Cellular adhesion dynamics: investigation of molecular clutch attachment and force transmission.

University of Minnesota Ph.D. dissertation. December 2008. Major: Biomedical Engineering. Advisor: David J. Odde. 1 computer file (PDF); ix, 120 pages, appendices A-B.As the major structural element of the cell, the cytoskeleton plays a vital role in response and transmission of forces in both extracellular and intracellular environments. For instance, in cell motility, the cell utilizes a host of proteins to physically link F-actin to the extracellular substrate, allowing the cell to exert traction forces as well as probe the mechanics of its local environment. During mitosis, the cell constructs a mitotic spindle, using microtubules and kinetochores to exert forces that segregate sister chromatids. Ultimately, understanding how cells build these robust molecular machines for unique tasks could one day lead to therapeutics that treat disease causing dysfunctions in these vital cellular processes. In order to explore how molecular clutches work in concert with the cytoskeleton to exert forces and maintain attachment under load, we developed a mechano-chemical cellular adhesion dynamics framework to simulate these processes. In the case of cellular motility, we find that a "motor-clutch" mechanism exhibits substrate-stiffness sensitive dynamics. On soft substrates, motor-clutch motility exhibits "load-and-fail" dynamics that lead to higher rates of retrograde flow and lower traction force transmission compared to stiff substrates. We confirm these predictions experimentally using embryonic chick forebrain neurons (ECFNs) plated on compliant polyacrylamide gels (PAGs) demonstrating that a motor clutch system could be the basis of cellular mechanosensing. We also use cellular adhesion dynamics to explore kinetochore-microtubule attachment during mitosis to identify what properties might be important in maintaining attachment during mitosis. We show that molecular clutch microtubule-lattice diffusion is important for relieving clutch stresses, prolonging bond life-times and minimizing detachment forces. Furthermore, molecular clutches that preferentially associate with interdimer interfaces, rather than with intradimer interfaces, promote robust kinetochore attachment by preventing the more distal, attachment-promoting linkers from becoming nonproductive. These findings help further our understanding of the mechanochemical basis of kinetochore attachment and mitosis, a process essential throughout development