che , 30 μ m ), or chelerythrine plus PMA at 37 °C for 30 min and lysed with cytoskeleton

Techniques Used:

Association of p57/coronin-1 with the actin cytoskeleton in HL60 cells. A ), and the lysate was separated into the detergent-soluble supernatant ( S ) and
Figure Legend Snippet: Association of p57/coronin-1 with the actin cytoskeleton in HL60 cells. A ), and the lysate was separated into the detergent-soluble supernatant ( S ) and

Techniques Used:

Analysis of p57/coronin-1 associated with the actin cytoskeleton by two-dimensional gel electrophoresis. PMA-treated HL60 cells were separated into cytosol ( Supernatant ) and cytoskeleton ( Precipitate ) fractions by extraction with cytoskeleton isolation
Figure Legend Snippet: Analysis of p57/coronin-1 associated with the actin cytoskeleton by two-dimensional gel electrophoresis. PMA-treated HL60 cells were separated into cytosol ( Supernatant ) and cytoskeleton ( Precipitate ) fractions by extraction with cytoskeleton isolation

Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Isolation

23) Product Images from "The Coordination Between B Cell Receptor Signaling and the Actin Cytoskeleton During B Cell Activation"

Article Title: The Coordination Between B Cell Receptor Signaling and the Actin Cytoskeleton During B Cell Activation

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.03096

Regulation of BCR signaling on the actin cytoskeleton. The association of the actin cytoskeleton with the plasma membrane is mediated by activated ERM proteins. The ERM proteins are first recruited to the plasma membrane by PIP2, and then phosphorylated by PKC, LOK, and effector proteins of RhoA, CDC42, and Nck. PLCγ2 induced inactivation of the ERM proteins through its down-regulation on PIP2. Activation of cofilin induces F-actin severing, which is regulated by the Rho family and Rap1 GTPase, and also intracellular calcium. BCR signaling regulates actin polymerization mainly through the actin-nucleation promotion factor WASP and WAVE, both of which can promote the nucleation effect of Arp2/3. Profilin and DIAPH1, which are regulated by RAP1 and RhoA, respectively, are suggested to participate in actin polymerization during B-cell activation. BCR signaling also influences contraction of the actin cytoskeleton through the regulation of RhoA on myosin.
Figure Legend Snippet: Regulation of BCR signaling on the actin cytoskeleton. The association of the actin cytoskeleton with the plasma membrane is mediated by activated ERM proteins. The ERM proteins are first recruited to the plasma membrane by PIP2, and then phosphorylated by PKC, LOK, and effector proteins of RhoA, CDC42, and Nck. PLCγ2 induced inactivation of the ERM proteins through its down-regulation on PIP2. Activation of cofilin induces F-actin severing, which is regulated by the Rho family and Rap1 GTPase, and also intracellular calcium. BCR signaling regulates actin polymerization mainly through the actin-nucleation promotion factor WASP and WAVE, both of which can promote the nucleation effect of Arp2/3. Profilin and DIAPH1, which are regulated by RAP1 and RhoA, respectively, are suggested to participate in actin polymerization during B-cell activation. BCR signaling also influences contraction of the actin cytoskeleton through the regulation of RhoA on myosin.

Techniques Used: Activation Assay

Overview of BCR signaling molecules involved in actin remodeling. CD19, PIP2, PLCγ2,PKC, the Rho family, and Rap GTPase, Btk, calcium, and WASP are major BCR signaling molecules involved in actin remodeling. These signaling molecules as well as their regulators form a network to participate in actin-cytoskeleton reorganization during B-cell activation.
Figure Legend Snippet: Overview of BCR signaling molecules involved in actin remodeling. CD19, PIP2, PLCγ2,PKC, the Rho family, and Rap GTPase, Btk, calcium, and WASP are major BCR signaling molecules involved in actin remodeling. These signaling molecules as well as their regulators form a network to participate in actin-cytoskeleton reorganization during B-cell activation.

Techniques Used: Activation Assay

24) Product Images from "Actin, RhoA, and Rab11 Participation during Encystment in Entamoeba invadens"

Article Title: Actin, RhoA, and Rab11 Participation during Encystment in Entamoeba invadens

Journal: BioMed Research International

doi: 10.1155/2013/919345

Disruption of the actin cytoskeleton with CD and Y27632 blocked the rearrangement of the actin cytoskeleton strongly affecting the cyst formation. (a) Percentage of inhibition of encystment by CD (1 μ M) and Y27632 (1 μ M) treatment during 96 h. ((b), (c)) Decrease in the intensity of F-actin fluorescence as a consequence of CD and Y27632 treatment. (d) Cysts ultrastructure at 96 h produced in the absence (control) or the presence of Cytochalasin D (CD) and ROCK-2 inhibitor (Y27632). Bar = 2 μ m.
Figure Legend Snippet: Disruption of the actin cytoskeleton with CD and Y27632 blocked the rearrangement of the actin cytoskeleton strongly affecting the cyst formation. (a) Percentage of inhibition of encystment by CD (1 μ M) and Y27632 (1 μ M) treatment during 96 h. ((b), (c)) Decrease in the intensity of F-actin fluorescence as a consequence of CD and Y27632 treatment. (d) Cysts ultrastructure at 96 h produced in the absence (control) or the presence of Cytochalasin D (CD) and ROCK-2 inhibitor (Y27632). Bar = 2 μ m.

Techniques Used: Inhibition, Fluorescence, Produced

Increase in RhoA-GTP levels at early times of encystment. RhoA-GTP increased at early times but decreased later on during encystment. RhoA-GTP levels were determined by G-LISA RhoA activation assay kit (Kit no. BK121, Cytoskeleton, Inc., Denver, CO). Results are the average of three independent experiments done in duplicate (* P
Figure Legend Snippet: Increase in RhoA-GTP levels at early times of encystment. RhoA-GTP increased at early times but decreased later on during encystment. RhoA-GTP levels were determined by G-LISA RhoA activation assay kit (Kit no. BK121, Cytoskeleton, Inc., Denver, CO). Results are the average of three independent experiments done in duplicate (* P

Techniques Used: Activation Assay

25) Product Images from "EspO1-2 Regulates EspM2-Mediated RhoA Activity to Stabilize Formation of Focal Adhesions in Enterohemorrhagic Escherichia coli-Infected Host Cells"

Article Title: EspO1-2 Regulates EspM2-Mediated RhoA Activity to Stabilize Formation of Focal Adhesions in Enterohemorrhagic Escherichia coli-Infected Host Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0055960

EspO1-1 and EspO1-2 stabilize FAs and the actin cytoskeleton in EHEC-infected cells. (A) Alignment of OspE2 homologs EspO1-1 and EspO1-2 in EHEC strain Sakai. EspO1-1 and EspO1-2 have 59% amino-acid sequence identity, and also have extensive homology with OspE2 (29% and 25% amino acid sequence identity to OspE2, respectively). Asterisks indicate amino acid residues that are conserved in either OspE1 and OspE2 or in EspO1-1 and EspO1-2. Black highlight indicates an amino-acid residue conserved in both OspE1 and OspE2 and either EspO1-1, EspO1-2, or both EspO1-1 and EspO1-2. Arrowhead indicates the tryptophan, which is involved in ILK binding, at amino acid residue 77 of EspO1s and residue 68 of OspEs. (B) Morphological change in HeLa cells uninfected or infected with EHEC. HeLa cells were infected with EHEC wild-type (WT), an Δ espO1-1 mutant (Δ espO1-1 ), an Δ espO1-2 mutant (Δ espO1-2 ), an Δ espO1-1 Δ espO1-2 double mutant (Δ espO1-1 Δ espO1-2 ), an Δ espO1-1 Δ espO1-2 /pEspO1-1 or an Δ espO1-1 Δ espO1-2 /pEspO1-2. At 4 h post-infection, the cells were fixed and Giemsa-stained. (C) Percent cell rounding in cells infected by WT, Δ espO1-1 Δ espO1-2 , Δ espO1-1 or Δ espO1-2 shown in (B) and quantified ( > 50 cells, n≥3). Data are the mean and S.D. * P
Figure Legend Snippet: EspO1-1 and EspO1-2 stabilize FAs and the actin cytoskeleton in EHEC-infected cells. (A) Alignment of OspE2 homologs EspO1-1 and EspO1-2 in EHEC strain Sakai. EspO1-1 and EspO1-2 have 59% amino-acid sequence identity, and also have extensive homology with OspE2 (29% and 25% amino acid sequence identity to OspE2, respectively). Asterisks indicate amino acid residues that are conserved in either OspE1 and OspE2 or in EspO1-1 and EspO1-2. Black highlight indicates an amino-acid residue conserved in both OspE1 and OspE2 and either EspO1-1, EspO1-2, or both EspO1-1 and EspO1-2. Arrowhead indicates the tryptophan, which is involved in ILK binding, at amino acid residue 77 of EspO1s and residue 68 of OspEs. (B) Morphological change in HeLa cells uninfected or infected with EHEC. HeLa cells were infected with EHEC wild-type (WT), an Δ espO1-1 mutant (Δ espO1-1 ), an Δ espO1-2 mutant (Δ espO1-2 ), an Δ espO1-1 Δ espO1-2 double mutant (Δ espO1-1 Δ espO1-2 ), an Δ espO1-1 Δ espO1-2 /pEspO1-1 or an Δ espO1-1 Δ espO1-2 /pEspO1-2. At 4 h post-infection, the cells were fixed and Giemsa-stained. (C) Percent cell rounding in cells infected by WT, Δ espO1-1 Δ espO1-2 , Δ espO1-1 or Δ espO1-2 shown in (B) and quantified ( > 50 cells, n≥3). Data are the mean and S.D. * P

Techniques Used: Infection, Sequencing, Binding Assay, Mutagenesis, Staining

Contribution of EspM2 to cell rounding and actin cytoskeleton structure disruption in the infected cells. (A) HeLa cells were infected with an Δ espO1-1 Δ espO1-2 Δ espM2 triple mutant and, at 4 h post-infection, stained with rhodamine-phalloidin to visualize actin filaments. (B) The percent of cells shown in (A) in which cell rounding was induced and the actin cytoskeleton was disrupted was quantified ( > 50 cells, n≥3). Data are the mean and S.D. * P
Figure Legend Snippet: Contribution of EspM2 to cell rounding and actin cytoskeleton structure disruption in the infected cells. (A) HeLa cells were infected with an Δ espO1-1 Δ espO1-2 Δ espM2 triple mutant and, at 4 h post-infection, stained with rhodamine-phalloidin to visualize actin filaments. (B) The percent of cells shown in (A) in which cell rounding was induced and the actin cytoskeleton was disrupted was quantified ( > 50 cells, n≥3). Data are the mean and S.D. * P

Techniques Used: Infection, Mutagenesis, Staining

26) Product Images from "Mechanobiological Modulation of Cytoskeleton and Calcium Influx in Osteoblastic Cells by Short-Term Focused Acoustic Radiation Force"

Article Title: Mechanobiological Modulation of Cytoskeleton and Calcium Influx in Osteoblastic Cells by Short-Term Focused Acoustic Radiation Force

Journal: PLoS ONE

doi: 10.1371/journal.pone.0038343

Pulsed ultrasound radiation affects the arrangement of actin cytoskeleton in MC3T3-E1 osteoblasts. Pulsed ultrasound was applied for 1 min, cells were then rinsed three times with fresh DPBS (total time,10 min), then fixed and stained with rhodamine-phalloidin. Actin stress fibers were imaged in control cells (A) and 10 min (B) after ultrasound radiation. Actin stress fibers increased following pulsed ultrasound radiation. Scale bar, 10 µm.
Figure Legend Snippet: Pulsed ultrasound radiation affects the arrangement of actin cytoskeleton in MC3T3-E1 osteoblasts. Pulsed ultrasound was applied for 1 min, cells were then rinsed three times with fresh DPBS (total time,10 min), then fixed and stained with rhodamine-phalloidin. Actin stress fibers were imaged in control cells (A) and 10 min (B) after ultrasound radiation. Actin stress fibers increased following pulsed ultrasound radiation. Scale bar, 10 µm.

Techniques Used: Staining

27) Product Images from "Regulation of Mammalian Mitochondrial Dynamics: Opportunities and Challenges"

Article Title: Regulation of Mammalian Mitochondrial Dynamics: Opportunities and Challenges

Journal: Frontiers in Endocrinology

doi: 10.3389/fendo.2020.00374

Proposed potential mechanisms driving mammalian mitochondrial fission, including Drp1-dependent and Drp1-independent mitochondrial fission. (Left) The Drp1-dependent mitochondrial fission as described in Figure 2B , including Drp1 and its four mitochondrial receptors Fis1, Mff, MIEF1, and MIEF2. (Right) Apart from the canonical Drp1-dependent mitochondrial fission, there may be multiple Drp1-independent mechanisms contributing to mitochondrial division in mammals although little is known about their details in the regulation of mitochondrial division. Emerging evidence implies that the Drp1-independent mitochondrial fission may involve: (1) Fis1 together with TBC1D15 and Rab7 regulate both mitochondrial and lysosomal dynamics, which likely is important in mitophagy; (2) Fis1 binds to the pro-fusion GTPases Mfn1/2 and OPA1 and inhibits the activity of the fusion machinery, thereby shifting mitochondrial morphology to a fission phenotype, probably via Drp1-dependent and -independent mechanisms (the details in this process still remain poorly understood); (3) The actin cytoskeleton and Dyn2, as well as the ER may have Drp1-independent functions in regulating mitochondrial division (the details are unclear).
Figure Legend Snippet: Proposed potential mechanisms driving mammalian mitochondrial fission, including Drp1-dependent and Drp1-independent mitochondrial fission. (Left) The Drp1-dependent mitochondrial fission as described in Figure 2B , including Drp1 and its four mitochondrial receptors Fis1, Mff, MIEF1, and MIEF2. (Right) Apart from the canonical Drp1-dependent mitochondrial fission, there may be multiple Drp1-independent mechanisms contributing to mitochondrial division in mammals although little is known about their details in the regulation of mitochondrial division. Emerging evidence implies that the Drp1-independent mitochondrial fission may involve: (1) Fis1 together with TBC1D15 and Rab7 regulate both mitochondrial and lysosomal dynamics, which likely is important in mitophagy; (2) Fis1 binds to the pro-fusion GTPases Mfn1/2 and OPA1 and inhibits the activity of the fusion machinery, thereby shifting mitochondrial morphology to a fission phenotype, probably via Drp1-dependent and -independent mechanisms (the details in this process still remain poorly understood); (3) The actin cytoskeleton and Dyn2, as well as the ER may have Drp1-independent functions in regulating mitochondrial division (the details are unclear).

Techniques Used: Activity Assay

28) Product Images from "The Syk Kinase Promotes Mammary Epithelial Integrity and Inhibits Breast Cancer Invasion by Stabilizing the E-Cadherin/Catenin Complex"

Article Title: The Syk Kinase Promotes Mammary Epithelial Integrity and Inhibits Breast Cancer Invasion by Stabilizing the E-Cadherin/Catenin Complex

Journal: Cancers

doi: 10.3390/cancers11121974

Enhanced Syk expression promotes the interaction between the E-cadherin/catenin complex, zonula occludens proteins, and the actin cytoskeleton. ( a ) Whole cell lysates (WCL) and immunoprecipitated proteins (IP) from HEK293T cells transiently transfected or not with a FLAG-Syk plasmid were analyzed by Western blotting (WB) with the indicated antibodies. ( b ) Immunofluorescence analysis of GFP-Syk MCF7 cells with anti-E-Cdh (TRITC/red), -ZO-1 (upper) and -ZO-3 antibodies (lower) (Cy5/purple). White arrows indicate GFP-Syk colocalization with E-Cdh and ZO-1 or ZO-3 at cell-cell junctions. DNA was stained with Hoechst (blue). Bar, 10 μm.
Figure Legend Snippet: Enhanced Syk expression promotes the interaction between the E-cadherin/catenin complex, zonula occludens proteins, and the actin cytoskeleton. ( a ) Whole cell lysates (WCL) and immunoprecipitated proteins (IP) from HEK293T cells transiently transfected or not with a FLAG-Syk plasmid were analyzed by Western blotting (WB) with the indicated antibodies. ( b ) Immunofluorescence analysis of GFP-Syk MCF7 cells with anti-E-Cdh (TRITC/red), -ZO-1 (upper) and -ZO-3 antibodies (lower) (Cy5/purple). White arrows indicate GFP-Syk colocalization with E-Cdh and ZO-1 or ZO-3 at cell-cell junctions. DNA was stained with Hoechst (blue). Bar, 10 μm.

Techniques Used: Expressing, Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Immunofluorescence, Staining

29) Product Images from "Changes in Gene Expression and Cellular Architecture in an Ovarian Cancer Progression Model"

Article Title: Changes in Gene Expression and Cellular Architecture in an Ovarian Cancer Progression Model

Journal: PLoS ONE

doi: 10.1371/journal.pone.0017676

PKCβII protein levels and interactions with cytoskeleton. Equal amounts of protein from MOSE-E and MOSE-L was fractionated into 1% triton X-100 soluble and non-soluble (pellet) portions and analyzed by western blot analysis ( A ). Protein levels are the mean of three measurements expressed as percent of the total PKCβII protein with standard deviations ≤1.5% for all samples, normalized by cell number. ( B ) Total protein levels (soluble + pellet) (mean of three measurements) are expressed as percent of MOSE-E levels. * p
Figure Legend Snippet: PKCβII protein levels and interactions with cytoskeleton. Equal amounts of protein from MOSE-E and MOSE-L was fractionated into 1% triton X-100 soluble and non-soluble (pellet) portions and analyzed by western blot analysis ( A ). Protein levels are the mean of three measurements expressed as percent of the total PKCβII protein with standard deviations ≤1.5% for all samples, normalized by cell number. ( B ) Total protein levels (soluble + pellet) (mean of three measurements) are expressed as percent of MOSE-E levels. * p

Techniques Used: Western Blot

Organization of the cytoskeleton and localization of actin regulating proteins with neoplastic progression. ( A ) Immunofluorescent staining of MOSE-E, MOSE-I and MOSE-L cells to visualize actin filaments (phalloidin, green), α- tubulin (2 nd column), β- tubulin (3 rd columns), or cytokeratin (4 th column) along with the nucleus (blue, DAPI). ( B and C ) Triple staining of MOSE-E and MOSE-L cells with DAPI (blue), phalloidin (f-actin, green), and antibodies against α-actinin (red, B) or vinculin (red, C). The confocal images shown are 0.6 µm apart within the cell, with image 1 starting at the base of the cell and image 2 towards the top of the cell. Co-localization appears as yellow in merged and confocal images. ( D ) Triple staining of MOSE-E and MOSE-L cells with DAPI (blue), antibody against FAK (green), and antibody against FAK phosphorylated tyrosine 861 (red, FAK Y861 ). Yellow in merged image indicates co-localization. (Original magnification X600)
Figure Legend Snippet: Organization of the cytoskeleton and localization of actin regulating proteins with neoplastic progression. ( A ) Immunofluorescent staining of MOSE-E, MOSE-I and MOSE-L cells to visualize actin filaments (phalloidin, green), α- tubulin (2 nd column), β- tubulin (3 rd columns), or cytokeratin (4 th column) along with the nucleus (blue, DAPI). ( B and C ) Triple staining of MOSE-E and MOSE-L cells with DAPI (blue), phalloidin (f-actin, green), and antibodies against α-actinin (red, B) or vinculin (red, C). The confocal images shown are 0.6 µm apart within the cell, with image 1 starting at the base of the cell and image 2 towards the top of the cell. Co-localization appears as yellow in merged and confocal images. ( D ) Triple staining of MOSE-E and MOSE-L cells with DAPI (blue), antibody against FAK (green), and antibody against FAK phosphorylated tyrosine 861 (red, FAK Y861 ). Yellow in merged image indicates co-localization. (Original magnification X600)

Techniques Used: Staining

Levels of cytoskeleton and actin regulating proteins in neoplastic progression. Whole cell extracts from MOSE-E (E, white bars), MOSE-I (I, grey bars), and MOSE-L (L, black bars) cells were subjected to Western blot analysis with antibodies directed against ( A ) actin regulating proteins and ( B ) microtubule proteins. Expression levels are expressed as percent MOSE-E levels normalization to ribosomal protein L19 (RPL19) or γ-tubulin for three biological replicates done in duplicate ± the standard deviation. A representative blot from the three biological replicates is shown. *p≤ 0.01.
Figure Legend Snippet: Levels of cytoskeleton and actin regulating proteins in neoplastic progression. Whole cell extracts from MOSE-E (E, white bars), MOSE-I (I, grey bars), and MOSE-L (L, black bars) cells were subjected to Western blot analysis with antibodies directed against ( A ) actin regulating proteins and ( B ) microtubule proteins. Expression levels are expressed as percent MOSE-E levels normalization to ribosomal protein L19 (RPL19) or γ-tubulin for three biological replicates done in duplicate ± the standard deviation. A representative blot from the three biological replicates is shown. *p≤ 0.01.

Techniques Used: Western Blot, Expressing, Standard Deviation

30) Product Images from "Alteration of actin dependent signaling pathways associated with membrane microdomains in hyperlipidemia"

Article Title: Alteration of actin dependent signaling pathways associated with membrane microdomains in hyperlipidemia

Journal: Proteome Science

doi: 10.1186/s12953-015-0087-0

Graphical overview of differentially expressed proteins identified in actin dependent signal transduction pathways. The proteins listed in Table 1 were found to be part of Regulation of actin cytoskeleton , Focal adhesion and Adherence junction over-represented signaling pathways targeted by hyperlipidemia and statin therapy
Figure Legend Snippet: Graphical overview of differentially expressed proteins identified in actin dependent signal transduction pathways. The proteins listed in Table 1 were found to be part of Regulation of actin cytoskeleton , Focal adhesion and Adherence junction over-represented signaling pathways targeted by hyperlipidemia and statin therapy

Techniques Used: Transduction

Actin dependent signal transduction pathways of identified and quantified detergent resistant membrane microdomains proteins. The interaction map comprises the currently discussed mass spectrometric identified proteins (red bordered boxes) and differentially expressed proteins (green filled red bordered boxes) and were integrated in the over-represented Regulation of actin cytoskeleton , Focal adhesion and Adherens junction signaling pathways. The complex signaling network represents the combination of map04810 , map 04510 and map 04520 KEGG signaling pathways. Abbreviations for the discussed proteins: Ras: Ras-related protein R-Ras; MEK: mitogen-activated protein kinase kinase 1; F2RCD14: coagulation factor II, CD14 antigen; DOCK180: dedicator of cytokinesis protein; VWF: von Willebrand factor; Cav: caveolin; Gβγ: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-12; Arp2/3: Actin-related protein 2/3 complex subunit 1B and 3; PIR121: Cytoplasmic FMR1-interacting protein 1; PFN: profilin; CFN: Cofilin-1; ZYX: Zyxin; MLC: Myosin regulatory light polypeptide 9; MLCK: Myosin light chain kinase 2; mDia: Protein diaphanous homolog 1; ACTN: actinin: VCL: vinculin; TLN: talin; ITG: integrin; Rap-1: Ras-related protein Rap-1A; Rac: Ras-related C3 botulinum toxin substrate 1; ILK: Integrin-linked protein kinase; MLCP: Serine/threonine-protein phosphatase PP1-beta catalytic subunit; RhoA: Transforming protein RhoA; ERM: ezrin/radixin/moesin; GIT1: ARF GTPase-activating protein GIT1; Yes: Tyrosine-protein kinase Yes; IQGAP1: RasGTPase-activating-like protein IQGAP1; CKII: Casein kinase II subunit alpha; Gα12,13: Guanine nucleotide-binding protein subunit alpha-13
Figure Legend Snippet: Actin dependent signal transduction pathways of identified and quantified detergent resistant membrane microdomains proteins. The interaction map comprises the currently discussed mass spectrometric identified proteins (red bordered boxes) and differentially expressed proteins (green filled red bordered boxes) and were integrated in the over-represented Regulation of actin cytoskeleton , Focal adhesion and Adherens junction signaling pathways. The complex signaling network represents the combination of map04810 , map 04510 and map 04520 KEGG signaling pathways. Abbreviations for the discussed proteins: Ras: Ras-related protein R-Ras; MEK: mitogen-activated protein kinase kinase 1; F2RCD14: coagulation factor II, CD14 antigen; DOCK180: dedicator of cytokinesis protein; VWF: von Willebrand factor; Cav: caveolin; Gβγ: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-12; Arp2/3: Actin-related protein 2/3 complex subunit 1B and 3; PIR121: Cytoplasmic FMR1-interacting protein 1; PFN: profilin; CFN: Cofilin-1; ZYX: Zyxin; MLC: Myosin regulatory light polypeptide 9; MLCK: Myosin light chain kinase 2; mDia: Protein diaphanous homolog 1; ACTN: actinin: VCL: vinculin; TLN: talin; ITG: integrin; Rap-1: Ras-related protein Rap-1A; Rac: Ras-related C3 botulinum toxin substrate 1; ILK: Integrin-linked protein kinase; MLCP: Serine/threonine-protein phosphatase PP1-beta catalytic subunit; RhoA: Transforming protein RhoA; ERM: ezrin/radixin/moesin; GIT1: ARF GTPase-activating protein GIT1; Yes: Tyrosine-protein kinase Yes; IQGAP1: RasGTPase-activating-like protein IQGAP1; CKII: Casein kinase II subunit alpha; Gα12,13: Guanine nucleotide-binding protein subunit alpha-13

Techniques Used: Transduction, Coagulation, Binding Assay

31) Product Images from "Cytoskeletal Dependence of Insulin Granule Movement Dynamics in INS-1 Beta-Cells in Response to Glucose"

Article Title: Cytoskeletal Dependence of Insulin Granule Movement Dynamics in INS-1 Beta-Cells in Response to Glucose

Journal: PLoS ONE

doi: 10.1371/journal.pone.0109082

Effect of glucose stimulation on microtubule structure and dynamics. Super-resolution STORM images of microtubules in INS-1 cells in low (A) and high (B) glucose conditions, and after perturbation of the microtubule cytoskeleton with taxol (C) and nocodazole (D). Insets in A–D show 3× magnification of area in the yellow box. (E) Total microtubule (MT) length per µm 2 of imaged cell area under various conditions. ** indicates p
Figure Legend Snippet: Effect of glucose stimulation on microtubule structure and dynamics. Super-resolution STORM images of microtubules in INS-1 cells in low (A) and high (B) glucose conditions, and after perturbation of the microtubule cytoskeleton with taxol (C) and nocodazole (D). Insets in A–D show 3× magnification of area in the yellow box. (E) Total microtubule (MT) length per µm 2 of imaged cell area under various conditions. ** indicates p

Techniques Used:

Effect of glucose stimulation on actin filament organization. Super-resolution STORM images of the actin cytoskeleton in INS-1 cells in low (A) and high (B) glucose conditions and after actin depolymerization with cytochalasin D (C). In each STORM image (A–C) yellow highlighted area is magnified 3x in the upper right image with skeletonized image below used for quantification of actin filament length and number of actin filament intersections in D. (D) Quantification of actin organization. Left: The length of actin per µm 2 of imaged cell area under various conditions. Right: The number of actin intersections per µm 2 of imaged cell area under various conditions. ** indicates p
Figure Legend Snippet: Effect of glucose stimulation on actin filament organization. Super-resolution STORM images of the actin cytoskeleton in INS-1 cells in low (A) and high (B) glucose conditions and after actin depolymerization with cytochalasin D (C). In each STORM image (A–C) yellow highlighted area is magnified 3x in the upper right image with skeletonized image below used for quantification of actin filament length and number of actin filament intersections in D. (D) Quantification of actin organization. Left: The length of actin per µm 2 of imaged cell area under various conditions. Right: The number of actin intersections per µm 2 of imaged cell area under various conditions. ** indicates p

Techniques Used:

32) Product Images from "Topography Design Concept of a Tissue Engineering Scaffold for Controlling Cell Function and Fate Through Actin Cytoskeletal Modulation"

Article Title: Topography Design Concept of a Tissue Engineering Scaffold for Controlling Cell Function and Fate Through Actin Cytoskeletal Modulation

Journal: Tissue Engineering. Part B, Reviews

doi: 10.1089/ten.teb.2013.0728

The mechanical and mechanochemical link of the cytoplasm to extracellular matrix (ECM) that contributes for cells to sense, integrate, transduce, and respond to the ECM architecture. The pathways mediated by the actin cytoskeleton are summarized based
Figure Legend Snippet: The mechanical and mechanochemical link of the cytoplasm to extracellular matrix (ECM) that contributes for cells to sense, integrate, transduce, and respond to the ECM architecture. The pathways mediated by the actin cytoskeleton are summarized based

Techniques Used:

33) Product Images from "Topography Design Concept of a Tissue Engineering Scaffold for Controlling Cell Function and Fate Through Actin Cytoskeletal Modulation"

Article Title: Topography Design Concept of a Tissue Engineering Scaffold for Controlling Cell Function and Fate Through Actin Cytoskeletal Modulation

Journal: Tissue Engineering. Part B, Reviews

doi: 10.1089/ten.teb.2013.0728

The mechanical and mechanochemical link of the cytoplasm to extracellular matrix (ECM) that contributes for cells to sense, integrate, transduce, and respond to the ECM architecture. The pathways mediated by the actin cytoskeleton are summarized based
Figure Legend Snippet: The mechanical and mechanochemical link of the cytoplasm to extracellular matrix (ECM) that contributes for cells to sense, integrate, transduce, and respond to the ECM architecture. The pathways mediated by the actin cytoskeleton are summarized based

Techniques Used:

34) Product Images from "A Legionella Effector Disrupts Host Cytoskeletal Structure by Cleaving Actin"

Article Title: A Legionella Effector Disrupts Host Cytoskeletal Structure by Cleaving Actin

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006186

Identification of L . pneumophila Dot/Icm substrates capable of altering the architecture of the actin cytoskeleton of mammalian cells. COS-1 cells were transfected to express GFP or GFP fusion of RavK, Lpg0944 and Lpg1290 for 24 h and fixed cells were subjected to staining with Texas-red-conjugated phalloidin. Images shown were from one representative experiment and similar results were seen in three independent experiments. Note that RavK severely reduced the phalloidin signals and that GFP-Lpg0944 caused a rearrangement of the actin cytoskeleton with an increase in cortical actin abundance. In contrast, expression of Lpg1290 did not cause any significant change in the actin cytoskeleton. The cells expressing GFP served as a control. Bar, 20 μm.
Figure Legend Snippet: Identification of L . pneumophila Dot/Icm substrates capable of altering the architecture of the actin cytoskeleton of mammalian cells. COS-1 cells were transfected to express GFP or GFP fusion of RavK, Lpg0944 and Lpg1290 for 24 h and fixed cells were subjected to staining with Texas-red-conjugated phalloidin. Images shown were from one representative experiment and similar results were seen in three independent experiments. Note that RavK severely reduced the phalloidin signals and that GFP-Lpg0944 caused a rearrangement of the actin cytoskeleton with an increase in cortical actin abundance. In contrast, expression of Lpg1290 did not cause any significant change in the actin cytoskeleton. The cells expressing GFP served as a control. Bar, 20 μm.

Techniques Used: Transfection, Staining, Expressing

35) Product Images from "Correlative STED and Atomic Force Microscopy on Live Astrocytes Reveals Plasticity of Cytoskeletal Structure and Membrane Physical Properties during Polarized Migration"

Article Title: Correlative STED and Atomic Force Microscopy on Live Astrocytes Reveals Plasticity of Cytoskeletal Structure and Membrane Physical Properties during Polarized Migration

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2017.00104

Actin cytoskeletal structure is reflected in cell topography and cellular stiffness. (A) STED image of the actin cytoskeleton overlaid with AFM topography (height) images (large image). AFM images were taken in regions 1 and 2. Zoomed AFM and the corresponding STED images are shown. STED/AFM reveals an agreement between the fluorescently labeled actin filaments and AFM topography. Both imaging modes show similar polarity. AFM color scale represents a range of heights of 455 nm and 464 nm in regions 1 and 2 respectively. (B) Bar graph representing the mean cell stiffness of control (non transfected, n = 38) and GFP transfected astrocytes ( n = 12), showing no significant effect of GFP transfection on stiffness ( p > 0.05, Mann Whitney test). (C) Line profile through the indicated region showing height (AFM) and signal (STED) showing good agreements between the locations of peaks in both profiles. (D) STED and AFM height images (height range: region 1; 1394 nm, region 2; 1320 nm) of an astrocyte with cytoskeleton depolymerized by cytochalasin D (30 μM, 1 h). STED images reveal actin depolymerization, while AFM images show reduced organization in cell topography. (E) Depolymerizing actin reduces cytoskeletal stiffness (GFP, n = 12; GFP + cytochalasin D, n = 10, p
Figure Legend Snippet: Actin cytoskeletal structure is reflected in cell topography and cellular stiffness. (A) STED image of the actin cytoskeleton overlaid with AFM topography (height) images (large image). AFM images were taken in regions 1 and 2. Zoomed AFM and the corresponding STED images are shown. STED/AFM reveals an agreement between the fluorescently labeled actin filaments and AFM topography. Both imaging modes show similar polarity. AFM color scale represents a range of heights of 455 nm and 464 nm in regions 1 and 2 respectively. (B) Bar graph representing the mean cell stiffness of control (non transfected, n = 38) and GFP transfected astrocytes ( n = 12), showing no significant effect of GFP transfection on stiffness ( p > 0.05, Mann Whitney test). (C) Line profile through the indicated region showing height (AFM) and signal (STED) showing good agreements between the locations of peaks in both profiles. (D) STED and AFM height images (height range: region 1; 1394 nm, region 2; 1320 nm) of an astrocyte with cytoskeleton depolymerized by cytochalasin D (30 μM, 1 h). STED images reveal actin depolymerization, while AFM images show reduced organization in cell topography. (E) Depolymerizing actin reduces cytoskeletal stiffness (GFP, n = 12; GFP + cytochalasin D, n = 10, p

Techniques Used: Labeling, Imaging, Transfection, MANN-WHITNEY

Astrocyte surface topography and stiffness is distinct from tubulin organization. (A) Correlative STED/AFM images of tubulin cytoskeleton and astrocyte topography. AFM images (height range: region 1; 279 nm, region 2; 389 nm) reveal a structure with different polarity to the corresponding STED images of tubulin region 1: AFM: 32.2°, STED: 51.3°; region 2: AFM: 29.0°, STED: 40.0°. (B) Line profile through the indicated region of the AFM (height) and STED (intensity) showing little agreement between peak locations and structures. (C) STED and AFM images of astrocytes (AFM height range: region 1; 680 nm, region 2; 388 nm) where tubulin has been depolymerized using nocodazole (16 μM, 1 h). Fibrilar structures remain present in the topography images. (D) No change in average stiffness was detected between control astrocytes ( n = 12) and astrocytes treated with nocodazole ( n = 7, p > 0.05, Mann Whitney test). Scale bars are 10 μm (large images) and 5 μm (zoomed images).
Figure Legend Snippet: Astrocyte surface topography and stiffness is distinct from tubulin organization. (A) Correlative STED/AFM images of tubulin cytoskeleton and astrocyte topography. AFM images (height range: region 1; 279 nm, region 2; 389 nm) reveal a structure with different polarity to the corresponding STED images of tubulin region 1: AFM: 32.2°, STED: 51.3°; region 2: AFM: 29.0°, STED: 40.0°. (B) Line profile through the indicated region of the AFM (height) and STED (intensity) showing little agreement between peak locations and structures. (C) STED and AFM images of astrocytes (AFM height range: region 1; 680 nm, region 2; 388 nm) where tubulin has been depolymerized using nocodazole (16 μM, 1 h). Fibrilar structures remain present in the topography images. (D) No change in average stiffness was detected between control astrocytes ( n = 12) and astrocytes treated with nocodazole ( n = 7, p > 0.05, Mann Whitney test). Scale bars are 10 μm (large images) and 5 μm (zoomed images).

Techniques Used: MANN-WHITNEY

36) Product Images from "All-Round Manipulation of the Actin Cytoskeleton by HIV"

Article Title: All-Round Manipulation of the Actin Cytoskeleton by HIV

Journal: Viruses

doi: 10.3390/v10020063

Actin-related changes in cellular behavior induced by HIV in relevant cell types. Coordinated manipulation of actin regulators results in global and cell-type specific changes in cellular morphology and motility that contribute to viral spread, impairment of immune function and HIV comorbidities. Many of these changes can be mapped to specific HIV accessory proteins, which act as master regulators of the cytoskeleton. Nef and Tat are expressed in HIV-infected cells but are also present extracellularly in serum and, thus, can affect both infected and uninfected cells. ( a ) Nef; leads to Rac1 activation in a wide range of cell types. In T-cells, this is associated with inactivation of Cofilin and severe cytoskeletal disorganization, which impairs cell migration and immunological synapse formation. In myeloid cells, Nef enhances formation of several membrane protrusions which promote cell motility and contacts with uninfected cells; ( b ) Tat modulates the expression of numerous genes involved in actin regulation. In T-cells, Tat interferes with chemotaxis and F-actin remodeling, whereas in monocytes it increases cell motility, chemotaxis and phagocytosis. Tat also induces expression of adhesion molecules and promotes leukocyte binding to the endothelium. Upward arrows represent enhancement of biological processes or increases in number of structures, whereas downward arrows represent impairment of processes. * = Effects also induced by HIV envelope glycoprotein (Env).
Figure Legend Snippet: Actin-related changes in cellular behavior induced by HIV in relevant cell types. Coordinated manipulation of actin regulators results in global and cell-type specific changes in cellular morphology and motility that contribute to viral spread, impairment of immune function and HIV comorbidities. Many of these changes can be mapped to specific HIV accessory proteins, which act as master regulators of the cytoskeleton. Nef and Tat are expressed in HIV-infected cells but are also present extracellularly in serum and, thus, can affect both infected and uninfected cells. ( a ) Nef; leads to Rac1 activation in a wide range of cell types. In T-cells, this is associated with inactivation of Cofilin and severe cytoskeletal disorganization, which impairs cell migration and immunological synapse formation. In myeloid cells, Nef enhances formation of several membrane protrusions which promote cell motility and contacts with uninfected cells; ( b ) Tat modulates the expression of numerous genes involved in actin regulation. In T-cells, Tat interferes with chemotaxis and F-actin remodeling, whereas in monocytes it increases cell motility, chemotaxis and phagocytosis. Tat also induces expression of adhesion molecules and promotes leukocyte binding to the endothelium. Upward arrows represent enhancement of biological processes or increases in number of structures, whereas downward arrows represent impairment of processes. * = Effects also induced by HIV envelope glycoprotein (Env).

Techniques Used: Activated Clotting Time Assay, Infection, Activation Assay, Migration, Expressing, Chemotaxis Assay, Binding Assay

Manipulation of the actin cytoskeleton by human immunodeficiency virus (HIV). ( a ) Actin regulators subjected to modulation by HIV. Mechanistically diverse strategies enable the virus to alter cellular cytoskeletal functions. Manipulation of host factors can be either direct, when mediated by physical interaction with viral proteins, or indirect, when requiring upstream cellular factors. Exploitation mechanisms increase native protein activity by upregulation of gene expression, or indirect activation within a cellular pathway. Neutralization of host factors is achieved by downregulation of gene expression or protein inactivation. Hijacking alters the functional outcome of host protein activity, either by overriding regulatory mechanisms (i.e., direct protein activation), changing protein subcellular localization, and/or modifying protein interaction partners. Note that some host factors can be manipulated by multiple strategies at diverse stages of the viral life cycle, as well as differentially in infected and uninfected cells. Examples of actin regulators corresponding to each strategy are provided, however this is not a complete list; ( b ) Functional consequences of actin-dependent changes induced by HIV. Normal immunological functions are compromised upon HIV infection, partly due to actin-remodeling changes orchestrated by viral proteins. CD4+ lymphocytes display severe impairment of chemotaxis and immunological synapse formation. Myeloid cells display aberrant enhancement of actin dependent structures, which alters cell motility and tissue distribution. Concurrent changes in actin remodeling in both cell types also promote viral spread via actin-dependent cell-cell contacts and support infection by inbound cell-free virus.
Figure Legend Snippet: Manipulation of the actin cytoskeleton by human immunodeficiency virus (HIV). ( a ) Actin regulators subjected to modulation by HIV. Mechanistically diverse strategies enable the virus to alter cellular cytoskeletal functions. Manipulation of host factors can be either direct, when mediated by physical interaction with viral proteins, or indirect, when requiring upstream cellular factors. Exploitation mechanisms increase native protein activity by upregulation of gene expression, or indirect activation within a cellular pathway. Neutralization of host factors is achieved by downregulation of gene expression or protein inactivation. Hijacking alters the functional outcome of host protein activity, either by overriding regulatory mechanisms (i.e., direct protein activation), changing protein subcellular localization, and/or modifying protein interaction partners. Note that some host factors can be manipulated by multiple strategies at diverse stages of the viral life cycle, as well as differentially in infected and uninfected cells. Examples of actin regulators corresponding to each strategy are provided, however this is not a complete list; ( b ) Functional consequences of actin-dependent changes induced by HIV. Normal immunological functions are compromised upon HIV infection, partly due to actin-remodeling changes orchestrated by viral proteins. CD4+ lymphocytes display severe impairment of chemotaxis and immunological synapse formation. Myeloid cells display aberrant enhancement of actin dependent structures, which alters cell motility and tissue distribution. Concurrent changes in actin remodeling in both cell types also promote viral spread via actin-dependent cell-cell contacts and support infection by inbound cell-free virus.

Techniques Used: Activity Assay, Expressing, Activation Assay, Neutralization, Functional Assay, Infection, Chemotaxis Assay

Functional protein interaction network of actin regulators targeted by HIV manipulation. Subversion of the actin cytoskeleton occurs throughout all layers of the actin cytoskeleton, including surface proteins, their linkers to actin filaments, diverse signaling nodes, their effectors, and proteins that directly mediate actin remodeling. Color coding of host factors indicates the main viral protein involved in their deregulation; Nef = green, Gag = yellow, Tat = pink, Env = blue, presently undefined = grey. So far, only proteins shown in bold have been experimentally confirmed to physically interact with the indicated viral proteins (i.e., direct manipulation). Network data was obtained from the STRING database [ 93 ] using the following settings: network edge meaning = evidence, active sources = databases, minimum confidence score = 0.9, number of interactors = query only (proteins covered in this review). The network nodes were rearranged using Cytoscape software [ 94 ], to represent functional groups within the cytoskeleton.
Figure Legend Snippet: Functional protein interaction network of actin regulators targeted by HIV manipulation. Subversion of the actin cytoskeleton occurs throughout all layers of the actin cytoskeleton, including surface proteins, their linkers to actin filaments, diverse signaling nodes, their effectors, and proteins that directly mediate actin remodeling. Color coding of host factors indicates the main viral protein involved in their deregulation; Nef = green, Gag = yellow, Tat = pink, Env = blue, presently undefined = grey. So far, only proteins shown in bold have been experimentally confirmed to physically interact with the indicated viral proteins (i.e., direct manipulation). Network data was obtained from the STRING database [ 93 ] using the following settings: network edge meaning = evidence, active sources = databases, minimum confidence score = 0.9, number of interactors = query only (proteins covered in this review). The network nodes were rearranged using Cytoscape software [ 94 ], to represent functional groups within the cytoskeleton.

Techniques Used: Functional Assay, Software

37) Product Images from "Azaspiracid substituent at C1 is relevant to in vitro toxicity"

Article Title: Azaspiracid substituent at C1 is relevant to in vitro toxicity

Journal: Chemical research in toxicology

doi: 10.1021/tx800165c

Effect of AZA-1, AZA-2, AZA-2-ME and biotin-AZA-2 on actin cytoskeleton of neuroblastoma BE(2)-M17 cells. Human neuroblastoma cells were incubated with concentrations of 1 and 10 nM of AZA-1 (A and B), AZA-2 (C and D), AZA-2-ME (E and F), biotin-AZA-2 (G and H) for 48 h. Control cells (I) were incubated with carrier in the same conditions as toxin-treated cells. Actin cytoskeleton was labelled with Oregon Green® 514 Phalloidin. Bar size: 10 μm. Representative of 3 experiments.
Figure Legend Snippet: Effect of AZA-1, AZA-2, AZA-2-ME and biotin-AZA-2 on actin cytoskeleton of neuroblastoma BE(2)-M17 cells. Human neuroblastoma cells were incubated with concentrations of 1 and 10 nM of AZA-1 (A and B), AZA-2 (C and D), AZA-2-ME (E and F), biotin-AZA-2 (G and H) for 48 h. Control cells (I) were incubated with carrier in the same conditions as toxin-treated cells. Actin cytoskeleton was labelled with Oregon Green® 514 Phalloidin. Bar size: 10 μm. Representative of 3 experiments.

Techniques Used: Incubation

Irreversible effects of AZA-2, AZA-2-ME and biotin-AZA-2 on actin cytoskeleton of neuroblastoma BE(2)-M17 cells. Human neuroblastoma cells were incubated with concentrations of 10, 25 and 50 nM of AZA-2 (A, B and C) and AZA-2-ME (D, E and F) and 100, 250 and 500 nM of biotin-AZA-2 (G, H and I) for 10 min. Then the cells were washed twice with a toxin-free medium and incubated in the absence of toxin for a total time of 48 h. Control cells (J) were incubated with carrier in the same conditions as toxin-treated cells. Actin cytoskeleton was labelled with Oregon Green® 514 Phalloidin. Bar size: 10 μm. Representative of 3 experiments.
Figure Legend Snippet: Irreversible effects of AZA-2, AZA-2-ME and biotin-AZA-2 on actin cytoskeleton of neuroblastoma BE(2)-M17 cells. Human neuroblastoma cells were incubated with concentrations of 10, 25 and 50 nM of AZA-2 (A, B and C) and AZA-2-ME (D, E and F) and 100, 250 and 500 nM of biotin-AZA-2 (G, H and I) for 10 min. Then the cells were washed twice with a toxin-free medium and incubated in the absence of toxin for a total time of 48 h. Control cells (J) were incubated with carrier in the same conditions as toxin-treated cells. Actin cytoskeleton was labelled with Oregon Green® 514 Phalloidin. Bar size: 10 μm. Representative of 3 experiments.

Techniques Used: Incubation

38) Product Images from "Evidence That Atypical Protein Kinase C-? and Atypical Protein Kinase C-? Participate in Ras-mediated Reorganization of the F-actin Cytoskeleton "

Article Title: Evidence That Atypical Protein Kinase C-? and Atypical Protein Kinase C-? Participate in Ras-mediated Reorganization of the F-actin Cytoskeleton

Journal: The Journal of Cell Biology

doi:

Treatment of Ras-expressing cells with the PI3K inhibitors wortmannin or LY294002 counteracts the effects of Ras on the actin cytoskeleton. Shown are representative fluorescence images of fibroblasts transiently expressing Ha-Ras L61. (A) Ha-Ras L61 alone, or (B) in the presence of 25 nM wortmannin, or (C) in the presence of 25 μM LY294002. 48 h posttransfection, cells were fixed and visualized as described in Materials and Methods. Representative cells of at least three different experiments are shown for all panels. Stacks of images were exported into Adobe Photoshop and printed as described.
Figure Legend Snippet: Treatment of Ras-expressing cells with the PI3K inhibitors wortmannin or LY294002 counteracts the effects of Ras on the actin cytoskeleton. Shown are representative fluorescence images of fibroblasts transiently expressing Ha-Ras L61. (A) Ha-Ras L61 alone, or (B) in the presence of 25 nM wortmannin, or (C) in the presence of 25 μM LY294002. 48 h posttransfection, cells were fixed and visualized as described in Materials and Methods. Representative cells of at least three different experiments are shown for all panels. Stacks of images were exported into Adobe Photoshop and printed as described.

Techniques Used: Expressing, Fluorescence

Reversion of Ras-induced disassembly of F-actin cytoskeleton by kinase-defective, DN as well as PKC isotype-specific antisense constructs. Shown are representative fluorescence images of fibroblasts transiently expressing Ha-Ras L61 (A–G). (A) Ha-Ras L61 alone, or (B) together with DN aPKC-λ; (C) antisense aPKC-λ; (D) DN aPKC-ζ; (E) antisense aPKC-ζ; (F) DN cPKC-α; and (G) antisense cPKC-α. 48 h posttransfection, cells were fixed and visualized as described in Materials and Methods. Representative cells of at least three different experiments are shown for all panels. The corresponding sense constructs were analyzed in parallel (data not shown). Stacks of images were exported into Adobe Photoshop and printed on a color laser copier system.
Figure Legend Snippet: Reversion of Ras-induced disassembly of F-actin cytoskeleton by kinase-defective, DN as well as PKC isotype-specific antisense constructs. Shown are representative fluorescence images of fibroblasts transiently expressing Ha-Ras L61 (A–G). (A) Ha-Ras L61 alone, or (B) together with DN aPKC-λ; (C) antisense aPKC-λ; (D) DN aPKC-ζ; (E) antisense aPKC-ζ; (F) DN cPKC-α; and (G) antisense cPKC-α. 48 h posttransfection, cells were fixed and visualized as described in Materials and Methods. Representative cells of at least three different experiments are shown for all panels. The corresponding sense constructs were analyzed in parallel (data not shown). Stacks of images were exported into Adobe Photoshop and printed on a color laser copier system.

Techniques Used: Construct, Fluorescence, Expressing

39) Product Images from "LI-Cadherin-mediated Cell-Cell Adhesion Does Not Require Cytoplasmic Interactions"

Article Title: LI-Cadherin-mediated Cell-Cell Adhesion Does Not Require Cytoplasmic Interactions

Journal: The Journal of Cell Biology

doi:

Actin cytoskeleton reorganization is not induced by LIcadherin expression. L cells expressing either LI-cadherin ( a and b ) or XB/U-cadherin ( c and d ) were fixed, permeabilized, and the actin cytoskeleton was stained with FITC-phalloidin ( b and d ). For double labeling, the same cells were incubated with anti–LIcadherin mAb 47.2 ( a ) or anti–XB/U-cadherin mAb 6D5 ( c ) followed by staining with secondary TRITC-labeled antibodies. In transfected L cells expressing XB/U-cadherin, the actin cytoskeleton was completely redistributed to sites of cell–cell contact ( d ). In contrast, expression of LI-cadherin did not promote any significant reorganization of the actin cytoskeleton and stress fibers were still visible ( b ). Bar, ( d ) 20 μm.
Figure Legend Snippet: Actin cytoskeleton reorganization is not induced by LIcadherin expression. L cells expressing either LI-cadherin ( a and b ) or XB/U-cadherin ( c and d ) were fixed, permeabilized, and the actin cytoskeleton was stained with FITC-phalloidin ( b and d ). For double labeling, the same cells were incubated with anti–LIcadherin mAb 47.2 ( a ) or anti–XB/U-cadherin mAb 6D5 ( c ) followed by staining with secondary TRITC-labeled antibodies. In transfected L cells expressing XB/U-cadherin, the actin cytoskeleton was completely redistributed to sites of cell–cell contact ( d ). In contrast, expression of LI-cadherin did not promote any significant reorganization of the actin cytoskeleton and stress fibers were still visible ( b ). Bar, ( d ) 20 μm.

Techniques Used: Expressing, Staining, Labeling, Incubation, Transfection

LI-cadherin mediates aggregation of transfected L cells. Aggregation of LI-cadherin expressing L cells was analyzed in the presence of 2 mM CaCl 2 ( a ), 2 mM EDTA ( b ) or anti–LI-cadherin pAb120 ( c ). For the disruption of the cytoskeleton ( d ), cells were preincubated with 1 μM cytochalasin D for 30 min at 37°C. LI-cadherin acted as a Ca 2+ -dependent cell adhesion molecule when expressed in L cells. Its function was not affected by the disruption of the actin cytoskeleton.
Figure Legend Snippet: LI-cadherin mediates aggregation of transfected L cells. Aggregation of LI-cadherin expressing L cells was analyzed in the presence of 2 mM CaCl 2 ( a ), 2 mM EDTA ( b ) or anti–LI-cadherin pAb120 ( c ). For the disruption of the cytoskeleton ( d ), cells were preincubated with 1 μM cytochalasin D for 30 min at 37°C. LI-cadherin acted as a Ca 2+ -dependent cell adhesion molecule when expressed in L cells. Its function was not affected by the disruption of the actin cytoskeleton.

Techniques Used: Transfection, Expressing

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    Cytoskeleton Inc f actin g actin in vivo assay kit
    GAP43 depletion decreased metastasis in vivo and triggered F-actin depolymerization. a Validation of stable GAP43 knockdown in NCI-H661 cells by western blotting. b Representative images of bioluminescence 4 and 8 weeks after left ventricular injection of NCI-H661-Luc-shNC and NCI-H661-Luc-shGAP43 cells. c Quantification of bioluminescence in the 8th week. Each group had 10 mice, while 2 mice in the NCI-H661-Luc-shNC group died soon after the injection of left ventricle. The P value was determined using a Mann–Whitney U test. d Representative images of F-actin immunofluorescence in NCI-H661-Luc-shNC and NCI-H661-Luc-shGAP43 cells. e Expression levels of F-actin and G-actin in NCI-H661-Luc-shNC and NCI-H661-Luc-shGAP43 cells examined by western blotting and a histogram of the <t>F-actin/G-actin</t> ratio. F-actin was depolymerized and decreased after GAP43 knockdown. The P value was estimated with Student’s t-test. f Detection of active and total Rac1 in NCI-H661-Luc-shNC and NCI-H661-Luc-shGAP43 cells by western blotting. Active Rac1 decreased and total Rac1 remained unchanged after GAP43 depletion. ** P
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    αN-catenin represses <t>Arp2/3-actin</t> association and polymerization (a) Elevated association between affinity-purified F-actin and ARP2/3 in CTNNA2- mutant cells. Input: basal expression. F-actin pulldown with phalloidin. Exaggerated ARP3 band in 1263A and knockout lines, compared MDS or Control line. EIF3D (does not bind actin) as a control for pulldown specificity. Cropped images shown. Repeated in triplicate, quantification and SEM below. Schematic depicts model (right). (b) Actin polymerization assay showing dose-dependent inhibition of Arp2/3 + VCA domain of WASP mediated actin polymerization by αN-catenin, measured by relative fluorescent units. In the absence of Arp2/3 (black) there was minimal polymerization, but when Arp2/3 + VCA was added (green) polymerization increased substantially, which was reversed upon dose escalation of αN-catenin. Repeated in triplicate. Schematic depicts model (right). (c) Loss of association between F-actin and ARP2/3 in CTNNA2 KO cells expressing the actin binding domain (ABD) of αN-catenin. Input: basal expression of ARP3 and Actin. F-actin pulldown with phalloidin. Exaggerated ARP3 band in knockout neurons, and loss of ARP3 in ABD-expressing knockout neurons, compared with band in Control line. Quantification by relative fluorescence of ARP3 to Actin. Cropped images shown. Repeated in duplicate. (d) ARP2/3 inhibition by CK-666 rescued neurite length defect in CTNNA2 mutant neurons, but not Control and MDS mutant neurons. Scale bar 50 μm. (e) Quantification of (d) with top and bottom quartiles and Median displayed. Whiskers represent the minimum and maximum values observed in the dataset. Repeated in three independent iPSC clones per patient or three CTNNA2 KO clones, 442 cells scored.*, ** and ***, significant P (see Statistics and Reproducibility).
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    Cytoskeleton Inc anterior anterior lateral cortical actin cytoskeleton
    The oocyte actin <t>cytoskeleton</t> is not maintained in orb . Egg chambers are stained with Cad99C (green), which labels the apical microvilli of the somatic follicle cells, and Phalloidin (red), which labels Actin on both the apical side of the follicle cells and the oocyte cortex, and DAPI (blue). (A and A’) Wild type organization of Actin along the oocyte cortex (n = 16). (B and B’) Defects in Actin organization in orb mel /orb 343 are observed in 72% of oocytes stages 9–11 (n = 18). (C and C’) Defects in actin organization when orb RNAi (#64002) is expressed during mid-stages of oogenesis (maternal α-tubulin Gal4, #7062) are observed in 65% of oocytes stages 9–11 (n = 20). We also observed a defect in actin structure at a frequency of 70% in HD19 , 343/mel oocytes stages 9–11 (n = 10). Arrows point to defects in cortical actin organization as described in the text. Top panel scale bars are 50 microns; bottom panel scale bars are 10 microns.
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    GAP43 depletion decreased metastasis in vivo and triggered F-actin depolymerization. a Validation of stable GAP43 knockdown in NCI-H661 cells by western blotting. b Representative images of bioluminescence 4 and 8 weeks after left ventricular injection of NCI-H661-Luc-shNC and NCI-H661-Luc-shGAP43 cells. c Quantification of bioluminescence in the 8th week. Each group had 10 mice, while 2 mice in the NCI-H661-Luc-shNC group died soon after the injection of left ventricle. The P value was determined using a Mann–Whitney U test. d Representative images of F-actin immunofluorescence in NCI-H661-Luc-shNC and NCI-H661-Luc-shGAP43 cells. e Expression levels of F-actin and G-actin in NCI-H661-Luc-shNC and NCI-H661-Luc-shGAP43 cells examined by western blotting and a histogram of the F-actin/G-actin ratio. F-actin was depolymerized and decreased after GAP43 knockdown. The P value was estimated with Student’s t-test. f Detection of active and total Rac1 in NCI-H661-Luc-shNC and NCI-H661-Luc-shGAP43 cells by western blotting. Active Rac1 decreased and total Rac1 remained unchanged after GAP43 depletion. ** P

    Journal: Journal of Translational Medicine

    Article Title: GAP43, a novel metastasis promoter in non-small cell lung cancer

    doi: 10.1186/s12967-018-1682-5

    Figure Lengend Snippet: GAP43 depletion decreased metastasis in vivo and triggered F-actin depolymerization. a Validation of stable GAP43 knockdown in NCI-H661 cells by western blotting. b Representative images of bioluminescence 4 and 8 weeks after left ventricular injection of NCI-H661-Luc-shNC and NCI-H661-Luc-shGAP43 cells. c Quantification of bioluminescence in the 8th week. Each group had 10 mice, while 2 mice in the NCI-H661-Luc-shNC group died soon after the injection of left ventricle. The P value was determined using a Mann–Whitney U test. d Representative images of F-actin immunofluorescence in NCI-H661-Luc-shNC and NCI-H661-Luc-shGAP43 cells. e Expression levels of F-actin and G-actin in NCI-H661-Luc-shNC and NCI-H661-Luc-shGAP43 cells examined by western blotting and a histogram of the F-actin/G-actin ratio. F-actin was depolymerized and decreased after GAP43 knockdown. The P value was estimated with Student’s t-test. f Detection of active and total Rac1 in NCI-H661-Luc-shNC and NCI-H661-Luc-shGAP43 cells by western blotting. Active Rac1 decreased and total Rac1 remained unchanged after GAP43 depletion. ** P

    Article Snippet: Proteins were extracted and analyzed using an F-actin/G-actin in vivo assay kit (BK037; Cytoskeleton, Denver, CO, USA) based on the manufacturer’s instructions.

    Techniques: In Vivo, Western Blot, Injection, Mouse Assay, MANN-WHITNEY, Immunofluorescence, Expressing

    αN-catenin represses Arp2/3-actin association and polymerization (a) Elevated association between affinity-purified F-actin and ARP2/3 in CTNNA2- mutant cells. Input: basal expression. F-actin pulldown with phalloidin. Exaggerated ARP3 band in 1263A and knockout lines, compared MDS or Control line. EIF3D (does not bind actin) as a control for pulldown specificity. Cropped images shown. Repeated in triplicate, quantification and SEM below. Schematic depicts model (right). (b) Actin polymerization assay showing dose-dependent inhibition of Arp2/3 + VCA domain of WASP mediated actin polymerization by αN-catenin, measured by relative fluorescent units. In the absence of Arp2/3 (black) there was minimal polymerization, but when Arp2/3 + VCA was added (green) polymerization increased substantially, which was reversed upon dose escalation of αN-catenin. Repeated in triplicate. Schematic depicts model (right). (c) Loss of association between F-actin and ARP2/3 in CTNNA2 KO cells expressing the actin binding domain (ABD) of αN-catenin. Input: basal expression of ARP3 and Actin. F-actin pulldown with phalloidin. Exaggerated ARP3 band in knockout neurons, and loss of ARP3 in ABD-expressing knockout neurons, compared with band in Control line. Quantification by relative fluorescence of ARP3 to Actin. Cropped images shown. Repeated in duplicate. (d) ARP2/3 inhibition by CK-666 rescued neurite length defect in CTNNA2 mutant neurons, but not Control and MDS mutant neurons. Scale bar 50 μm. (e) Quantification of (d) with top and bottom quartiles and Median displayed. Whiskers represent the minimum and maximum values observed in the dataset. Repeated in three independent iPSC clones per patient or three CTNNA2 KO clones, 442 cells scored.*, ** and ***, significant P (see Statistics and Reproducibility).

    Journal: Nature genetics

    Article Title: Bi-allelic loss of human CTNNA2, encoding αN-catenin, leads to ARP2/3 over-activity and disordered cortical neuronal migration

    doi: 10.1038/s41588-018-0166-0

    Figure Lengend Snippet: αN-catenin represses Arp2/3-actin association and polymerization (a) Elevated association between affinity-purified F-actin and ARP2/3 in CTNNA2- mutant cells. Input: basal expression. F-actin pulldown with phalloidin. Exaggerated ARP3 band in 1263A and knockout lines, compared MDS or Control line. EIF3D (does not bind actin) as a control for pulldown specificity. Cropped images shown. Repeated in triplicate, quantification and SEM below. Schematic depicts model (right). (b) Actin polymerization assay showing dose-dependent inhibition of Arp2/3 + VCA domain of WASP mediated actin polymerization by αN-catenin, measured by relative fluorescent units. In the absence of Arp2/3 (black) there was minimal polymerization, but when Arp2/3 + VCA was added (green) polymerization increased substantially, which was reversed upon dose escalation of αN-catenin. Repeated in triplicate. Schematic depicts model (right). (c) Loss of association between F-actin and ARP2/3 in CTNNA2 KO cells expressing the actin binding domain (ABD) of αN-catenin. Input: basal expression of ARP3 and Actin. F-actin pulldown with phalloidin. Exaggerated ARP3 band in knockout neurons, and loss of ARP3 in ABD-expressing knockout neurons, compared with band in Control line. Quantification by relative fluorescence of ARP3 to Actin. Cropped images shown. Repeated in duplicate. (d) ARP2/3 inhibition by CK-666 rescued neurite length defect in CTNNA2 mutant neurons, but not Control and MDS mutant neurons. Scale bar 50 μm. (e) Quantification of (d) with top and bottom quartiles and Median displayed. Whiskers represent the minimum and maximum values observed in the dataset. Repeated in three independent iPSC clones per patient or three CTNNA2 KO clones, 442 cells scored.*, ** and ***, significant P (see Statistics and Reproducibility).

    Article Snippet: Actin polymerization assays were performed with pyrene actin (Cytoskeleton, Inc., AP05), α-actinin, GST-tagged VCA domain of human WASP protein (#VCG03), and Arp2/3 protein complex from porcine brain (Cytoskeleton, Inc., RP01P) according to manufacturer’s recommendations, with the addition of recombinant αN-catenin.

    Techniques: Affinity Purification, Mutagenesis, Expressing, Knock-Out, Polymerization Assay, Inhibition, Binding Assay, Fluorescence, Clone Assay

    Cortactin T24 is required for binding and activation of Arp2/3 complex. A. Diagram representing cortactin functional domains. NTA, N-terminal acidic domain; R1-R6, repeats regions with F-actin binding site indicated; Helix, alpha helical domain, PRR, proline rich region; SH3, Src homology 3 domain. NTA domain with position of S11 and T24 in context of the DDW region is shown. B. Immunoprecipitation analysis of Arp2/3 binding to cortactin NTA mutants. HEK293T/17 cells transfected with FLAG-empty vector (EV), FLAG-wild-type cortactin (WT) or the indicated FLAG-cortactin mutants. Immune complexes were Western blotted with antibodies against cortactin (top) and Arp3 (bottom). 1:10 diluted total cell lysates were Western blotted as indicated. C. Coomassie blue staining of the indicated purified recombinant human cortactin proteins. D. Effect of cortactin T24A on Arp2/3 complex activation. Fluorometric evaluation of actin polymerization over time with the indicated cortactin mutants incubated with Arp2/3 complex and pyrene-labeled actin. Polymerization curves: WT cortactin (blue); T24A cortactin (dark green) and ΔDDW cortactin (purple). N-WASp VCA domain (black) was used as a positive control; negative controls include Arp2/3 complex plus actin (pale green) and actin alone (red). Polymerization curves are representative from three independent experiments.

    Journal: Molecular cancer research : MCR

    Article Title: Cortactin Phosphorylation by Casein Kinase 2 Regulates Actin-Related Protein 2/3 Complex Activity, Invadopodia Function and Tumor Cell Invasion

    doi: 10.1158/1541-7786.MCR-18-0391

    Figure Lengend Snippet: Cortactin T24 is required for binding and activation of Arp2/3 complex. A. Diagram representing cortactin functional domains. NTA, N-terminal acidic domain; R1-R6, repeats regions with F-actin binding site indicated; Helix, alpha helical domain, PRR, proline rich region; SH3, Src homology 3 domain. NTA domain with position of S11 and T24 in context of the DDW region is shown. B. Immunoprecipitation analysis of Arp2/3 binding to cortactin NTA mutants. HEK293T/17 cells transfected with FLAG-empty vector (EV), FLAG-wild-type cortactin (WT) or the indicated FLAG-cortactin mutants. Immune complexes were Western blotted with antibodies against cortactin (top) and Arp3 (bottom). 1:10 diluted total cell lysates were Western blotted as indicated. C. Coomassie blue staining of the indicated purified recombinant human cortactin proteins. D. Effect of cortactin T24A on Arp2/3 complex activation. Fluorometric evaluation of actin polymerization over time with the indicated cortactin mutants incubated with Arp2/3 complex and pyrene-labeled actin. Polymerization curves: WT cortactin (blue); T24A cortactin (dark green) and ΔDDW cortactin (purple). N-WASp VCA domain (black) was used as a positive control; negative controls include Arp2/3 complex plus actin (pale green) and actin alone (red). Polymerization curves are representative from three independent experiments.

    Article Snippet: Kazazian K, Go C, Wu H, Brashavitskaya O, Xu R, Dennis JW, et al. Plk4 Promotes Cancer Invasion and Metastasis through Arp2/3 Complex Regulation of the Actin Cytoskeleton .

    Techniques: Binding Assay, Activation Assay, Functional Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Staining, Purification, Recombinant, Incubation, Labeling, Positive Control

    CK2α phosphorylation of cortactin T24 inhibits Arp2/3 complex binding and activation. A. Cortactin T24 is a CK2α phosphorylation site. Autoradiogram of active CK2α incubated with the increasing amounts (0, 0.25, 0.5, and 1 μg) of GST-WT-NTA or GST-T24A-NTA cortactin fusion proteins. GST (1 μg) and the GST-VCA domain of N-WASp (0.25 μg) were used as respective negative and positive phosphorylation controls. Positions of autophosphorylated CK2α, GST-VCA and cortactin NTA proteins are indicated on the left; autoradiogram is representative of three independent experiments. B. CK2α phosphorylation at cortactin T24 ablates binding to Arp2/3 complex. Purified recombinant human WT and T24A cortactin proteins (2.5 μg) were bound with an anti-cortactin antibody to protein G beads. Immune complexes were preincubated with or without 75 ng active CK2α, washed and incubated with 50 ng purified Arp2/3 complex. Co-immunoprecipitated complexes were Western blotted for cortactin (top) and Arp3 (bottom). 4F11-bound protein G beads were used as a negative control for non-specific binding (Beads). Arp2/3 complex (5 ng) was used as positive control for Arp3 immunoblotting. Blot is representative of two independent experiments. C. Cortactin phosphorylation by CK2α inhibits cortactin-mediated Arp2/3 actin polymerization. WT human cortactin or GST-VCA proteins were preincubated with the indicated amounts of active CK2α and evaluated for effects on Arp2/3 activity. Polymerization curves are representative from three independent experiments. D. Phosphorylation of T24 is responsible for the inhibitory effect of CK2α on cortactin-mediated Arp2/3 activation. Human WT and T24A cortactin proteins were preincubated with or without 30 ng active CK2α and evaluated for effects on Arp2/3-mediated actin assembly. Polymerization curves are representative from three independent experiments.

    Journal: Molecular cancer research : MCR

    Article Title: Cortactin Phosphorylation by Casein Kinase 2 Regulates Actin-Related Protein 2/3 Complex Activity, Invadopodia Function and Tumor Cell Invasion

    doi: 10.1158/1541-7786.MCR-18-0391

    Figure Lengend Snippet: CK2α phosphorylation of cortactin T24 inhibits Arp2/3 complex binding and activation. A. Cortactin T24 is a CK2α phosphorylation site. Autoradiogram of active CK2α incubated with the increasing amounts (0, 0.25, 0.5, and 1 μg) of GST-WT-NTA or GST-T24A-NTA cortactin fusion proteins. GST (1 μg) and the GST-VCA domain of N-WASp (0.25 μg) were used as respective negative and positive phosphorylation controls. Positions of autophosphorylated CK2α, GST-VCA and cortactin NTA proteins are indicated on the left; autoradiogram is representative of three independent experiments. B. CK2α phosphorylation at cortactin T24 ablates binding to Arp2/3 complex. Purified recombinant human WT and T24A cortactin proteins (2.5 μg) were bound with an anti-cortactin antibody to protein G beads. Immune complexes were preincubated with or without 75 ng active CK2α, washed and incubated with 50 ng purified Arp2/3 complex. Co-immunoprecipitated complexes were Western blotted for cortactin (top) and Arp3 (bottom). 4F11-bound protein G beads were used as a negative control for non-specific binding (Beads). Arp2/3 complex (5 ng) was used as positive control for Arp3 immunoblotting. Blot is representative of two independent experiments. C. Cortactin phosphorylation by CK2α inhibits cortactin-mediated Arp2/3 actin polymerization. WT human cortactin or GST-VCA proteins were preincubated with the indicated amounts of active CK2α and evaluated for effects on Arp2/3 activity. Polymerization curves are representative from three independent experiments. D. Phosphorylation of T24 is responsible for the inhibitory effect of CK2α on cortactin-mediated Arp2/3 activation. Human WT and T24A cortactin proteins were preincubated with or without 30 ng active CK2α and evaluated for effects on Arp2/3-mediated actin assembly. Polymerization curves are representative from three independent experiments.

    Article Snippet: Kazazian K, Go C, Wu H, Brashavitskaya O, Xu R, Dennis JW, et al. Plk4 Promotes Cancer Invasion and Metastasis through Arp2/3 Complex Regulation of the Actin Cytoskeleton .

    Techniques: Binding Assay, Activation Assay, Incubation, Purification, Recombinant, Immunoprecipitation, Western Blot, Negative Control, Positive Control, Activity Assay

    The oocyte actin cytoskeleton is not maintained in orb . Egg chambers are stained with Cad99C (green), which labels the apical microvilli of the somatic follicle cells, and Phalloidin (red), which labels Actin on both the apical side of the follicle cells and the oocyte cortex, and DAPI (blue). (A and A’) Wild type organization of Actin along the oocyte cortex (n = 16). (B and B’) Defects in Actin organization in orb mel /orb 343 are observed in 72% of oocytes stages 9–11 (n = 18). (C and C’) Defects in actin organization when orb RNAi (#64002) is expressed during mid-stages of oogenesis (maternal α-tubulin Gal4, #7062) are observed in 65% of oocytes stages 9–11 (n = 20). We also observed a defect in actin structure at a frequency of 70% in HD19 , 343/mel oocytes stages 9–11 (n = 10). Arrows point to defects in cortical actin organization as described in the text. Top panel scale bars are 50 microns; bottom panel scale bars are 10 microns.

    Journal: PLoS Genetics

    Article Title: The CPEB translational regulator, Orb, functions together with Par proteins to polarize the Drosophila oocyte

    doi: 10.1371/journal.pgen.1008012

    Figure Lengend Snippet: The oocyte actin cytoskeleton is not maintained in orb . Egg chambers are stained with Cad99C (green), which labels the apical microvilli of the somatic follicle cells, and Phalloidin (red), which labels Actin on both the apical side of the follicle cells and the oocyte cortex, and DAPI (blue). (A and A’) Wild type organization of Actin along the oocyte cortex (n = 16). (B and B’) Defects in Actin organization in orb mel /orb 343 are observed in 72% of oocytes stages 9–11 (n = 18). (C and C’) Defects in actin organization when orb RNAi (#64002) is expressed during mid-stages of oogenesis (maternal α-tubulin Gal4, #7062) are observed in 65% of oocytes stages 9–11 (n = 20). We also observed a defect in actin structure at a frequency of 70% in HD19 , 343/mel oocytes stages 9–11 (n = 10). Arrows point to defects in cortical actin organization as described in the text. Top panel scale bars are 50 microns; bottom panel scale bars are 10 microns.

    Article Snippet: Defects in the expression of these proteins would interfere with the remodeling of the anterior/anterior-lateral cortical actin cytoskeleton and consequently disrupt Par dependent MT polarization.

    Techniques: Staining