Analysis of cdGAP in Extracellular Matrix Rigidity Sensing and Cell Migration

Abstract

CdGAP is a Rac1/Cdc42 specific GTPase activating protein that localizes to cell–matrix adhesions through an interaction with the adhesion scaffold α-parvin/actopaxin to regulate lamellipodia formation and cell spreading. In chapter 2 of this thesis, using a combination of siRNA-mediated silencing and over expression, I show that cdGAP negatively regulates directed and random migration by controlling adhesion maturation and dynamics through the regulation of both adhesion assembly and disassembly. Interestingly, cdGAP was also localized to adhesions formed in three-dimensional matrix environments and cdGAP depletion promoted cancer cell migration and invasion through 3D matrices. Cell migration in 3D CDMs more closely approximates the topography of in vitroconnective tissues, suggesting that cdGAP likely plays an important regulatory role in cell migration in vivo. Other aspects of the extracellular matrix also influence cell migration. Specifically, motile cells are capable of sensing the stiffness of the surrounding extracellular matrix through integrin-mediated focal adhesions and migrate towards regions of higher rigidity in a process known as durotaxis. Durotaxis plays an important role in normal development and disease progression, including tumor invasion and metastasis. However, the signaling mechanisms underlying focal adhesion-mediated rigidity sensing and durotaxis are poorly understood. In chapter three of this thesis, I utilizefibronectin-coated polydimethoxysiloxane gelsto manipulate substrate compliance, and show that cdGAP is necessary for U2OS osteosarcoma cells to coordinate cell shape changes and migration as a function of extracellular matrix stiffness. CdGAP regulated rigidity-dependent motility by controlling membrane protrusion and adhesion dynamics, as well as by modulating Rac1 activity. I also found that CdGAP was necessary for U2OS cell durotaxis. Taken together, these data identify cdGAP as an important component of an integrin-mediated signaling pathway that senses and responds to mechanical cues in the extracellular matrix in order to coordinate directed cell motility.These findings highlight the importance of GAP proteins in the regulation of Rho family GTPases andprovide insight into how GAPs co-ordinate the cell migration machinery.