Journal: Biology Open

Article Title: Depletion or over-expression of Sh3px1 results in dramatic changes in cell morphology

doi: 10.1242/bio.013755

Figure Lengend Snippet: Localization of endogenous Sh3px1. (A,B) Drosophila S2 cells were treated with dsRNAs against gfp (A) or sh3px1 (B). Four days after dsRNA treatment, the cells were spotted onto concanavalin A (con A) coated coverslips and allowed to adhere for 2 h. The cells were then fixed and analyzed using an antibody against Sh3px1 (green). The cells were also counterstained with DAPI to reveal nuclei (red). (C) S2 cells were treated with dsRNAs against gfp (lane 1) or sh3px1 (lane 2). Lysates were prepared from these cells and run on an SDS-PAGE gel. The proteins were transferred to nitrocellulose and processed for western blot analysis using the indicated antibodies. (D) S2 cells were spotted onto con A coverslips and allowed to adhere for 2 h. The cells were then fixed and processed for immunofluorescence using an antibody against Sh3px1 (green). The cells were also counterstained with TRITC conjugated Phalloidin to reveal F-actin (red). Sh3px1 localized adjacent to the cortical actin network (arrow) as well as to internal foci. (E) S2 cells were transfected with a plasmid expressing RFP-tagged Wasp. Four days after transfection, the cells were spotted onto Con A coverslips and allowed to adhere for 2 h. The cells were then fixed and processed for immunofluorescence using an antibody against Sh3px1 (green). Sh3px1 co-localized with RFP-Wasp at the cell cortex (arrow). (F) S2 cells were spotted onto con A coverslips and allowed to adhere for 2 h. The cells were then fixed and processed for immunofluorescence using an antibody against Sh3px1 (green) and Scar (red). Sh3px1 co-localized with endogenous Scar at the cell cortex (arrow). In addition, a perinuclear enrichment of Sh3px1 could be detected in approximately 45% of cells (arrowhead). Scale bars: 15 μm.

Article Snippet: Soluble lysates were prepared by resuspending S2 cell pellets in RIPA buffer (50 mM Tris.Cl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA) containing protease inhibitors (Halt protease inhibitor cocktail kit, Pierce).

Techniques: SDS Page, Western Blot, Immunofluorescence, Transfection, Plasmid Preparation, Expressing

Journal: Biology Open

Article Title: Depletion or over-expression of Sh3px1 results in dramatic changes in cell morphology

doi: 10.1242/bio.013755

Figure Lengend Snippet: Depletion of Sh3px1 results in defective lamellipodia formation . (A,B) S2 cells were transfected with a plasmid expressing a control shRNA targeting the yeast gal80 gene (A) or with a plasmid expressing shRNA targeting sh3px1 (B). The cells were also transfected with the Act5c-Gal4 plasmid in order to induce expression of the shRNA. Three days after transfection, the cells were spotted onto con A coverslips and allowed to adhere for 2 h. The cells were then fixed and processed for immunofluorescence using an antibody against Sh3px1 (green). The cells were also counterstained to reveal F-actin (red). The percentage of Sh3px1 depleted cells displaying the lamellipodia defect is indicated in panel B. (C,D) S2 cells were transfected with a plasmid expressing the gal80 control shRNA (C) or with a plasmid expressing an shRNA targeting scar (D). Three days after transfection, the cells were fixed, processed using an antibody against Scar (green), and were counterstained to reveal F-actin (red). (E,F) S2 cells were transfected with a plasmid expressing the gal80 control shRNA (E) or with a plasmid expressing an shRNA targeting sh3px1 (F). Three days post transfection, the cells were fixed and processed using antibodies against Scar (red) and Sh3px1 (green). (G,H) S2 cells were transfected with a plasmid expressing the gal80 control shRNA (G) or with a plasmid expressing an shRNA targeting scar (H). Three days later, the cells were fixed and processed using an antibody against Sh3px1 (green). The cells were also counterstained to reveal F-actin (red). Scale bars: 15 μm.

Article Snippet: Soluble lysates were prepared by resuspending S2 cell pellets in RIPA buffer (50 mM Tris.Cl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA) containing protease inhibitors (Halt protease inhibitor cocktail kit, Pierce).

Techniques: Transfection, Plasmid Preparation, Expressing, Control, shRNA, Immunofluorescence

Journal: Biology Open

Article Title: Depletion or over-expression of Sh3px1 results in dramatic changes in cell morphology

doi: 10.1242/bio.013755

Figure Lengend Snippet: The PX-BAR domain is required for tubule and protrusion formation. (A) Schematic of the domain organization of Sh3px1. The amino acid residues that comprise the domains are listed. Also shown is a comparison of the percent identity and similarity in the BAR domains between Sh3px1 and Snx9, Snx18 and Snx33. (B) S2 cells were transfected with a GFP-BAR construct. Two days after transfection, the cells were spotted onto con A coverslips, allowed to adhere, fixed and counterstained to reveal F-actin (red). (C,D) S2 cells were co-transfected with a GFP-PX-BAR construct and a control shRNA (C) or an shRNA targeting endogenous sh3px1 (D). Three days after transfection, the cells were treated as in panel B. 64.7% (±8) cells expressing the control shRNA formed protrusions in comparison to 63% (±3) for cells expressing shRNA targeting sh3px1 . This difference is not statistically significant ( P =0.7477). (E,F) S2 cells were transfected with GFP-PX-BAR mutant Y256A (E) or with GFP-PX-BAR mutant K496E, R500E (F). Two days after transfection, the cells were treated as in panel B. Scale bars: 15 μm. (G) Cells from the experiments represented in panels B-F were quantified. As before, cells in which GFP-Sh3px1 displayed no particular localization pattern were counted as ‘Diffuse’. Cells in which membrane protrusions were observed were scored as ‘Protrusions’. Cells containing tubular structures were scored as ‘Tubules’. Cells containing a strong enrichment of GFP signal close to the cytoplasmic face of the nuclear envelope were scored as ‘Perinuclear’. For each experiment, 100 cells were counted for each construct. The experiment was done three times.

Article Snippet: Soluble lysates were prepared by resuspending S2 cell pellets in RIPA buffer (50 mM Tris.Cl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA) containing protease inhibitors (Halt protease inhibitor cocktail kit, Pierce).

Techniques: Comparison, Transfection, Construct, Control, shRNA, Expressing, Mutagenesis, Membrane

Journal: Biology Open

Article Title: Depletion or over-expression of Sh3px1 results in dramatic changes in cell morphology

doi: 10.1242/bio.013755

Figure Lengend Snippet: Formation of tubules and protrusions by GFP-Sh3px1. (A-E) S2 cells were transfected with a plasmid encoding GFP (A) or GFP-Sh3px1 (B-E) and the Act5c-Gal4 plasmid that was required for driving expression of the fusion proteins. Two days after transfection, the cells were spotted onto con A coverslips and allowed to adhere for 2 h. The cells were then fixed, counterstained with Phalloidin-TRITC, and imaged. (F) S2 cells were transfected with a plasmid encoding GFP-Sh3px1 and spotted onto con A coverslips as noted above. The cells were then fixed in methanol at −20°C and stained with an antibody against Alpha-tubulin to reveal the microtubule network (red). Panel F′ represents a magnified view of the region outlined by the rectangle in F. (G) S2 cells were transfected with a plasmid encoding GFP-Sh3px1. Two days after transfection, the cells were spotted onto con A coated glass bottom dishes and allowed to adhere. The cells were then imaged live. Select time points (0 s, 168 s, and 300 s) from one such imaging experiment are shown. The panel on the far right represents a temporal color coded image. Images at the start of the experiment are depicted in purple and those at the end of the experiment are shown in white. The arrows indicate accumulation of GFP-Sh3px1 in small membrane protrusions that continue to elongate during the course of the experiment. (H) Similar treatment as panel G. Select time points (0 s, 72 s, and 150 s) from a live imaging experiment are shown here. The panel on the far right represents a temporal color-coded image. Arrows indicate membrane protrusions that continue to elongate during the time course of the experiment. Scale bars: 15 μm. (I) Quantification of the phenotypes observed in panels A-E. Cells in which no particular localization pattern was detected were counted as ‘Diffuse’. Cells in which membrane protrusions were observed were scored as ‘Protrusions’. Cells containing tubular structures were scored as ‘Tubules’. The graph represents data from three independent experiments. For each experiment, 100 cells were counted for each construct. The error bars represent standard deviation. ** P =0.0007, *** P ≤0.0001, Unpaired t -test. (J-M) S2 cells were transfected with the following expression constructs, Cip4-GFP (J), Syndapin-mCherry (K), Mim-GFP (L), and Nwk-GFP (M). Two days after transfection, the cells were spotted onto con A coated coverslips and allowed to adhere. The cells were then fixed, counterstained to reveal F-actin, and imaged.

Article Snippet: Soluble lysates were prepared by resuspending S2 cell pellets in RIPA buffer (50 mM Tris.Cl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA) containing protease inhibitors (Halt protease inhibitor cocktail kit, Pierce).

Techniques: Transfection, Plasmid Preparation, Expressing, Staining, Imaging, Membrane, Construct, Standard Deviation

Journal: Biology Open

Article Title: Depletion or over-expression of Sh3px1 results in dramatic changes in cell morphology

doi: 10.1242/bio.013755

Figure Lengend Snippet: Localization of Snx9, Snx18 and Snx33 in S2 cells. (A,B) S2 cells were transfected with a plasmid encoding GFP-Snx9. Two days after transfection, the cells were spotted onto con A coverslips and allowed to adhere. The cells were then fixed and counterstained with Phalloidin to visualize F-actin (red). Panel A represents an example of a tubule containing cell and panel B represent a cell that has formed protrusions. (C,D) S2 cells were transfected with a plasmid encoding GFP-Snx18. The cells were treated as in the above panels. C is an example of a GFP-Snx18 cell that has formed tubules and panel D is an example of a cell with long protrusions. (E,F) S2 cells were transfected with a plasmid encoding GFP-Snx33 and treated as above. E is an example of a tubule containing cell and panel F is an example of a cell that has formed protrusions. Scale bars: 15 μm. (G) Quantification of phenotypes observed upon over-expressing GFP-Snx9, Sxn18 and Snx33. The quantification criteria for determining Diffuse, Tubule or Protrusion was the same as previously described. The graph represents data from three independent experiments. For each experiment, 100 cells were counted per construct. The error bars represent standard deviation.

Article Snippet: Soluble lysates were prepared by resuspending S2 cell pellets in RIPA buffer (50 mM Tris.Cl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA) containing protease inhibitors (Halt protease inhibitor cocktail kit, Pierce).

Techniques: Transfection, Plasmid Preparation, Expressing, Construct, Standard Deviation

Journal: Biology Open

Article Title: Depletion or over-expression of Sh3px1 results in dramatic changes in cell morphology

doi: 10.1242/bio.013755

Figure Lengend Snippet: Co-localization between endogenous Sh3px1 and BAR domain constructs. (A-G) S2 cells were transfected with GFP-Snx9 (A,B), GFP-Snx18 (C,D), GFP-Snx33 (E), GFP-Mim (F) or GFP-Nwk (G). Two days after the transfection, the cells were fixed and processed for immunofluorescence using an antibody against endogenous Sh3px1 (red). Scale bars: 15 μm. (H) The Pearson's co-localization coefficient was quantified for endogenous Sh3px1 and the indicated GFP-tagged constructs. For each construct, 25 cells were imaged and quantified. The co-localization was quantified for the entire cell rather than using a specific region within the cell. The error bars represent standard deviation. The P values for comparing the level of co-localization observed between the indicated GFP-tagged constructs and endogenous Sh3px1 are shown in Fig. S4 .

Article Snippet: Soluble lysates were prepared by resuspending S2 cell pellets in RIPA buffer (50 mM Tris.Cl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA) containing protease inhibitors (Halt protease inhibitor cocktail kit, Pierce).

Techniques: Construct, Transfection, Immunofluorescence, Standard Deviation

Journal: Biology Open

Article Title: Depletion or over-expression of Sh3px1 results in dramatic changes in cell morphology

doi: 10.1242/bio.013755

Figure Lengend Snippet: The potential involvement of F-actin in protrusion formation. (A,B) S2 cells were transfected with a plasmid encoding GFP-Sh3px1. Two days after transfection, the cells were spotted onto con A coverslips, allowed to adhere, and then fixed. Next, the cells were counterstained with Phalloidin-TRITC (red) to reveal F-actin. Panel A depicts a cell with long protrusions and panel B depicts a cell that is just starting to form protrusions. A′ and B′ represent magnified views of the regions outlined by rectangles in A and B, respectively. The arrows in A′ indicate that signal for Sh3px1 could be detected on the membrane of the long protrusions, whereas F-actin was observed more internally. The arrow in B′ indicates a short protrusion that contains more signal for Sh3px1 than F-actin. (C,D) Panels C and D represent select time frames from two independent live imaging experiments. S2 cells were co-transfected with plasmids encoding GFP-Sh3px1 (green) and mRuby2-Lifeact (red). Individual and merged images are shown. The arrows indicate protrusions that form and elongate during the course of the experiment. (E,F) S2 cells were co-transfected with the following combination of plasmids; GFP-Sh3px1 and the gal80 control shRNA (E) and GFP-Sh3px1 and wasp shRNA-2 (F). Three days after transfection, the cells were spotted onto con A coverslips, allowed to adhere, and then fixed. Scale bars: 15 μm. (G) Quantification of phenotypes from the experiment depicted in panels E and F. The quantification criteria for determining Diffuse, Tubule, and Protrusion were as described previously. The graph represents data from three independent experiments. For each experiment, 100 cells were counted per construct. The error bars represent standard deviation. *** P =0.0018, unpaired t -test.

Article Snippet: Soluble lysates were prepared by resuspending S2 cell pellets in RIPA buffer (50 mM Tris.Cl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA) containing protease inhibitors (Halt protease inhibitor cocktail kit, Pierce).

Techniques: Transfection, Plasmid Preparation, Membrane, Imaging, Control, shRNA, Construct, Standard Deviation