Journal: The Journal of Cell Biology

Article Title: WISp39 binds phosphorylated Coronin 1B to regulate Arp2/3 localization and Cofilin-dependent motility

doi: 10.1083/jcb.201410095

Figure Lengend Snippet: Loss of WISp39 results in elongated cell morphology. (A) Subconfluent U2OS cells were transfected with either control (left) or human WISp39 siRNA (WISp39 KD; right) for 48 h and representative 20× phase images were taken. Western blot shows KD of WISp39 at 48 h. Vinculin is a loading control. (B) Depletion of WISp39 changes cell polarity. Cells were scored as either apolar (round)/unipolar (one lamellipodium) or bipolar/multipolar (two or more lamellipodia) and are presented as a percentage of total cells scored from more than five independent experiments. n = 1,165 control cells; n = 1,483 WISp39 KD cells. Data represent the means ± SD. Student’s t test; ***, P ≤ 0.001. (C) Quantitation of the maximum length to maximum width ratio of control cells versus WISp39 KD cells from an experiment performed as in A. Number of cells scored per group is 50 from more than three independent experiments. Data represent the means ± SD. Student’s t test; ***, P ≤ 0.001. a.u., arbitrary unit. (D) Cytoplasmic WISp39 localizes to actin filaments at the leading edge. U2OS cells were transfected with GFP-WISp39, fixed with paraformaldehyde, and stained with rhodamine-phalloidin (actin). Bars, 10 µm.

Article Snippet: Confluent U2OS monolayers, grown in each chamber of a LabTek 8-chamber glass slide in DMEM (Mediatech) supplemented with 10% fetal bovine serum, were wounded with a pipette tip, and filming was initiated 1 h after wounding.

Techniques: Transfection, Control, Western Blot, Quantitation Assay, Staining

Journal: The Journal of Cell Biology

Article Title: WISp39 binds phosphorylated Coronin 1B to regulate Arp2/3 localization and Cofilin-dependent motility

doi: 10.1083/jcb.201410095

Figure Lengend Snippet: WISp39 KD cells exhibit decreased directional motility. (A) WISp39 KD leads to chaotic migration. U2OS cells plated on glass coverslips were transfected with either control or WISp39 siRNA. After 48 h, the cells were confluent, and migration was initiated by creating a wound with a pipette tip. 20× phase images were taken over after a 24-h period as described in the Materials and methods. The movements of individual representative cells, as shown, were obtained using MetaMorph nuclear tracking. WISp39 KD cells lacked directional movement toward the wound, which is on the right side of the frame. The data shown are from a single representative experiment ( n = 10), out of three independent experiments. (B) Time-lapse videos were used to calculate protrusion differences as described in Materials and methods. Green areas are the protruded areas, and red areas are the retracted areas calculated between four successive frames of 7-min intervals each (i.e., the 28 min preceding the tile image) beginning at the 112-min mark. Arrows indicate the direction of cell movement averaged from four successive frames. Time between tiles is 140 min. Expected direction of migration toward wound closure is on the right side of each frame. Note that WISp39 KD cells did not consistently move in a defined direction relative to the protrusion. (C) Quantitation of directionality in actively migrating cells collected using DIAS software. Directionality is a measure of path variance from the central axis of the wound. Number of control and WISp39 KD cells scored is 25 each from three independent experiments. a.u., arbitrary units. (D) Quantitation of persistence in actively migrating cells. Persistence is the ratio of net translocation distance to the total length of migration. Number of control and WISp39 KD cells scored is 25 each from three independent experiments. deg, degree. (E) Quantitation of velocity in actively migrating cells. Velocity was measured using the central difference method as described in the DIAS manual. The number of control and WISp39 KD cells scored is 25 each from three independent experiments. (F) Quantitation of total path length, the distance the cell traveled from the first frame to the last frame taken as a straight line approximation every four frames. The number of control and WISp39 KD cells scored is 25 each from three independent experiments. (G) Quantitation of net path, the distance from the starting frame to the last frame. The number of control and WISp39 KD cells scored is 25 each from three independent experiments. Data shown in C–G represent the means ± SD. Student’s t test; ***, P ≤ 0.001.

Article Snippet: Confluent U2OS monolayers, grown in each chamber of a LabTek 8-chamber glass slide in DMEM (Mediatech) supplemented with 10% fetal bovine serum, were wounded with a pipette tip, and filming was initiated 1 h after wounding.

Techniques: Migration, Transfection, Control, Transferring, Quantitation Assay, Software, Translocation Assay

Journal: The Journal of Cell Biology

Article Title: WISp39 binds phosphorylated Coronin 1B to regulate Arp2/3 localization and Cofilin-dependent motility

doi: 10.1083/jcb.201410095

Figure Lengend Snippet: WISp39 KD effect on motility is rescued by WT WISp39 and requires Hsp90. (A) Images of WISp39 KD rescue by WT WISp39. U2OS cells were cotransfected with human WISp39 siRNA and GFP-mouse WISp39 WT plasmid. 20× phase-contrast images were taken after 48 h of transfection. Top images show a cell transfected with rescue GFP plasmid (yellow arrows), whereas the neighboring cell is not. This cell (yellow arrows) retains normal apolar/unipolar morphology throughout the experiment unlike its neighbor (red arrows), which becomes elongated and multipolar. post trans., posttransfection. Bar, 10 µm. (B) Images of failure of WISp39 KD rescue by WISp39ΔTPR. U2OS cells were cotransfected with human WISp39 siRNA and mouse GFP-WISp39ΔTPR (which does not bind Hsp90) for 48 h and imaged as in A. The cell containing GFP-WISp39ΔTPR (top, red arrows) gradually loses its normal appearance and becomes elongated. Bar, 10 µm. (C) Quantitation of WISp39 KD rescue experiments. U2OS cells were cotransfected with human WISp39 siRNA and a plasmid expressing GFP-tagged mouse WISp39 WT or WISp39ΔTPR. In additional experiments, cells were transfected with WISp39 siRNA, HA-tagged mWISp39-WT or -ΔTPR, and GFP-H2B to mark transfected cells. Abnormal cell polarity of WISp39 KD cells is rescued by WT WISp39 but not by WISp39ΔTPR. Cells were scored as either apolar/unipolar or bipolar/multipolar and presented as a percentage of total cells scored from more than three independent experiments. Number of rescued cells scored: GFP-mouse WISp39 WT (30); GFP-mouse WISp39ΔTPR (75); HA-mouse WISp39 WT (172); HA-mouse WISp39ΔTPR (105). Note that only fluorescent cells were scored. (D) Migration assay of WISp39 KD rescue with WT WISp39 versus WISp39ΔTPR. Migration was initiated by wounding and the experiment was conducted as in . GFP-WISp39 WT rescues the WISp39 KD phenotype, whereas WISp39ΔTPR does not. The data shown are from a single representative experiment ( n = 10), out of three independent experiments. (E and F) Quantitation of cell motility parameters in U2OS cells cotransfected with human WISp39 siRNA and either GFP-mouse WISp39 WT or GFP-mouse WISp39ΔTPR plasmid. Controls were transfected with control siRNA. Measurements of wound directionality and persistence were performed as described in . Number of control and WISp39 KD cells scored is 25 each from three independent experiments. Data shown in C, E, and F represent the means ± SD. ANOVA followed by Tukey’s test; ***, P ≤ 0.001. a.u., arbitrary unit; deg, degree.

Article Snippet: Confluent U2OS monolayers, grown in each chamber of a LabTek 8-chamber glass slide in DMEM (Mediatech) supplemented with 10% fetal bovine serum, were wounded with a pipette tip, and filming was initiated 1 h after wounding.

Techniques: Plasmid Preparation, Transfection, Quantitation Assay, Expressing, Migration, Control

Journal: The Journal of Cell Biology

Article Title: WISp39 binds phosphorylated Coronin 1B to regulate Arp2/3 localization and Cofilin-dependent motility

doi: 10.1083/jcb.201410095

Figure Lengend Snippet: Coronin 1B binds to WISp39 and colocalizes with WISp39. (A) Coronin 1B (Coro 1B) binds WISp39. Extracts from U2OS cells transfected with FLAG-WISp39 were immunoprecipitated (IP) with either anti-FLAG or mouse IgG and subsequently Western blotted for associated endogenous Coronin 1B (top) and WISp39 (bottom). The experiment was repeated three times with similar results. endog., endogenous; IP, immunoprecipitation. (B) Mapping of the Coronin 1B binding region in WISp39. HEK293 cells were transfected with HA-tagged WISp39 WT or WISp39 truncation mutants. WISp39 and its associated proteins were isolated as described in the Materials and methods. The bound proteins were detected by Western blotting. Coronin 1B binds residues 1–98 of WISp39. Mutant 99–347 (asterisk) is only present in the nucleus, whereas the other WISp39 mutants and the WT WISp39 are both nuclear and cytoplasmic. Mutant Δ99–205 lacks residues 99–205 of WISp39. Arrow indicates a nonspecific band. Minus sign indicates another control lane. (C) In vitro binding of WISp39 to Coronin 1B. (left top) HeLa cell lysates were incubated with bacterially expressed GST-WISp39 WT or GST-WISp39(1–205) bound to glutathione-Sepharose, and the associated Coronin 1B was detected by Western blotting with Coronin 1B–specific antibodies. (right top) In vitro transcribed and translated (IVT) HA–Coronin 1B was incubated with GST-WISp39 WT or GST-WISp39(1–205) bound to glutathione-Sepharose, and the associated HA–Coronin 1B was detected by Western blotting with HA-specific antibodies. Ponceau staining of the nitrocellulose membranes is shown. The association of IVT HA–Coronin 1B with GST-WISp39 WT or GST-WISp39(1–205) is not notably different (right), perhaps because cellular proteins that associate with WT WISp39–Hsp90 reduce the fraction of WISp90–Hsp90 complex available for binding Coronin 1B. The experiments were repeated more than three times with similar results. (D) WISp39 colocalizes with Coronin 1B at the leading edge. HeLa cells were transfected with GFP–Coronin 1B and HA-WISp39 or HA–Coronin 1B and GFP-WISp39. Cells were fixed and stained with the anti-HA antibody. Bars, 10 µm. (E) Loss of Coronin 1B yields a morphology similar to WISp39 KD. U2OS cells transfected with either control siRNA or Coronin 1B siRNA or WISp39 siRNA for 48 h were wounded as in and then imaged. Phase-contrast images (20×) of Coronin 1B KD and WISp39 KD show elongated and multipolar morphology. Western blot shows KD of Coronin 1B at 48 h. Actin is a loading control. Bars, 10 µm. (F) Quantitation of Coronin 1B KD morphology. U2OS cells transfected with either control siRNA or Coronin 1B siRNA or WISp39 siRNA for 48 h were wounded as in . Cells were scored from phase-contrast images (20×) as either apolar/unipolar or bipolar/multipolar and are presented as a percentage of total cells scored from more than three independent experiments (control = 200, Coronin 1B KD = 404, and WISp39 KD = 258). Note that the cells were scored after wounding; therefore, they are somewhat constrained from producing multipolar morphology by the surrounding cells, unlike sparsely plated cells in . Thus, there are fewer multipolar cells in comparison to . (G and H) Decrease in directionality and persistence of movement in Coronin 1B KD cells. Quantitation of motility parameters was performed as in . Number of cells scored from three independent experiments: control (20); Coronin 1B KD (20); WISp39 KD (20). a.u., arbitrary unit. (I) Kymograph analysis. (left) A magnified region of the cell is outlined in the rectangular box. Sample kymographs for control and WISp39 KD cells are shown on the right. Experimental parameters are detailed in J–L. (J–L) Protrusion parameters of control and WISp39 KD cells. Kymographs were constructed along a 1-pixel-wide axis oriented in the direction of the protrusion and perpendicular to the edge of the lamellipodia using MetaMorph software. Rate, distance, and persistence of protrusions were calculated from the distance of protrusion (y axis) and time (x axis). Δy = distance of lamellipodia protrusions; Δx = persistence (time) of lamellipodia protrusions; Δy/Δx = rate of lamellipodia protrusions. 10 cells from three independent experiments (8–10 protrusions/cell) were analyzed. deg, degree. Data shown in F–H and J–L represent the means ± SD. (F–H) ANOVA followed by Tukey’s test; ***, P ≤ 0.001. (J–L) Student’s t test; ***, P ≤ 0.001.

Article Snippet: Confluent U2OS monolayers, grown in each chamber of a LabTek 8-chamber glass slide in DMEM (Mediatech) supplemented with 10% fetal bovine serum, were wounded with a pipette tip, and filming was initiated 1 h after wounding.

Techniques: Transfection, Immunoprecipitation, Western Blot, Binding Assay, Isolation, Mutagenesis, Control, In Vitro, Incubation, Staining, Quantitation Assay, Comparison, Construct, Software

Journal: The Journal of Cell Biology

Article Title: WISp39 binds phosphorylated Coronin 1B to regulate Arp2/3 localization and Cofilin-dependent motility

doi: 10.1083/jcb.201410095

Figure Lengend Snippet: Phosphorylated Coronin 1B associates with WISp39 and SSH. (A) Coronin 1B (Coro 1B) associates with WISp39 and SSH when it is phosphorylated. HEK293 cells were transfected with GFP-tagged WT, S2A, or S2D Coronin 1B. Lysates were immunoprecipitated with anti-GFP antibody and analyzed by Western blotting with indicated antibodies. The experiments were repeated three times with similar results. (B) Coronin 1B associates with SSH only in the presence of WISp39. U2OS cells were cotransfected with control or WISp39 siRNA and GFP-tagged WT Coronin 1B. Lysates were immunoprecipitated with anti-GFP antibody and analyzed for association with SSH by Western blotting. The experiments were repeated more than three times with similar results. (C) Coronin 1B phosphorylation increases in the absence of WISp39. HeLa cells were cotransfected with control or WISp39 siRNA and HA-tagged WT Coronin 1B. Lysates were immunoprecipitated with the anti-HA antibody (HA–Coronin 1B) and analyzed for phosphorylation of Coronin 1B on Ser2 by Western blotting with phospho-PKC substrate-specific antibody (phospho-Ser). Quantitation of the phospho-Ser Coronin 1B in control and WISp39 KD cells from three experiments is shown. Student’s t test; **, P ≤ 0.01. (D) WISp39 KD increases phosphorylated Cofilin. U2OS cells were transfected with control or WISp39 siRNA, and the cell lysates were analyzed for phosphorylation by Western blotting with antibodies recognizing phospho-Cofilin. Quantitation of the phospho-Cofilin in control and WISp39 KD cells from three experiments is shown. Student’s t test; **, P ≤ 0.01. (E) WISp39 KD does not influence the levels of other motility-associated proteins. U2OS cells were transfected with control or WISp39 siRNA, and the cell lysates were analyzed by Western blotting with the indicated antibodies. Ctrl, control; IP, immunoprecipitation.

Article Snippet: Confluent U2OS monolayers, grown in each chamber of a LabTek 8-chamber glass slide in DMEM (Mediatech) supplemented with 10% fetal bovine serum, were wounded with a pipette tip, and filming was initiated 1 h after wounding.

Techniques: Transfection, Immunoprecipitation, Western Blot, Control, Phospho-proteomics, Quantitation Assay

Journal: The Journal of Cell Biology

Article Title: WISp39 binds phosphorylated Coronin 1B to regulate Arp2/3 localization and Cofilin-dependent motility

doi: 10.1083/jcb.201410095

Figure Lengend Snippet: WISp39 KD decreases the localization of Arp2/3 complex and increases the generation of free barbed ends at the cell leading edge. (A) WISp39 KD decreases Arp2/3 complex localization at the cell leading edge. Immunolocalization of the Arp2/3 subunit p34-Arc and F-actin phalloidin staining in cells transfected for 48 h with control or WISp39 siRNA. U2OS cells were transfected with WISp39 siRNA in combination with a GFP plasmid expressing histone H2B to visualize transfected cells. After 48 h, the cells were fixed, and immunofluorescence images were acquired as described in Materials and methods. Insets in the first image show a cell expressing GFP-H2B, cotransfected with the siRNA. Magnified regions are shown in the right-most image. (B) Quantification of fluorescence intensity (±SEM) of F-actin, p34-Arc, and ratio of p34-Arc/F-actin in control and WISp39-depleted cells, measured from the cell edge (0) to the cell center (7 µm). Bands of constant distance to the cell edge were constructed, and individual fluorescence intensities were accumulated and averaged in each band to produce graphs of fluorescence intensities versus distance to the cell edge ( Fig. S5 ). The data shown represent one experiment and are averaged from n ≥ 11 cells for each condition. Only cells with a spread morphology have been included. Student’s t test; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 compared with control siRNA cells. The experiment was repeated three times, with similar results. (C) G-actin incorporation marking free barbed ends and F-actin phalloidin staining in cells transfected for 48 h with control or WISp39 siRNA. U2OS cells were transfected with WISp39 siRNA in combination with GFP plasmid expressing histone H2B for 48 h, and immunofluorescence localization of barbed ends was performed by live-cell microscopy. Insets in the first image show the expression of H2B, cotransfected with the siRNA. Magnified regions are shown in the right-most image. WISp39 depletion induces the formation of free barbed filament ends. (D) Quantification of fluorescence intensity of F-actin, free barbed ends, and the ratio of free barbed ends: F-actin in control and WISp39 KD cells, measured from the cell edge (distance = 0) to the cell center (7 µm). Quantitation was performed as in B. The data shown represent one experiment and are averaged from n ≥ 15 cells for each condition (±SEM). Only cells with a spread morphology have been included. Student’s t test; *, P ≤ 0.05; ***, P ≤ 0.001 compared with control siRNA cells. The experiment was repeated four times, with similar results. The mean fluorescent F-actin intensity is elevated slightly relative to B, and this variation may arise because the analysis in B was performed on fixed cells. a.u., arbitrary unit. Bars: (left images and insets) 15 µm; (zoom images) 5 µm.

Article Snippet: Confluent U2OS monolayers, grown in each chamber of a LabTek 8-chamber glass slide in DMEM (Mediatech) supplemented with 10% fetal bovine serum, were wounded with a pipette tip, and filming was initiated 1 h after wounding.

Techniques: Staining, Transfection, Control, Plasmid Preparation, Expressing, Immunofluorescence, Fluorescence, Construct, Microscopy, Quantitation Assay

Journal: The Journal of Cell Biology

Article Title: WISp39 binds phosphorylated Coronin 1B to regulate Arp2/3 localization and Cofilin-dependent motility

doi: 10.1083/jcb.201410095

Figure Lengend Snippet: WISp39 KD can be rescued by expression of Cofilin(S3A) and WT Coronin 1B. (A and B) Images of control and WISp39 KD cells. U2OS cells were transfected with control (A) or WISp39 (B) siRNA and with GFP plasmid to visualize transfected cells. 20× DIC images were taken at 48 h after transfection over a period of 16 h. A control cell (A) retains normal apolar/unipolar morphology throughout the experiment, unlike a WISp39 KD cell (B), which becomes elongated and multipolar. (C) Images of failure of Cofilin(S3A) to rescue WISp39 KD. U2OS cells were cotransfected with human WISp39 siRNA and GFP-Cofilin(S3A) for 48 h, and one cell was imaged as in A. WISp39 KD cell transfected with GFP-Cofilin(S3A) shows abnormal cell polarity similar to that of WISp39 KD cells. (D) Images of rescue of WISp39 KD by Cofilin(S3A) and Coronin 1B. U2OS cells were cotransfected with human WISp39 siRNA and GFP-Cofilin(S3A) and HA–Coronin 1B WT for 48 h, and 20× DIC images were taken. WISp39 KD cell transfected with GFP-Cofilin(S3A) and HA–Coronin 1B (Coro 1B) retains normal apolar/unipolar morphology throughout the experiment. (E) Quantitation of WISp39 KD rescue by Cofilin(S3A) alone or Cofilin(S3A) and Coronin 1B WT. U2OS cells were cotransfected for 48 h. Cells were scored as either apolar/unipolar or bipolar/multipolar and presented as a percentage of total cells scored. Number of rescued cells scored: control siRNA + GFP (22); WISp39 KD + GFP (21); WISp39KD + GFP-Cofilin(S3A) (25); WISp39KD + GFP-Cofilin(S3A) + HA–Coronin 1B WT (25). Note that only fluorescent cells were scored. Data represent the means ± SD. Student’s t test; **, P ≤ 0.01; ns, not significant. CTRL, control; post trans., posttransfection. Bars, 50 µm.

Article Snippet: Confluent U2OS monolayers, grown in each chamber of a LabTek 8-chamber glass slide in DMEM (Mediatech) supplemented with 10% fetal bovine serum, were wounded with a pipette tip, and filming was initiated 1 h after wounding.

Techniques: Expressing, Control, Transfection, Plasmid Preparation, Quantitation Assay