latrunculin  (Cayman Chemical)

 
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    Name:
    Latrunculin A
    Description:
    Actin disruption is used to study cell functions in vitro e g migration endocytosis and in vivo e g tumor cell invasion Latrunculin A is a bioactive 2 thiazolidinone macrolide derived from sponges that sequesters G actin and prevents F actin assembly It binds monomeric actin with 1 1 stoichiometry and can be used to block actin polymerization both in vitro K 0 2 μM and in cells 0 5 μM 30 min Latrunculin A 1 10 μM causes depolymerization of tumor cell cytoskeleton within ten minutes Overnight treatment of cells with latrunculin A 10 μM strongly suppresses actin synthesis Prolonged cell treatment blocks dexamethasone induced changes in actin cytoskeleton with no effect on cell viability
    Catalog Number:
    10010630
    Price:
    $39
    Purity:
    ≥95%
    Size:
    25 µg
    Product Aliases:
    NSC 613011
    Formula:
    A solution in ethanol
    Buy from Supplier


    Structured Review

    Cayman Chemical latrunculin
    Inflammasome priming by Francisella is independent of bacteria internalization. Human monocytes were pretreated for 30 min with <t>latrunculin</t> A (Latr) (200 nM) and cytochalasin D (Cyto D) (5 μg/ml) and infected with F . novicida for 16 h. CFU of internalized bacteria ( A ) and IL-18 release ( B ) were counted. Monocytes treated with latrunculin and cytochalasin D as in A, B and then primed with Francisella for 30 min followed by ATP (5mM) for 30 min were analyzed for IL-18 in cell culture media by ELISA ( C ). Data represent mean ± SEM, n = 3 independent experiments. * p
    Actin disruption is used to study cell functions in vitro e g migration endocytosis and in vivo e g tumor cell invasion Latrunculin A is a bioactive 2 thiazolidinone macrolide derived from sponges that sequesters G actin and prevents F actin assembly It binds monomeric actin with 1 1 stoichiometry and can be used to block actin polymerization both in vitro K 0 2 μM and in cells 0 5 μM 30 min Latrunculin A 1 10 μM causes depolymerization of tumor cell cytoskeleton within ten minutes Overnight treatment of cells with latrunculin A 10 μM strongly suppresses actin synthesis Prolonged cell treatment blocks dexamethasone induced changes in actin cytoskeleton with no effect on cell viability
    https://www.bioz.com/result/latrunculin/product/Cayman Chemical
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    latrunculin - by Bioz Stars, 2021-09
    98/100 stars

    Images

    1) Product Images from "Inflammasome Priming Is Similar for Francisella Species That Differentially Induce Inflammasome Activation"

    Article Title: Inflammasome Priming Is Similar for Francisella Species That Differentially Induce Inflammasome Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0127278

    Inflammasome priming by Francisella is independent of bacteria internalization. Human monocytes were pretreated for 30 min with latrunculin A (Latr) (200 nM) and cytochalasin D (Cyto D) (5 μg/ml) and infected with F . novicida for 16 h. CFU of internalized bacteria ( A ) and IL-18 release ( B ) were counted. Monocytes treated with latrunculin and cytochalasin D as in A, B and then primed with Francisella for 30 min followed by ATP (5mM) for 30 min were analyzed for IL-18 in cell culture media by ELISA ( C ). Data represent mean ± SEM, n = 3 independent experiments. * p
    Figure Legend Snippet: Inflammasome priming by Francisella is independent of bacteria internalization. Human monocytes were pretreated for 30 min with latrunculin A (Latr) (200 nM) and cytochalasin D (Cyto D) (5 μg/ml) and infected with F . novicida for 16 h. CFU of internalized bacteria ( A ) and IL-18 release ( B ) were counted. Monocytes treated with latrunculin and cytochalasin D as in A, B and then primed with Francisella for 30 min followed by ATP (5mM) for 30 min were analyzed for IL-18 in cell culture media by ELISA ( C ). Data represent mean ± SEM, n = 3 independent experiments. * p

    Techniques Used: Infection, Cell Culture, Enzyme-linked Immunosorbent Assay

    2) Product Images from "Dysregulation in Actin Cytoskeletal Organization Drives Increased Stiffness and Migratory Persistence in Polyploidal Giant Cancer Cells"

    Article Title: Dysregulation in Actin Cytoskeletal Organization Drives Increased Stiffness and Migratory Persistence in Polyploidal Giant Cancer Cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-29817-5

    Biophysical phenotype of PGCCs is driven by actin cytoskeletal organization and mediated via the RhoA-ROCK1 pathway. ( A ) Total averaged MSD ensembles of particles embedded in non-PGCCs and ( B ) PGCCs tracked after 2-hour pretreatment with ML7 (MLCK inhibitor), H1152 (ROCK inhibitor) and latrunculin A (Actin cytoskeleton disruptor). ( C ) Total averaged MSD ensembles of chromatin granule motion in non-PGCCs and ( D ) PGCCs tracked after 2-hour pretreatment with ML7, H1152 and latrunculin A. ( E ) Summary table of inhibitor studies detailing MSD (at 1 second time lag), CV (coefficient of variation) and alpha (logarithmic slope of MSD as a function of time, for cytoplasmic mechanics) and RT (relaxation time, for nuclear mechanics). All results are normalized to either non-PGCC or PGCC untreated counterparts. Experiments were performed in triplicate.
    Figure Legend Snippet: Biophysical phenotype of PGCCs is driven by actin cytoskeletal organization and mediated via the RhoA-ROCK1 pathway. ( A ) Total averaged MSD ensembles of particles embedded in non-PGCCs and ( B ) PGCCs tracked after 2-hour pretreatment with ML7 (MLCK inhibitor), H1152 (ROCK inhibitor) and latrunculin A (Actin cytoskeleton disruptor). ( C ) Total averaged MSD ensembles of chromatin granule motion in non-PGCCs and ( D ) PGCCs tracked after 2-hour pretreatment with ML7, H1152 and latrunculin A. ( E ) Summary table of inhibitor studies detailing MSD (at 1 second time lag), CV (coefficient of variation) and alpha (logarithmic slope of MSD as a function of time, for cytoplasmic mechanics) and RT (relaxation time, for nuclear mechanics). All results are normalized to either non-PGCC or PGCC untreated counterparts. Experiments were performed in triplicate.

    Techniques Used:

    3) Product Images from "Dysregulation in Actin Cytoskeletal Organization Drives Increased Stiffness and Migratory Persistence in Polyploidal Giant Cancer Cells"

    Article Title: Dysregulation in Actin Cytoskeletal Organization Drives Increased Stiffness and Migratory Persistence in Polyploidal Giant Cancer Cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-29817-5

    Biophysical phenotype of PGCCs is driven by actin cytoskeletal organization and mediated via the RhoA-ROCK1 pathway. ( A ) Total averaged MSD ensembles of particles embedded in non-PGCCs and ( B ) PGCCs tracked after 2-hour pretreatment with ML7 (MLCK inhibitor), H1152 (ROCK inhibitor) and latrunculin A (Actin cytoskeleton disruptor). ( C ) Total averaged MSD ensembles of chromatin granule motion in non-PGCCs and ( D ) PGCCs tracked after 2-hour pretreatment with ML7, H1152 and latrunculin A. ( E ) Summary table of inhibitor studies detailing MSD (at 1 second time lag), CV (coefficient of variation) and alpha (logarithmic slope of MSD as a function of time, for cytoplasmic mechanics) and RT (relaxation time, for nuclear mechanics). All results are normalized to either non-PGCC or PGCC untreated counterparts. Experiments were performed in triplicate.
    Figure Legend Snippet: Biophysical phenotype of PGCCs is driven by actin cytoskeletal organization and mediated via the RhoA-ROCK1 pathway. ( A ) Total averaged MSD ensembles of particles embedded in non-PGCCs and ( B ) PGCCs tracked after 2-hour pretreatment with ML7 (MLCK inhibitor), H1152 (ROCK inhibitor) and latrunculin A (Actin cytoskeleton disruptor). ( C ) Total averaged MSD ensembles of chromatin granule motion in non-PGCCs and ( D ) PGCCs tracked after 2-hour pretreatment with ML7, H1152 and latrunculin A. ( E ) Summary table of inhibitor studies detailing MSD (at 1 second time lag), CV (coefficient of variation) and alpha (logarithmic slope of MSD as a function of time, for cytoplasmic mechanics) and RT (relaxation time, for nuclear mechanics). All results are normalized to either non-PGCC or PGCC untreated counterparts. Experiments were performed in triplicate.

    Techniques Used:

    Related Articles

    other:

    Article Title: ERK-mediated Curvature Feedback Regulates Branching Morphogenesis in Lung Epithelial Tissue
    Article Snippet: Scale bar, 30 µm. (A) (D) Relative change in cell height after treatment with cytoskeletal inhibitors, such as 1 µM Latrunculin A, 1 µM Nocodazole, and 30 µM Blebbistatin.

    Article Title: Rapid propagation of membrane tension at a presynaptic terminal
    Article Snippet: In some cases, 20 μM Latrunculin-A (LatA, Cayman Chemicals) was added to the bath during cell plating to disrupt the F-actin cytoskeleton.

    Article Title: Serotonin 2A (5-HT2A) receptor affects cell–matrix adhesion and the formation and maintenance of stress fibers in HEK293 cells
    Article Snippet: Calphostin C (Cayman, 121263-19-2), CCG-1423 (Cayman 285986-88-1), SU6656, W7 (Cayman, 61714-27-0), latrunculin-A (Cayman, 76343-93-6), were purchased from Cayman Chemical, USA.

    Recombinant:

    Article Title: ERK-mediated Curvature Feedback Regulates Branching Morphogenesis in Lung Epithelial Tissue
    Article Snippet: .. Antibodies, small molecules, and recombinant proteinsWe used the following primary antibodies at a dilution ratio of 1:200 unless stated otherwise: rat monoclonal anti-E-cadherin (Cell Signaling Technology, #3195), rabbit polyclonal anti- phospho-myosin light chain (pMLC) (Abcam, #ab2480), Alexa Fluor 546-conjugated goat anti-rat IgG (H+L) antibody (Thermo Fisher Scientific, #A11081, 1:1000), Alexa Fluor 647- conjugated goat anti-rabbit IgG (H+L) antibody (Abcam, #ab150079, or Thermo Fisher Scientific, #A21247, 1:1000), Blebbistatin (Merck Millipore, #203391), CK-666 (Sigma- Aldrich, #SML0006), Cytochalasin D (Merck Millipore, #250255), FGF1 (R & D Systems, #232-FA), FGF7 (R & D Systems, #251-KG), Jasplakinolide (Abcam, #ab141409), Latrunculin A (Cayman Chemical, #10010630), Nocodazole (Merck Millipore, #487928), PD0325901 (FUJIFILM Wako Pure Chemical Corporation, #162-25291), SMIFH2 (Sigma-Aldrich, #S4826), Y-27632 (Merck Millipore, #SCM075). ..

    Concentration Assay:

    Article Title: Vav2 catalysis-dependent pathways contribute to skeletal muscle growth and metabolic homeostasis
    Article Snippet: .. When indicated, cells were treated for 1 h with the indicated concentration of Wortmannin (100 nM, Cat. #19545-26-7, Calbiochem), PIK-75 (200 nM, Cat. #372196-77-5, Cayman Chemical), TGX-221 (500 nM, Cat. #663619-89-4, Cayman Chemical), latrunculin A (200 nM, Cat. #L5163, Sigma-Aldrich) and cytochalasin D (2 μM, Cat. #C8273, Sigma-Aldrich). ..

    Imaging:

    Article Title: Targeted millisecond-scale activation of cells using non-invasive Sonogenetics
    Article Snippet: .. For cytoskeleton experiments, nocodazole (5μM; Tocris, #1228), jasplakinolide (200μM; ThermoFisher # J7473), paclitaxel (600nM; Sigma-Aldrich # T7191), cytochalasin D (5μM; Cayman Chemicals, #11330) or latrunculin A (1μM; Cayman Chemicals, # 10630) in 0.1% DMSO were added to the culture media 45 minutes prior to imaging . ..

    In Vitro:

    Article Title: Surface Engineering of FLT4-Targeted Nanocarriers Enhances Cell-Softening Glaucoma Therapy
    Article Snippet: .. For uptake studies in vitro MCs were prepared to load DiI hydrophobic dye (Invitrogen), whereas formulations intended for IOP studies in vivo were prepared to co-load Latrunculin A (LatA; Cayman Chemical Company) and DiI. ..

    In Vivo:

    Article Title: Surface Engineering of FLT4-Targeted Nanocarriers Enhances Cell-Softening Glaucoma Therapy
    Article Snippet: .. For uptake studies in vitro MCs were prepared to load DiI hydrophobic dye (Invitrogen), whereas formulations intended for IOP studies in vivo were prepared to co-load Latrunculin A (LatA; Cayman Chemical Company) and DiI. ..

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    Cayman Chemical latrunculin a
    PLMs induce caspase-1 activation independent on ROS production. ( A - C ). J774 macrophages were primed with LPS (100 ng/ml) for 2h. Then, indicated stimuli were added and cells were cultivated for additional 4h. ( A ). Caspase-1 activation was revealed by addition of fluorescent FAM-YVAD-FMK and examined by fluorescent microscopy. Adenosine triphosphate (ATP) and Aluminum hydroxide (Alum) were used as positive control for caspase-1 activation. ( B ). Total pro-caspase-1 and cleaved caspase-1 levels were revealed by western blot analysis. ( C ). Production of IL-1β after stimulation with PLMs of LPS-primed cells was measured after 12- and 24h-incubation by ELISA. ( D ). Macrophages were pretreated or not with <t>Latrunculin-A</t> and then stimulated with indicated PLMs. Total pro-caspase-1 and cleaved caspase-1 levels were revealed by western blot analysis. Data are representative of 4 independent experiments. ( E and F ). Macrophages were pretreated or not with Latrunculin A for 30min. Live yeast cells at 5 yeast: 1 cell ratio ( E ) or PLMs (50µg/ml) ( F ) were then added. Chemiluminescence was monitored for 2h in the presence of 66 µM of 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol). 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 µM) was used as a positive control. Results are representative of 2 independent experiments.
    Latrunculin A, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/latrunculin a/product/Cayman Chemical
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    latrunculin a - by Bioz Stars, 2021-09
    98/100 stars
      Buy from Supplier

    86
    Cayman Chemical latrunculin treatment
    Exocytosis of Cryptococcus neoformans from Dictyostelium is dependent on WASH and the actin cytoskeleton. (A) Heat killed S. cerevisiae are not exocytosed from WASH null D. discoideum . Example from 12 h time lapse imaging of heat killed S. cerevisiae in WASH null D. discoideum representative of 60 phagosomes containing heat killed S. cerevisiae from three independent experiments (images start at 500 min after phagocytosis). (B) C. neoformans are exocytosed from WASH null D. discoideum . Example from 12 h time lapse imaging representative of 62 phagosomes containing C. neoformans from three independent experiments (images start at 200 min after phagocytosis). Scale bars 5 μm. (C) Quantification of the percentage of exocytosis of C. neoformans and heat killed S. cerevisiae from WASH null D. discoideum from three independent 12 h time lapses. (D) Frequency histogram of combined 35 transit times measured from three independent 12 h time lapses. (E) Exocytosis of heat killed S. cerevisiae from wild type Ax2 but not WASH null or <t>latrunculin</t> A treated Ax2 D. discoideum . Quantification of the percentage of exocytosis from three independent 12 h time lapses. Total of 60 phagosomes were analyzed from each condition. P -values are Fishers test. (F) Exocytosis of C. neoformans from wild type Ax2, WASH null, and latrunculin A treated D. discoideum . Quantification of the percentage of exocytosis from three independent 12 h time lapses. Total of 60 phagosomes were analyzed from each condition. P -values are Fishers test. (G) Transit times of C. neoformans through WASH null and latrunculin A treated Ax2 D. discoideum are not significantly different. P -values are Mann-Whitney test.
    Latrunculin Treatment, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/latrunculin treatment/product/Cayman Chemical
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    latrunculin treatment - by Bioz Stars, 2021-09
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    Image Search Results


    PLMs induce caspase-1 activation independent on ROS production. ( A - C ). J774 macrophages were primed with LPS (100 ng/ml) for 2h. Then, indicated stimuli were added and cells were cultivated for additional 4h. ( A ). Caspase-1 activation was revealed by addition of fluorescent FAM-YVAD-FMK and examined by fluorescent microscopy. Adenosine triphosphate (ATP) and Aluminum hydroxide (Alum) were used as positive control for caspase-1 activation. ( B ). Total pro-caspase-1 and cleaved caspase-1 levels were revealed by western blot analysis. ( C ). Production of IL-1β after stimulation with PLMs of LPS-primed cells was measured after 12- and 24h-incubation by ELISA. ( D ). Macrophages were pretreated or not with Latrunculin-A and then stimulated with indicated PLMs. Total pro-caspase-1 and cleaved caspase-1 levels were revealed by western blot analysis. Data are representative of 4 independent experiments. ( E and F ). Macrophages were pretreated or not with Latrunculin A for 30min. Live yeast cells at 5 yeast: 1 cell ratio ( E ) or PLMs (50µg/ml) ( F ) were then added. Chemiluminescence was monitored for 2h in the presence of 66 µM of 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol). 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 µM) was used as a positive control. Results are representative of 2 independent experiments.

    Journal: PLoS ONE

    Article Title: Deficient Beta-Mannosylation of Candida albicans Phospholipomannan Affects the Proinflammatory Response in Macrophages

    doi: 10.1371/journal.pone.0084771

    Figure Lengend Snippet: PLMs induce caspase-1 activation independent on ROS production. ( A - C ). J774 macrophages were primed with LPS (100 ng/ml) for 2h. Then, indicated stimuli were added and cells were cultivated for additional 4h. ( A ). Caspase-1 activation was revealed by addition of fluorescent FAM-YVAD-FMK and examined by fluorescent microscopy. Adenosine triphosphate (ATP) and Aluminum hydroxide (Alum) were used as positive control for caspase-1 activation. ( B ). Total pro-caspase-1 and cleaved caspase-1 levels were revealed by western blot analysis. ( C ). Production of IL-1β after stimulation with PLMs of LPS-primed cells was measured after 12- and 24h-incubation by ELISA. ( D ). Macrophages were pretreated or not with Latrunculin-A and then stimulated with indicated PLMs. Total pro-caspase-1 and cleaved caspase-1 levels were revealed by western blot analysis. Data are representative of 4 independent experiments. ( E and F ). Macrophages were pretreated or not with Latrunculin A for 30min. Live yeast cells at 5 yeast: 1 cell ratio ( E ) or PLMs (50µg/ml) ( F ) were then added. Chemiluminescence was monitored for 2h in the presence of 66 µM of 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol). 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 µM) was used as a positive control. Results are representative of 2 independent experiments.

    Article Snippet: PLM-induced secretion of TNF-α is independent of internalization Taking into consideration that the molecular differences between the three different types of PLMs ( ) may affect their intermolecular interactions and lead to a different pattern of internalization and/or interaction with cell surface receptors, we next tested whether Latrunculin A would affect PLM-induced TNF-α.

    Techniques: Activation Assay, Microscopy, Positive Control, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay

    Long chain oligomannosides are required for PLM-induced TNF-α production. ( A ). J774 macrophages were incubated with increasing concentration of PLM-A (●), PLM-B (■) or PLM-BMT6∆ (○) and cultivated for 5h at 37°C. Cell culture supernatants were harvested and TNF-α production was evaluated by ELISA. ( B ). Alternatively, cells were stimulated with PLM-A (50 µg/ml) or with curdlan (100 µg/ml) and Pam3CSK4 (500 ng/ml) as positive controls. Data represents the mean ± standard deviation from 3 independent experiments, each one performed in triplicates. ( C and D ). J774 macrophages were pretreated (■) or not (□) with Latrunculin A for 30 min prior to PLMs addition at the indicated concentrations ( C ) or live C. albicans serotype A and B yeast cells at a 5 yeast : 1 cell ratio ( D ). Curdlan and Pam3CSK4 were used as positive controls. TNF-α concentration in cell-free supernatants was determined by ELISA after 4h incubation. Data are representative of two independent experiments performed in triplicates. *p

    Journal: PLoS ONE

    Article Title: Deficient Beta-Mannosylation of Candida albicans Phospholipomannan Affects the Proinflammatory Response in Macrophages

    doi: 10.1371/journal.pone.0084771

    Figure Lengend Snippet: Long chain oligomannosides are required for PLM-induced TNF-α production. ( A ). J774 macrophages were incubated with increasing concentration of PLM-A (●), PLM-B (■) or PLM-BMT6∆ (○) and cultivated for 5h at 37°C. Cell culture supernatants were harvested and TNF-α production was evaluated by ELISA. ( B ). Alternatively, cells were stimulated with PLM-A (50 µg/ml) or with curdlan (100 µg/ml) and Pam3CSK4 (500 ng/ml) as positive controls. Data represents the mean ± standard deviation from 3 independent experiments, each one performed in triplicates. ( C and D ). J774 macrophages were pretreated (■) or not (□) with Latrunculin A for 30 min prior to PLMs addition at the indicated concentrations ( C ) or live C. albicans serotype A and B yeast cells at a 5 yeast : 1 cell ratio ( D ). Curdlan and Pam3CSK4 were used as positive controls. TNF-α concentration in cell-free supernatants was determined by ELISA after 4h incubation. Data are representative of two independent experiments performed in triplicates. *p

    Article Snippet: PLM-induced secretion of TNF-α is independent of internalization Taking into consideration that the molecular differences between the three different types of PLMs ( ) may affect their intermolecular interactions and lead to a different pattern of internalization and/or interaction with cell surface receptors, we next tested whether Latrunculin A would affect PLM-induced TNF-α.

    Techniques: Incubation, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Actin- and cholesterol-dependent EGFR recycling in  Nf2 −/−  cells. ( A ) Distribution ( left ) and quantitation ( right ) of TR-EGF in  Nf2 −/−  and  Nf2 WT -expressing cells at 2 and 30 min after stimulation. Quantitation at 2 min was limited to a 5-μm 2  box at the cell edge to capture the spatial difference in TR-EGF ( top ), and, at 30 min, internalized TR-EGF across the entire cell area was quantitated ( bottom ). ( B ) Confocal images ( left ) and quantitation ( right ) showing the pattern of internalized TR-EGF and F-actin in  Nf2 −/−  cells treated with 10 μM latrunculin A. ( C ) Distribution ( left ) and quantitation ( right ) of TR-EGF 2 min after stimulation in  Nf2 −/−  or  Nf2 WT -expressing cells treated with vehicle or 10 mM MβCD. ( D ) Quantitation of TR-EGF +  vesicles following 30 min of stimulation in  Nf2 −/−  cells treated with vehicle (DMSO) or 50 μM EIPA. ( E ) Confocal images ( left ) and quantitation ( right ) depicting total EGFR levels in  Nf2 −/−  or  Nf2 WT -expressing cells pretreated with cyclohexamide and stimulated with EGF for 30 min at 4°C ( left ) and then transferred for 30 min or 2 h to 37°C. Error bars represent mean ± SEM. (**)  P

    Journal: Genes & Development

    Article Title: Merlin/ERM proteins regulate growth factor-induced macropinocytosis and receptor recycling by organizing the plasma membrane:cytoskeleton interface

    doi: 10.1101/gad.317354.118

    Figure Lengend Snippet: Actin- and cholesterol-dependent EGFR recycling in Nf2 −/− cells. ( A ) Distribution ( left ) and quantitation ( right ) of TR-EGF in Nf2 −/− and Nf2 WT -expressing cells at 2 and 30 min after stimulation. Quantitation at 2 min was limited to a 5-μm 2 box at the cell edge to capture the spatial difference in TR-EGF ( top ), and, at 30 min, internalized TR-EGF across the entire cell area was quantitated ( bottom ). ( B ) Confocal images ( left ) and quantitation ( right ) showing the pattern of internalized TR-EGF and F-actin in Nf2 −/− cells treated with 10 μM latrunculin A. ( C ) Distribution ( left ) and quantitation ( right ) of TR-EGF 2 min after stimulation in Nf2 −/− or Nf2 WT -expressing cells treated with vehicle or 10 mM MβCD. ( D ) Quantitation of TR-EGF + vesicles following 30 min of stimulation in Nf2 −/− cells treated with vehicle (DMSO) or 50 μM EIPA. ( E ) Confocal images ( left ) and quantitation ( right ) depicting total EGFR levels in Nf2 −/− or Nf2 WT -expressing cells pretreated with cyclohexamide and stimulated with EGF for 30 min at 4°C ( left ) and then transferred for 30 min or 2 h to 37°C. Error bars represent mean ± SEM. (**) P

    Article Snippet: Drug pretreatment was as follows: 50 µM EIPA (Sigma-Aldrich) for 60 min, 25 µM HCQ (Sigma-Aldrich) for 48 h, 2.5 µM FTY720 (Cayman) for 48 h, 1 µM GDC0941 (Selleck) for 1 h, 40 µM ketoconazole (Sigma-Aldrich) for 72 h (sensitivity) or 24 h (recycling), 1 µM BIBW2992 (Selleck) for 1 h, 10 mM MβCD (Sigma-Aldrich) for 30 min, 10 µM latrunculin A (Cayman) for15 min, 5 µM jasplakinolide for 15 min, and 50 µg/mL cyclohexamide (Sigma-Aldrich) for 1 h. These doses were maintained throughout the experiment.

    Techniques: Quantitation Assay, Expressing

    Multiplexed SIFTER detects subpopulations of cells with altered cytoskeletal protein complexes in response to F-actin destabilization. a) Schematic of the cell cytoskeleton composed of F-actin, intermediate filaments (IF) and microtubules (MT), and the unknown effects of Latrunculin A (LatA) on IF and MT. (b) Representative false-color fluorescence micrographs and intensity profiles from SIFTER. Monomeric proteins (e.g., β-Tubulin, β-Tub) are electrophoresed left of the microwell while F-actin, MT and IF are electrophoresed to the right of the microwell. Protein quantification is performed by peak area integration. Scale bar is 100 μm. (c) Heat maps with dendrograms from agglomerative hierarchical clustering with Euclidean distance metric and Ward linkage for U2OS cells incubated in DMSO (n = 92 cells, four SIFTER gels) or 2 μM Latrunculin A (LatA, n = 134 cells, four SIFTER gels). Distinct sub-lineages used as bait groups A-F for CellFishing are shown with colored bars. Heatmap is standardized by row (mean at 0, and color gradations at units of standard deviation). (d) Spectral clustering projections and heatmaps depicting LatA treatment bait group cells (blue), DMSO control cells (grey) and fished out DMSO control cells (yellow).

    Journal: bioRxiv

    Article Title: Quantifying cytoskeletal heterogeneity via single-cell protein-complex fractionation

    doi: 10.1101/2020.09.12.294801

    Figure Lengend Snippet: Multiplexed SIFTER detects subpopulations of cells with altered cytoskeletal protein complexes in response to F-actin destabilization. a) Schematic of the cell cytoskeleton composed of F-actin, intermediate filaments (IF) and microtubules (MT), and the unknown effects of Latrunculin A (LatA) on IF and MT. (b) Representative false-color fluorescence micrographs and intensity profiles from SIFTER. Monomeric proteins (e.g., β-Tubulin, β-Tub) are electrophoresed left of the microwell while F-actin, MT and IF are electrophoresed to the right of the microwell. Protein quantification is performed by peak area integration. Scale bar is 100 μm. (c) Heat maps with dendrograms from agglomerative hierarchical clustering with Euclidean distance metric and Ward linkage for U2OS cells incubated in DMSO (n = 92 cells, four SIFTER gels) or 2 μM Latrunculin A (LatA, n = 134 cells, four SIFTER gels). Distinct sub-lineages used as bait groups A-F for CellFishing are shown with colored bars. Heatmap is standardized by row (mean at 0, and color gradations at units of standard deviation). (d) Spectral clustering projections and heatmaps depicting LatA treatment bait group cells (blue), DMSO control cells (grey) and fished out DMSO control cells (yellow).

    Article Snippet: F-actin cell staining with phalloidin and Latrunculin A and Jasplakinolide drug treatmentLatrunculin A (Cayman Chemicals 10010630) was dissolved in DMSO as a 2 mM stock solution and stored at −20 °C until use.

    Techniques: Fluorescence, Incubation, Standard Deviation

    Exocytosis of Cryptococcus neoformans from Dictyostelium is dependent on WASH and the actin cytoskeleton. (A) Heat killed S. cerevisiae are not exocytosed from WASH null D. discoideum . Example from 12 h time lapse imaging of heat killed S. cerevisiae in WASH null D. discoideum representative of 60 phagosomes containing heat killed S. cerevisiae from three independent experiments (images start at 500 min after phagocytosis). (B) C. neoformans are exocytosed from WASH null D. discoideum . Example from 12 h time lapse imaging representative of 62 phagosomes containing C. neoformans from three independent experiments (images start at 200 min after phagocytosis). Scale bars 5 μm. (C) Quantification of the percentage of exocytosis of C. neoformans and heat killed S. cerevisiae from WASH null D. discoideum from three independent 12 h time lapses. (D) Frequency histogram of combined 35 transit times measured from three independent 12 h time lapses. (E) Exocytosis of heat killed S. cerevisiae from wild type Ax2 but not WASH null or latrunculin A treated Ax2 D. discoideum . Quantification of the percentage of exocytosis from three independent 12 h time lapses. Total of 60 phagosomes were analyzed from each condition. P -values are Fishers test. (F) Exocytosis of C. neoformans from wild type Ax2, WASH null, and latrunculin A treated D. discoideum . Quantification of the percentage of exocytosis from three independent 12 h time lapses. Total of 60 phagosomes were analyzed from each condition. P -values are Fishers test. (G) Transit times of C. neoformans through WASH null and latrunculin A treated Ax2 D. discoideum are not significantly different. P -values are Mann-Whitney test.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Cryptococcus neoformans Escape From Dictyostelium Amoeba by Both WASH-Mediated Constitutive Exocytosis and Vomocytosis

    doi: 10.3389/fcimb.2018.00108

    Figure Lengend Snippet: Exocytosis of Cryptococcus neoformans from Dictyostelium is dependent on WASH and the actin cytoskeleton. (A) Heat killed S. cerevisiae are not exocytosed from WASH null D. discoideum . Example from 12 h time lapse imaging of heat killed S. cerevisiae in WASH null D. discoideum representative of 60 phagosomes containing heat killed S. cerevisiae from three independent experiments (images start at 500 min after phagocytosis). (B) C. neoformans are exocytosed from WASH null D. discoideum . Example from 12 h time lapse imaging representative of 62 phagosomes containing C. neoformans from three independent experiments (images start at 200 min after phagocytosis). Scale bars 5 μm. (C) Quantification of the percentage of exocytosis of C. neoformans and heat killed S. cerevisiae from WASH null D. discoideum from three independent 12 h time lapses. (D) Frequency histogram of combined 35 transit times measured from three independent 12 h time lapses. (E) Exocytosis of heat killed S. cerevisiae from wild type Ax2 but not WASH null or latrunculin A treated Ax2 D. discoideum . Quantification of the percentage of exocytosis from three independent 12 h time lapses. Total of 60 phagosomes were analyzed from each condition. P -values are Fishers test. (F) Exocytosis of C. neoformans from wild type Ax2, WASH null, and latrunculin A treated D. discoideum . Quantification of the percentage of exocytosis from three independent 12 h time lapses. Total of 60 phagosomes were analyzed from each condition. P -values are Fishers test. (G) Transit times of C. neoformans through WASH null and latrunculin A treated Ax2 D. discoideum are not significantly different. P -values are Mann-Whitney test.

    Article Snippet: This differed for latrunculin treatment experiments: amoebae were mixed with yeast at a ratio of 1:1 and left for 1 h for phagocytosis to occur before addition of 5 μm Latrunculin A (Cayman chemical Co.).

    Techniques: Imaging, MANN-WHITNEY

    Exocytosis and vomocytosis of C. neoformans cap59 and plb1 mutants are indistinguishable from wild type H99. (A) Quantification of the percentage of non-constitutive exocytosis of C. neoformans mutants from wild-type Ax2 D. discoideum treated with latrunculin A from three independent 12 h time lapses. Total of 45 phagosomes were analyzed for wild type H99, 90 for cap59 and 90 for plb1 . P -values are Fisher's exact test. (B) Transit times of C. neoformans H99 wild type, mutant cap59 and plb1 . Phagosomes analyzed the same as (A) . P -values for between cryptococcal strain significance tests are non-significant in all cases. P -values are Mann-Whitney test.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Cryptococcus neoformans Escape From Dictyostelium Amoeba by Both WASH-Mediated Constitutive Exocytosis and Vomocytosis

    doi: 10.3389/fcimb.2018.00108

    Figure Lengend Snippet: Exocytosis and vomocytosis of C. neoformans cap59 and plb1 mutants are indistinguishable from wild type H99. (A) Quantification of the percentage of non-constitutive exocytosis of C. neoformans mutants from wild-type Ax2 D. discoideum treated with latrunculin A from three independent 12 h time lapses. Total of 45 phagosomes were analyzed for wild type H99, 90 for cap59 and 90 for plb1 . P -values are Fisher's exact test. (B) Transit times of C. neoformans H99 wild type, mutant cap59 and plb1 . Phagosomes analyzed the same as (A) . P -values for between cryptococcal strain significance tests are non-significant in all cases. P -values are Mann-Whitney test.

    Article Snippet: This differed for latrunculin treatment experiments: amoebae were mixed with yeast at a ratio of 1:1 and left for 1 h for phagocytosis to occur before addition of 5 μm Latrunculin A (Cayman chemical Co.).

    Techniques: Mutagenesis, MANN-WHITNEY