Molecular determinants of cell mechanics

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Our long-term objective is to determine how the cytoskeleton governs cellular organization and mechanics. The cytoskeleton is a set of self-assembling, dynamic polymers that give the cell its shape and structure. The organization of these polymers changes in response to mechanical and biochemical signals, allowing the cell to rapidly tune its mechanics from highly-crosslinked, elastic networks to weakly-crosslinked, viscous networks. Precise control of the cell's mechanical properties is required for cells to divide, move, and differentiate to form tissues and organs. We develop single-molecule methods for imaging cytoskeletal organization, controlling protein activity, and measuring intracellular forces on the nanometer and pico-Newton scales relevant to cell biology. To quantify the viscoelasticity of living cells, we manipulate micron-sized beads in the cytoplasm of living cells using optical tweezers. In parallel, we use single-molecule localization microscopy to map the organization of the cytoskeleton with nanometer resolution. Aim 1. To uncover how the microtubule cytoskeleton controls cell mechanics. The cytoskeleton consists of three types of filaments: microtubules, actin, and intermediate filaments. These cytoskeletal filaments interact to govern cell mechanics. While the field has largely focused on actin and intermediate filaments, recent studies demonstrate an important role for microtubules in governing the mechanics of cardiac muscle. We hypothesize that microtubules are a key regulator of cell mechanics not only in muscle cells, but rather this is a general mechanism used by many cell types. Microtubule rigidity is modulated by post-translational modifications and microtubule-associated proteins that bind along the microtubule lattice. We will use new optogenetic tools to modulate microtubule post-translational modifications, and quantify the impact on cytoskeletal organization and mechanics with single-molecule resolution. Aim 2. To dissect the molecular mechanisms that govern the assembly and mechanics of membraneless organelles. While many cellular organelles are separated from the cytoplasm by a membrane, others compartmentalize through liquid-liquid phase separation (LLPS). LLPS is central to many cell processes including gene expression, neurodegeneration, spindle assembly, and endocytosis. We will (1) trigger the formation of membraneless organelles in the cytoplasm using optogenetics and measure the effect on cell mechanical properties, and (2) use optical tweezers to probe the viscoelastic behavior of phase-separated membraneless organelles to uncover how weak, multivalent interactions between multiple proteins control assembly and disassembly. Together, this program will provide a mechanistic understanding of how changes in the mechanics of single polymers and phase-separated condensates tune the viscoelastic properties of the cell in response to chemical, mechanical, and developmental cues.