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Theses Doctoral

Integrin-Linked Kinase, ECM Composition, and Substrate Rigidity Regulate Focal Adhesion - Actin Coupling, Modulating Survival, Proliferation and Migration: Towards a Biophysical Cancer Biomarker

Chander, Ashok Coil

The extracellular matrix (ECM) has been implicated in numerous physiological and pathogenic processes. Integrins are thought to be the primary receptors that cells use to transduce biochemical and physical signals from the ECM. Integrin - ligand binding is specific for ECM molecules and is regulated by specific protein-protein interactions that further regulate downstream cellular activity such as motility, survival, growth, and proliferation. Termed outside-in signaling, the engagement of integrins results in protein recruitment to sites of cell - ECM contacts known as focal adhesions. Focal adhesions (FAs) are central to cell spreading, motility, survival and growth and serve as both physical linkages between the ECM and cytoskeleton as well as signaling centers for a cell on 2D substrates. Termed focal adhesion-actin coupling, FAs physically link the cytoskeleton with the ECM via actin binding proteins and are involved in mechanically coupling the cell to the ECM. To date, FAs' signaling properties and FA- actin coupling have been unrelated and independent mechanisms. This study provides data that suggests the amount, or level, of focal adhesion coupling in addition to regulating traction force generation, motility events and the rigidity response, also regulates the amount of biochemical signaling towards survival, growth and proliferation. First, via a knockout cell line system I demonstrate that Integrin-Linked Kinase is involved in coupling Beta1 integrins to collagen and FAs. I then demonstrate that lack of coupling results in altered rigidity sensing, defects in spreading of the cytoplasm, lower force generation and collagen contraction, as well as altered localization and activation of MAP kinases. Specifically, when ILK null cells were plated on collagen coated glass they were unable to reinforce Beta1 integrin mediated interactions nor spread their cytoplasm or undergo contractile activity. In contrast, when ILK null cells were plated on fibronectin coated glass, ILK null cells progressed to the contractile phase of spreading and then retracted their adhesions, losing the ability to stabilize late stage Beta1 integrin mediated fibronectin interactions. Moreover, I demonstrate that actin retrograde flow regulates the localization and modification state of FA signaling molecules that regulate survival, growth, and proliferation. Secondly, via changing ECM composition and rigidity of the substrate, I demonstrate that the engagement of both Beta1 and Beta3 integrins via collagen type I and fibronectin increases focal adhesion size, focal adhesion-actin coupling, and activation of signaling molecules involved in translation, survival, growth, and proliferation. This investigation presents data that supports the idea that the degree of focal adhesion mediated ECM-cytoskeletal coupling correlates with the ability to activate signaling molecules and suggests a model in which focal adhesion-actin coupling regulates the localization and modification state of scaffold and signaling proteins that result in the modulation of survival, growth and proliferation. Finally, I propose the use of an experimentally derived metric to describe ECM-FA-actin coupling and present preliminary data that the proposed metric can also be used as a biomarker for specific disease states such as cancer.


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More About This Work

Academic Units
Biological Sciences
Thesis Advisors
Sheetz, Michael P.
Ph.D., Columbia University
Published Here
February 27, 2012