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Cell attachment also leads to dramatic cytoskeleton reorganization. While FA formation was only induced by fibronectinmediated attachment, cytoskeleton reorganization as well as cytoplasmic translocation of YAP were also seen in polylysinemediated attachments.Cells attached to fibronectincoated coverglasses spread and formed cortical actin networks and stress fibers. We did not observe any obvious difference in the microtubule cytoskeleton between cells attached to fibronectin and polylysinecoated coverglasses, although we saw microtubule extending into filopodia in cells on polylysine. These observations raise the possibility that cytoskeleton reorganization may mediate the regulation of YAP by cell attachment.Cell attachment induces Hematoxylin dephosphorylation of YAP.MCFA cells were trypsinized and attached onto fibronectincoated petri dishes for the indicated time.Phostagcontaining SDSPAGE gels were used as indicated.Cell detachment strongly induces YAP phosphorylation.Cell attachment induces Acalabrutinib nuclear translocation of YAP.MCFA cells were trypsinized and attached onto fibronectincoated coverglasses for the indicated time. Cells were then fixed and stained with antiYAP antibody.Both XY views and XZ views from confocal imaging are presented in the left panel.Attachment to fibronectin or polylysinecoated supporting materials similarly induces nuclear translocation of YAP.MCFA cells were trypsinized and attached onto fibronectin or polylysinecoated coverglasses for the indicated time in serumfree medium.Phostagcontaining SDSPAGE gels were used as indicated.A similar effect was also observed when cells were treated with cytochalasin D, another inhibitor that disrupts the actin cytoskeleton by capping filament plus ends. The microtubule cytoskeleton is also reorganized during cell attachment.However, disruption of microtubule polymerization by nocodazole or vinblastine as well as induction of microtubule polymerization by taxol did not block attachmentinduced YAP dephosphorylation. Interestingly, pretreatment of cells with nocodazole strongly blocked detachmentinduced YAP phosphorylation. This effect was specific to microtubule disruption because concomitant treatment with microtubulestabilizing reagent taxol neutralized the effect of nocodazole.Again, detachmentinduced YAP phosphorylation was not abolished by PP, PF, or blebbistatin treatment. These data indicate that both actin and microtubule cytoskeletons are involved in YAP regulation by the cell attachment status. We next examined whether cytoskeletons could play a role in YAP regulation in a context other than cell attachment.When cultured at high cell density, YAP phosphorylation is relatively high.In these cells, disruption of microtubule by nocodazole or vinblastine also induced YAP dephosphorylation. However, blebbistatin and ROCK inhibitors Y and H did not repress YAP phosphorylation. In lowdensity cell culture, YAP phosphorylation is at a low level.Disruption of the actin cytoskeleton by latrunculin B or cytochalasin D strongly induced YAP phosphorylation, while relieving tension on the actin cytoskeleton by blebbistatin had a minor effect. Actin and microtubule cytoskeletons have extensive physical and functional interactions.Interestingly, the dephosphorylation of YAP by disruption of microtubule was largely blocked by codisruption of the actin cytoskeleton, indicating that these two cytoskeleton systems coordinately regulate YAP phosphorylation.At low cell density, endogenous YAP mainly localized to the nucleus, while disruption of the actin cytoskeleton by latrunculin B induced YAP cytoplasmic translocation. YAP mainly resided in the cytoplasm at high cell confluence.Interestingly, disruption of the microtubule cytoskeleton by nocodazole led to nuclear translocation of YAP, which was enhanced by cotreatment with leptomycin B. LMB by itself did not significantly induce translocation of YAP under the condition tested.

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