Figure 3.
P-YAP1 facilitates cytoskeletal activation by binding its WW2 domain to MYH9. (A) Confocal images of MKs treated with a YAP1 activator (XMU-MP-1, X) or YAP1 inhibitor (Peptide 17, P) after immunostaining for tubulin (green, tubulin), F-actin (yellow, phalloidin), and MKs (red, CD41). Scale bar, 10 μm. Proplatelet formation is indicated by the red arrows. Assessment of percentage of stress fiber formation (middle) and spreading area (right) in the 3 groups (n ≥ 5). (B) Confocal images of MKs from patients with ITP and healthy controls after immunostaining for YAP1 (green, YAP1), F-actin (yellow, phalloidin), and MKs (red, CD41). Scale bar, 10 μm. Right: analysis of the MK spreading area in the ITP group vs the control group (n = 10). (C) MKs were isolated from WT and YAP1+/− mice to observe the cytoskeleton (left) and the spreading area (right; n = 10). (D) Coimmunoprecipitation assay to verify the interaction between YAP1 and MYH9 (n = 3). (E) Confocal images of PCDH-YAP1-flag-DAMI (YAP1-flag) after immunostaining for MYH9 (yellow, MYH9) and YAP1 (red, CD41). Scale bar, 10 μm. (F) P-YAP1 binding to MYH9 was confirmed by coimmunoprecipitation using flag antibodies (n ≥ 3). (G) P-YAP1 was responsible for MYH9 binding in MKs. After P-YAP1 coimmunoprecipitation, the total binding protein complex and the protein supernatant that did not bind to P-YAP1 were collected. Flag coimmunoprecipitation was performed to verify the interaction between unphosphorylated YAP1 and MYH9 in the MKs (n ≥ 3). (H) Compared with full-length YAP1, WW1 deletion (WW1-del) led to P-YAP1 reduction, whereas WW2 deletion (WW2-del) and WW1-WW2 double deletion (WW1-WW2-del) showed no decrease or even an increase in P-YAP1. The cooperation between MYH9 and the 4 isoforms of YAP1 was demonstrated by coimmunoprecipitation. (I) The WW2 domain fragment plays a critical role in MYH9 binding, and WW2 deletion leads to the upregulation of dysfunctional P-YAP1, leading to severe defects in MYH9 binding (n ≥ 3). ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Flag, PCDH-flag-DAMI; IP, immunoprecipitation.

P-YAP1 facilitates cytoskeletal activation by binding its WW2 domain to MYH9. (A) Confocal images of MKs treated with a YAP1 activator (XMU-MP-1, X) or YAP1 inhibitor (Peptide 17, P) after immunostaining for tubulin (green, tubulin), F-actin (yellow, phalloidin), and MKs (red, CD41). Scale bar, 10 μm. Proplatelet formation is indicated by the red arrows. Assessment of percentage of stress fiber formation (middle) and spreading area (right) in the 3 groups (n ≥ 5). (B) Confocal images of MKs from patients with ITP and healthy controls after immunostaining for YAP1 (green, YAP1), F-actin (yellow, phalloidin), and MKs (red, CD41). Scale bar, 10 μm. Right: analysis of the MK spreading area in the ITP group vs the control group (n = 10). (C) MKs were isolated from WT and YAP1+/− mice to observe the cytoskeleton (left) and the spreading area (right; n = 10). (D) Coimmunoprecipitation assay to verify the interaction between YAP1 and MYH9 (n = 3). (E) Confocal images of PCDH-YAP1-flag-DAMI (YAP1-flag) after immunostaining for MYH9 (yellow, MYH9) and YAP1 (red, CD41). Scale bar, 10 μm. (F) P-YAP1 binding to MYH9 was confirmed by coimmunoprecipitation using flag antibodies (n ≥ 3). (G) P-YAP1 was responsible for MYH9 binding in MKs. After P-YAP1 coimmunoprecipitation, the total binding protein complex and the protein supernatant that did not bind to P-YAP1 were collected. Flag coimmunoprecipitation was performed to verify the interaction between unphosphorylated YAP1 and MYH9 in the MKs (n ≥ 3). (H) Compared with full-length YAP1, WW1 deletion (WW1-del) led to P-YAP1 reduction, whereas WW2 deletion (WW2-del) and WW1-WW2 double deletion (WW1-WW2-del) showed no decrease or even an increase in P-YAP1. The cooperation between MYH9 and the 4 isoforms of YAP1 was demonstrated by coimmunoprecipitation. (I) The WW2 domain fragment plays a critical role in MYH9 binding, and WW2 deletion leads to the upregulation of dysfunctional P-YAP1, leading to severe defects in MYH9 binding (n ≥ 3). ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Flag, PCDH-flag-DAMI; IP, immunoprecipitation.

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