Journal: PLoS ONE

Article Title: The Rho-Family GTPase Rac1 Regulates Integrin Localization in Drosophila Immunosurveillance Cells

doi: 10.1371/journal.pone.0019504

Figure Lengend Snippet: (A) Lamellocytes were bled from at least 24 control ( w 1118 ), homozygous Rac1 J11 , and homozygous Rac2 Δ larvae approximately 40 hours post-parasitization and stained for Myospheroid protein expression (red), as well as actin (green). (B) Anti-Mys antibody was bound to the surface of lamellocytes bled from control ( w 1118 ) larvae, followed by internalization for various times as indicated (n = 12 larvae/time-point). The cells were not permeabilized (C) Graph indicating percentage of surface Myospheroid expressed on control ( w 1118 ) haemocytes. Fluorescent intensity was measured using ImageJ to calculate the amount of cell surface Myospheroid on lamellocytes. Asterisk indicates significant difference (One-way Anova, P <0.01), errors bars indicate ± SE; n = 12 larvae/time-point, 0 minutes n = 1256 haemocytes, 30 minutes n = 1294, 60 minutes n = 1175). (D) Anti-Mys antibody was bound to the surface of lamellocytes bled from control ( w 1118 ) larvae, followed by internalization for 120 minutes as indicated (n = 12 larvae/time-point). Cells were permeabilized to allow for visualization of internalized Myospheroid. Size bars always indicate 20 µm.

Article Snippet: Mouse monoclonal anti-Myospheroid (Developmental Studies Hybridoma Bank, Iowa, USA), lamellocyte-specific mouse monoclonal antibody (L1a) and plasmatocyte-specific monoclonal mouse anti-Nimrod , were all used undiluted, mouse monoclonal anti-Rac1 (BD Biosciences) diluted 1∶250, mouse monoclonal antibody anti-α-Tubulin (Sigma) diluted 1∶1,000, rabbit polyclonal anti-α-Tubulin (Abcam) diluted 1∶500, rat monoclonal anti-Hsp90 (Abcam) diluted 1∶300.

Techniques: Staining, Expressing

Journal: PLoS ONE

Article Title: The Rho-Family GTPase Rac1 Regulates Integrin Localization in Drosophila Immunosurveillance Cells

doi: 10.1371/journal.pone.0019504

Figure Lengend Snippet: (A) Control ( w 1118 ) and homozygous Rac1 J11 larvae were raised at 22°C or 29°C, lamellocytes were recovered from at least 12 larvae during three experiments approximately 40 hours post-parasitization and stained for Myospheroid protein expression (green), as well as α-Tubulin (red). Arrowheads indicate Myospheroid protein localization. Size bar indicates 20 µm. (B) Lamellocytes were recovered from at least 24 non-parasitized late third instar larvae and stained for Myospheroid protein expression (green), as well as α-Tubulin (red). Size bar indicates 20 µm (C) Anti-Mys antibody was bound to the surface of lamellocytes bled from either non-parasitized or parasitized control ( w 1118 ), homozygous Rac1 J11 , or homozygous UAS-Rac1;He-GAL4,Rac1 j11 larvae raised at the indicated temperature, followed by internalization for 30 minutes (n = 12 larvae/time-point).

Article Snippet: Mouse monoclonal anti-Myospheroid (Developmental Studies Hybridoma Bank, Iowa, USA), lamellocyte-specific mouse monoclonal antibody (L1a) and plasmatocyte-specific monoclonal mouse anti-Nimrod , were all used undiluted, mouse monoclonal anti-Rac1 (BD Biosciences) diluted 1∶250, mouse monoclonal antibody anti-α-Tubulin (Sigma) diluted 1∶1,000, rabbit polyclonal anti-α-Tubulin (Abcam) diluted 1∶500, rat monoclonal anti-Hsp90 (Abcam) diluted 1∶300.

Techniques: Staining, Expressing

Journal: PLoS ONE

Article Title: The Rho-Family GTPase Rac1 Regulates Integrin Localization in Drosophila Immunosurveillance Cells

doi: 10.1371/journal.pone.0019504

Figure Lengend Snippet: (A) Control ( w 1118 ), homozygous Rac1 J11 , homozygous Rac1 J11 ,P(Hsp83+) larvae were raised at 22°C, lamellocytes were recovered from at least 24 larvae approximately 40 hours post-parasitization and stained for Myospheroid protein expression (green), as well as α-Tubulin (red). Size bar indicates 20 µm. (B) Control ( w 1118 ), homozygous Rac1 J11 , homozygous Rac1 J11 ,P(Hsp83+) , and mys nj42 ;Rac1 j11 ,P(Hsp83) larvae were raised at 22°C and an encapsulation assay in response to parasitization by L. boulardi G486 was performed. Values for proper encapsulation were calculated by the following equation [(Number of properly encapsulated wasp eggs/number of parasitized larvae) x 100]. Numbers above the bars indicate the number of wasp-parasitized larvae examined.

Article Snippet: Mouse monoclonal anti-Myospheroid (Developmental Studies Hybridoma Bank, Iowa, USA), lamellocyte-specific mouse monoclonal antibody (L1a) and plasmatocyte-specific monoclonal mouse anti-Nimrod , were all used undiluted, mouse monoclonal anti-Rac1 (BD Biosciences) diluted 1∶250, mouse monoclonal antibody anti-α-Tubulin (Sigma) diluted 1∶1,000, rabbit polyclonal anti-α-Tubulin (Abcam) diluted 1∶500, rat monoclonal anti-Hsp90 (Abcam) diluted 1∶300.

Techniques: Staining, Expressing

Journal: Frontiers in Physiology

Article Title: Specialized Positioning of Myonuclei Near Cell-Cell Junctions

doi: 10.3389/fphys.2018.01531

Figure Lengend Snippet: Effects of larval stretching on MJM positioning. (A) Max intensity projections of confocal images of anterior and posterior MTJs for the indicated genotypes stained for β-integrin (gray) and nuclei (green). White asterisks mark trachea branches, which are not part of the muscle. Scale bar is 10 μm (B-F) Tukey boxplots showing the distance between MJMs and the MTJ in larval that have not been stretched (not stretched), larvae dissected immediately after stretching (stretched), larva that have been stretched and dissected after a 2 h recovery (stretched +2 h). (G) Cartoon of MJM position in stretching experiments. Summary of the stretching experiments from (A-F) . For sake of simplicity, only one MJM is represented for each MTJ. Black arrows indicate the direction of the change in nuclear positioning. * p < 0.05, *** p < 0.0005, **** p < 0.0001.

Article Snippet: Antibodies for larva filet staining were used at the following final dilutions: rat anti- tyrosinated tubulin, 1:400 (MAB1864; Millipore), mouse anti-myospheroid (β-integrin), 1:100 (CF.6G11, Developmental Studies Hybridoma Bank).

Techniques: Staining

Journal: Journal of Biology

Article Title: A functional genomic analysis of cell morphology using RNA interference

doi: 10.1186/1475-4924-2-27

Figure Lengend Snippet: High-throughput RNAi screens by cell imaging. (a) Cellular phenotypes were visualized 3 days after the addition of dsRNA. In the example shown Kc 167 cells changed shape from round to polarized, with F-actin puncta (arrowhead) and extended microtubules (arrow), in response to Cdc42 dsRNA. (b) Kc 167 and (c) S2R + cells at low (left) and high (far right) magnifications, fluorescently labeled for F-actin (red), α-tubulin (green) and DNA (blue). Cell-shape changes could be induced using drugs that affect the cytoskeleton or using extracellular signals, as seen upon treatment of Kc 167 cells with (d) latrunculin A or (e) 20-hydroxyecdysone (20-H-ecdysone). Scale bar, 30 μm.

Article Snippet: Semi-dry transfer to nitrocellulose membrane was probed with rabbit anti-Myospheroid (185E; gift of R. Hynes), mouse anti-Enabled (gift of D. Van Vactor) and mouse anti-α-tubulin (DM1A; Sigma) and detected with HRP anti-rabbit or anti-mouse (Jackson Labs, Bar Harbor, USA) with ECL Western Blotting Analysis System (Amersham Bioscience Corp., Piscataway, USA).

Techniques: High Throughput Screening Assay, Imaging, Labeling

Journal: Journal of Biology

Article Title: A functional genomic analysis of cell morphology using RNA interference

doi: 10.1186/1475-4924-2-27

Figure Lengend Snippet: A test of RNAi screen efficacy: identifying genes involved in cell-cycle progression. (a) Gene identity and phenotypic annotation for RNAi phenotypes identifying predicted cell-cycle regulators. The 'Profile' column provides a summary of the phenotypic profiles distinguishing sets of genes involved in specific stages of the cell cycle. The 'Classification' column gives a single predicted functional category assigned to each targeted gene on the basis of primary sequence and/or known functional data. The 'FlyBase ID' and 'Gene name' columns are information as annotated at FlyBase . The 'Predicted function' column provides detail on the putative molecular function of each specific gene. 'Cell type' refers to whether the phenotype was observed in Kc 167 (Kc) and/or S2R + (S2R) cells. Profile I: RNAi phenotypes resulting in an increase in cell size, uniform or disorganized microtubules, irregular cell shapes and decreased cell numbers identified genes involved in cell-cycle progression through G1 to S and G2 to M stages. Phenotypes were further distinguished on the basis of levels of F-actin accumulation and DNA morphology. Profile II: RNAi phenotypes resulting in aberrant morphology or increased frequency of microtubule-based mitotic spindles identified genes involved in mitosis. Profile III: RNAi phenotypes observed in S2R + cells identified additional genes with putative roles in cell cycle/mitosis progression. (b-g) Kc 167 cells stained for F-actin (red), α-tubulin (green), DNA (blue), imaged using automated microscopy and scored visually. (b) Control. (c, d) Profile I: Dp and string RNAi resulting in big cells. (e, f) Profile II: fizzy and polo RNAi resulting in increased frequency of cells with mitotic spindles. (g) Cdk5 RNAi resulting in smaller cells and disorganized microtubules (and increased spindles in S2R + cells; not shown). Scale bar, 30 μm.

Article Snippet: Semi-dry transfer to nitrocellulose membrane was probed with rabbit anti-Myospheroid (185E; gift of R. Hynes), mouse anti-Enabled (gift of D. Van Vactor) and mouse anti-α-tubulin (DM1A; Sigma) and detected with HRP anti-rabbit or anti-mouse (Jackson Labs, Bar Harbor, USA) with ECL Western Blotting Analysis System (Amersham Bioscience Corp., Piscataway, USA).

Techniques: Functional Assay, Sequencing, Staining, Microscopy

Journal: Journal of Biology

Article Title: A functional genomic analysis of cell morphology using RNA interference

doi: 10.1186/1475-4924-2-27

Figure Lengend Snippet: RNAi screens identified a wide range of gene functions based on diverse morphological phenotypes. Cells were stained for F-actin (red), α-tubulin (green) and DNA (blue), imaged using automated microscopy and scored visually. (a) Control Kc 167 cells. (b-e) Kc 167 cells with RNAi phenotypes. (f) Control S2R + cells. (g-j) S2R + cells with RNAi phenotypes. (b) F-actin accumulation; CG13503 RNAi (encoding a predicted WH2-containing actin-binding protein). (c, h) Flatter, polarized cells with actin-rich lamellipodia (arrows); CG5169 RNAi (a predicted kinase). (d) Opposing protrusions rich in F-actin (arrow) or microtubules (arrowhead), Hsp83 RNAi (chaperone). (e) Flat cells; puckered RNAi (JNK phosphatase). (g) Widely-distributed F-actin puncta; capping protein beta RNAi (component of CapZ). (i) Radial protrusions (arrows) and reduced cortical actin (asterisk); CG31536 RNAi (predicted Rho-GEF with FERM domain). (j) Rounder cells, decreased in size; CG4629 RNAi (predicted kinase). Scale bar, 30 μm.

Article Snippet: Semi-dry transfer to nitrocellulose membrane was probed with rabbit anti-Myospheroid (185E; gift of R. Hynes), mouse anti-Enabled (gift of D. Van Vactor) and mouse anti-α-tubulin (DM1A; Sigma) and detected with HRP anti-rabbit or anti-mouse (Jackson Labs, Bar Harbor, USA) with ECL Western Blotting Analysis System (Amersham Bioscience Corp., Piscataway, USA).

Techniques: Staining, Microscopy, Binding Assay

Journal: Journal of Biology

Article Title: A functional genomic analysis of cell morphology using RNA interference

doi: 10.1186/1475-4924-2-27

Figure Lengend Snippet: Similar phenotypic profiles identified genes in pathways and protein complexes. Cells were stained for F-actin (red), α-tubulin (green) and DNA (blue). Distinct phenotypes were observed with dsRNAs targeting different members of the same functional family (for example, GTPases, in the left panels). (a, b) Phenotypes observed in both cell types. (a) RNAi-induced binucleate cell phenotypes identified genes required for cytokinesis, including Rho1 (encoding a GTPase), pebble (a Rho-GEF) and CG10522 (a predicted citron kinase). Kc 167 cells are shown. (b) RNAi resulting in loss of actin filaments from the cell cortex identified regulators of actin-filament formation, including Cdc42 (GTPase), enabled (actin-binding protein) and SCAR (actin-binding, Arp2/3 regulator). Kc 167 cells (shown) also formed microtubule extensions and a polarized cell shape. (c, d) Some phenotypes were unique to one cell type. (c) RNAi resulting in round, non-adherent S2R + cells identified genes required for cell-matrix adhesion, including Roughened (a Rap1 GTPase), Tenascin-major (an adhesion protein with a laminin domain) and myospheroid (β integrin). (d) An RNAi-induced amorphous S2R + cell phenotype identified genes in the mitogen-activated protein (MAP) kinase pathway, including Ras85D (a GTPase), Downstream of raf1 (a MAP kinase kinase, or MEK) and kinase suppressor of Ras (a MAP kinase scaffold protein).

Article Snippet: Semi-dry transfer to nitrocellulose membrane was probed with rabbit anti-Myospheroid (185E; gift of R. Hynes), mouse anti-Enabled (gift of D. Van Vactor) and mouse anti-α-tubulin (DM1A; Sigma) and detected with HRP anti-rabbit or anti-mouse (Jackson Labs, Bar Harbor, USA) with ECL Western Blotting Analysis System (Amersham Bioscience Corp., Piscataway, USA).

Techniques: Staining, Functional Assay, Binding Assay

Journal: Journal of Biology

Article Title: A functional genomic analysis of cell morphology using RNA interference

doi: 10.1186/1475-4924-2-27

Figure Lengend Snippet: Levels of gene expression do not necessarily correlate with gene function. Immunoblot detection of anti-βPS-integrin (Mys, top panels) and anti-Enabled (Ena, middle panels) after 3 days RNAi. Columns represent Kc 167 cells (left) and S2R + cells (right) treated with different dsRNAs ( gfp , ena , mys , R , talin ). Both cell types expressed Mys and Ena in cells treated with a nonspecific dsRNA. The respective proteins were completely and specifically depleted by treatment with mys or ena dsRNAs. Anti-α-tubulin (bottom panels; Tub) shows a loading comparison.

Article Snippet: Semi-dry transfer to nitrocellulose membrane was probed with rabbit anti-Myospheroid (185E; gift of R. Hynes), mouse anti-Enabled (gift of D. Van Vactor) and mouse anti-α-tubulin (DM1A; Sigma) and detected with HRP anti-rabbit or anti-mouse (Jackson Labs, Bar Harbor, USA) with ECL Western Blotting Analysis System (Amersham Bioscience Corp., Piscataway, USA).

Techniques: Expressing, Western Blot

Journal: Journal of Biology

Article Title: A functional genomic analysis of cell morphology using RNA interference

doi: 10.1186/1475-4924-2-27

Figure Lengend Snippet: A co-RNAi screen for modifiers of Pten -dsRNA phenotype. Microtubules are visualized by α-tubulin immunostaining. (a) Control Kc 167 cells exhibited normal, round morphology. (b) In response to Pten dsRNA at the same concentration as the original screening conditions, Kc 167 cells were bipolar and spindle-shaped with microtubule extensions (arrows). (c) In response to a relatively low concentration of Pten dsRNA, the conditions used for the modifier screen, Kc 167 cells exhibited a less pronounced phenotype with asymmetric microtubule accumulation (arrowheads). Specific dsRNA suppressors of the Pten -RNAi-induced cell shape restored the normal, round cell morphology and microtubule organization, identifying (d) Pi3K92E , (e) Akt1 and (f) LIMK1 .

Article Snippet: Semi-dry transfer to nitrocellulose membrane was probed with rabbit anti-Myospheroid (185E; gift of R. Hynes), mouse anti-Enabled (gift of D. Van Vactor) and mouse anti-α-tubulin (DM1A; Sigma) and detected with HRP anti-rabbit or anti-mouse (Jackson Labs, Bar Harbor, USA) with ECL Western Blotting Analysis System (Amersham Bioscience Corp., Piscataway, USA).

Techniques: Immunostaining, Concentration Assay