| My studies focused on identifying the importance of protein interactions with the integrin beta subunit cytoplasmic domain in regulating adhesion-dependent processes, including cell spreading and the connection of integrins with actin filaments at adhesion sites. In Section 1 of my thesis, I characterized the role of talin-beta tail interactions downstream of integrin activation in the context of recombinant integrins containing either the wild type (WT) or the (YA) mutant beta1A tail. In addition to inhibiting cytokinesis, the YA mutation also suppresses integrin signaling, cell spreading and focal adhesion and stress fiber formation (Nieves, 2010). Importantly, constitutively activating the mutant integrin restores these integrin-dependent processes, bringing into question the importance of the NPIY motif and talin1 downstream of integrin activation. By depleting talin1 using siRNA, I demonstrate that talin1 is required for cell spreading, focal adhesion and stress fiber formation, as well as microtubule assembly, even when cells are adhered by constitutively activated WT integrins. Depletion of talin1 does not inhibit these processes when cells are adhered by constitutively activated mutant integrins, suggesting that the binding of an inhibitory protein to the NPIY motif negatively regulates integrin function when talin1 is depleted. My studies identified filamin A (FLNa) as this inhibitory protein; it binds to the beta1A tail in an NPIY-dependent manner and inhibiting of FLNa expression in talin1-depleted cells restores integrin function when cells are adhered by constitutively activated WT integrins. FLNa binds FilGAP, which is a negative regulator of Rac1 activation. Expression of the dominant inhibitory mutant, FilGAPDeltaGAP, which lacks GAP activity restores spreading in cells adhered by constitutively activated integrins containing the beta1A tail, but not by integrins containing the beta1D tail, known to bind poorly to FLNa. Together, these results suggest that the binding of talin1 to the NPIY motif is required downstream of integrin activation to promote cell spreading by preventing the inappropriate recruitment of FLNa and FilGAP to the beta1A tail. Our studies emphasize the importance of understanding the mechanisms that regulate the differential binding FLNa and talin to the beta1 tail downstream of integrin activation in promoting integrin function. These studies demonstrate that integrin signaling, cell spreading, and/or focal adhesion and stress fiber formation may be required for successful cytokinesis. Furthermore, these findings suggest that talin binding to the membrane-proximal NPIY motif of the beta1 tail is not strictly required for cytokinesis.;In Section 2, my studies are focused at understanding which aspects of integrin function are require for successful cytokinesis and to identify the molecular mechanism(s) involved. The results demonstrate that the YA mutation inhibits ERK activity, suppresses PI(4,5)P2 concentration at the cleavage furrow, delays cytokinesis and results in significant cytokinesis failure. Interestingly, pharmacological inhibition of MEK/ERK signaling in cells adhered by WT integrins results in similar phenotypes. Activating the YA mutant integrin with manganese treatment promoted successful cytokinesis by a MEK/ERK-dependent mechanism. In addition, MEK/ERK signaling was implicated as a critical regulator of PI(4,5)P2 concentration at the cleavage furrow for timely and successful progression through cytokinesis. Furthermore, expression of constitutively active Raf1 was sufficient to promote PI(4,5)P2 concentration at the cleavage furrow, but did not rescue cell spreading. These findings suggest that integrin-regulated MEK-ERK signaling is sufficient to promote successful cytokinesis, independent of cell spreading. Taken together, our results demonstrate a novel role for integrin-mediated MEK/ERK signaling in regulating the enhanced concentration of PI(4,5)P2 at the cleavage furrow for successful progression and completion of cytokinesis. (Abstract shortened by UMI.)... |
