Biophysical Mechanisms of Force Transmission in Cytoskeletal Ensembles

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Project Summary: Actin filaments and microtubules are cytoskeletal polymers essential for cell division, motility, and intracellular transport, and deficiencies in these proteins are implicated in cancer, heart disease, and other disorders. In order to facilitate vital tasks that span the entire cell, these filaments coordinate with each other through motor proteins, such as kinesin and myosin, and associated binding proteins. The molecular basis for this communication through tension and compression forces and how these signals propagate through the cytoskeleton is not well understood. Approaches to study such cytoskeletal phenomena have traditionally been either at the single molecule level or whole cell level, and the actin and microtubule cytoskeletons have generally been evaluated as separate systems in vitro. While single molecule experiments, with methods such as optical trapping, have been invaluable in deciphering the mechanics of individual motors, a completely reductionist approach with one filament and one motor protein does not accurately represent the structural hierarchy in which crosslinking motors and proteins function. On the other hand, cell level studies take place in a quite complex environment. In this research plan, we will bridge the gap in scale and assay control by engineering novel, physiologically relevant cytoskeletal environments, or nanocells, in which to probe motor protein mechanics and cytoskeletal crosstalk. Much like LEGOs, we can choose which cytoskeletal elements to incorporate in our nanocell’s architecture and tune the building blocks accordingly to understand how changes at the molecular level propagate to system level force generation and network stiffness. Using this innovative approach, our overarching goal is to provide a fundamental molecular understanding of how motors, crosslinkers, filaments, and signaling factors communicate with each other in ensembles and to the local cytoskeletal environment utilizing optical trapping, quartz crystal microbalance with dissipation, and spectroscopic techniques. Specifically, we will investigate how myosins work together in ensembles in actin assemblies and what molecular components dictate productive force generation. Hybrid nanocells that consist of elements from both the actin and microtubule cytoskeleton will be probed to understand how polymers of different stiffnesses, crosslinking proteins with different pliability, and motor proteins with varying processivity and force generation capability affect cytoskeletal crosstalk. As E-hooks are the diversity site of tubulin and uniquely influence motility in disparate kinesin families, we will interrogate how E- hook structure affects ensemble kinesin force generation in nanocells. The proposed research will pave the way to our long-term goal, which is not only to understand fundamental mechanisms that sustain life, but ultimately be able to reconstitute physiologically realistic models of cellular processes in vitro, providing an enormous potential for developing diagnostic and treatment strategies for cytoskeletal diseases.