Date of Award
2024
Document Type
Open Access Dissertation
Degree Name
Doctor of Philosophy in Biomedical Engineering (PhD)
Administrative Home Department
Department of Biomedical Engineering
Advisor 1
Sangyoon J. Han
Committee Member 1
Jeremy Goldman
Committee Member 2
Smitha Rao Hatti
Committee Member 3
Tarun Dam
Abstract
Mechanical stiffness of the extracellular matrix (ECM) impacts many cellular functions such as proliferation, migration, and differentiation. ECM stiffness is sensed by a cell via integrin-based focal adhesions (FAs) by changing conformation and biochemical activities of molecules within FAs by the exchange of the force between the ECM and filamentous actin (F-actin). Cells in turn respond to this stiffness by generating traction force that plays an important role in many biological events such as tissue morphogenesis, stem cell differentiation, wound healing, and cancer cell metastasis. The stiffness of the extracellular matrix induces differential tension within integrin-based adhesions. Understanding stiffness sensing mechanism can thus help in identification of treatments against developmental disorders and cancer progression and metastasis as well as better designs of functional tissue scaffolds for tissue transplantations. FAs have been shown to transmit force in a differential manner in response to varying stiffness i.e., increasing force transmission with increasing stiffness. Nevertheless, the differential force transmission phenomenon has been studied mostly in matured FAs where the main source of tension is contractility by a non-muscle myosin-II protein. Myosin-II pulls the actin retrograde flow generating observed traction in response to stiffness. However, it has been unclear if the stiffness-dependent differential tension is induced solely by myosin activity and might be sensed during the early adhesion assembly in the absence of myosin. The initial adhesion assembly which forms nascent adhesion (NA) occurs in the lamellipodia of the cell which is myosin-independent and mediated only by the actin polymerization driving actin retrograde flow. During this, RIAM (Rap1-GTP-interacting adaptor molecule), an effector of small GTPase Rap1 is involved in the adhesion initiation and formation. RIAM binds to mechanosensitive protein talin and activates integrin during early adhesion formation. This protein is solely present in the nascent adhesion and gets replaced by other protein, vinculin in mature focal adhesion. However, it is present in an autoinhibited state and has to be activated before the adhesion can form. Therefore, understanding RIAM-mediated adhesion dynamics can elicit the process of nascent adhesion and role of RIAM in their formation.
Here, I report that in the absence of myosin contractility, 3T3 fibroblasts still transmit stiffness-dependent differential levels of traction. This myosin-independent differential traction is regulated by polymerizing actin assisted by actin nucleators Arp2/3 and formin where formin has a stronger contribution than Arp2/3. Interestingly, I report a four-fold reduction in traction of cells when both Arp2/3 and myosin were inhibited, compared to cells with only myosin inhibition, while there was only a slight reduction in F-actin flow speed in those cells. I show that the conventional rigid-actin-based clutch model is insufficient to explain this force-flow behavior and requires the inclusion of F-actin’s elasticity into consideration. This new model suggests that Arp2/3 and formin modulate stiffness sensing via stiffening the F-actin network and transmitting the flow-driven force more effectively. I also report that the presence of RIAM for longer period to Talin accelerate the adhesion assembly and disassembly rate. Its increased presence in the focal adhesion also increases the traction force generation. These stiffness-sensing and molecular adhesion dynamic studies help in understanding nascent adhesions. This will lead to new insight on their importance in physiology and pathophysiology including tissue development, regeneration, and cancer progression.
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Recommended Citation
Mittal, Nikhil, "MATRIX STIFFNESS SENSING BY NASCENT ADHESIONS AND THE ROLE OF RIAM IN ADHESION ASSEMBLY", Open Access Dissertation, Michigan Technological University, 2024.
Included in
Biological Engineering Commons, Biology and Biomimetic Materials Commons, Biomaterials Commons, Biomechanics and Biotransport Commons, Molecular, Cellular, and Tissue Engineering Commons