Limits...
WISp39 binds phosphorylated Coronin 1B to regulate Arp2/3 localization and Cofilin-dependent motility.

Howell M, Brickner H, Delorme-Walker VD, Choi J, Saffin JM, Miller D, Panopoulos A, DerMardirossian C, Fotedar A, Margolis RL, Fotedar R - J. Cell Biol. (2015)

Bottom Line: WISp39 knockdown (KD) resulted in the loss of directional motility of mammalian cells and profound changes in cell morphology, including the loss of a single leading edge.WISp39 KD-induced morphological changes could be rescued by overexpression of Coronin 1B together with a constitutively active Cofilin mutant.We conclude that WISp39 associates with Hsp90, Coronin 1B, and SSH to regulate Cofilin activation and Arp2/3 complex localization at the leading edge.

View Article: PubMed Central - HTML - PubMed

Affiliation: Sanford-Burnham Medical Research Institute, La Jolla, CA 92037.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4384738&req=5

fig7: 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.

Mentions: Because WISp39 promotes the dephosphorylation of Coronin 1B and Cofilin, the profound changes in cell morphology and directional migration observed in WISp39 KD cells may ultimately be a result of the loss of proper Coronin 1B and Cofilin activation and function, which in turn would affect Arp2/3 complex function. To test this, we attempted to rescue the phenotype of WISp39 KD by expressing constitutively active Cofilin(S3A) alone or in combination with Coronin 1B WT (Fig. 7). Cofilin(S3A) alone was unable to rescue the phenotype created by depletion of WISp39, as measured by morphology changes from apolar to bi- and multipolar cells (Fig. 7, C and E). In contrast, Cofilin(S3A) expressed together with Coronin 1B WT was able to rescue the elongated, irregular shape and multipolarity typical of WISp39 KD cells to the extent that they appeared similar to control cells (Fig. 7, D and E). Collectively, these results suggest that WISp39 plays an important role in regulating actin-dependent lamellipodial dynamics at the leading edge, by binding phosphorylated Coronin 1B in a complex with SSH and coordinating the activation of Cofilin and the regulation of the Arp2/3 complex.


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

Howell M, Brickner H, Delorme-Walker VD, Choi J, Saffin JM, Miller D, Panopoulos A, DerMardirossian C, Fotedar A, Margolis RL, Fotedar R - J. Cell Biol. (2015)

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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4384738&req=5

fig7: 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.
Mentions: Because WISp39 promotes the dephosphorylation of Coronin 1B and Cofilin, the profound changes in cell morphology and directional migration observed in WISp39 KD cells may ultimately be a result of the loss of proper Coronin 1B and Cofilin activation and function, which in turn would affect Arp2/3 complex function. To test this, we attempted to rescue the phenotype of WISp39 KD by expressing constitutively active Cofilin(S3A) alone or in combination with Coronin 1B WT (Fig. 7). Cofilin(S3A) alone was unable to rescue the phenotype created by depletion of WISp39, as measured by morphology changes from apolar to bi- and multipolar cells (Fig. 7, C and E). In contrast, Cofilin(S3A) expressed together with Coronin 1B WT was able to rescue the elongated, irregular shape and multipolarity typical of WISp39 KD cells to the extent that they appeared similar to control cells (Fig. 7, D and E). Collectively, these results suggest that WISp39 plays an important role in regulating actin-dependent lamellipodial dynamics at the leading edge, by binding phosphorylated Coronin 1B in a complex with SSH and coordinating the activation of Cofilin and the regulation of the Arp2/3 complex.

Bottom Line: WISp39 knockdown (KD) resulted in the loss of directional motility of mammalian cells and profound changes in cell morphology, including the loss of a single leading edge.WISp39 KD-induced morphological changes could be rescued by overexpression of Coronin 1B together with a constitutively active Cofilin mutant.We conclude that WISp39 associates with Hsp90, Coronin 1B, and SSH to regulate Cofilin activation and Arp2/3 complex localization at the leading edge.

View Article: PubMed Central - HTML - PubMed

Affiliation: Sanford-Burnham Medical Research Institute, La Jolla, CA 92037.

Show MeSH
Related in: MedlinePlus