Regulation of the adaptive actin response by force-dependent bonds

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Abstract Force transmission through the actin cytoskeleton is fundamental to how cells sense the geometric and mechanical constraints of their environments, move through tissues, remodel the extracellular matrix, and regulate signaling receptors at the plasma membrane (PM) to determine cell fate. In systems such as the leading edge of migrating cells or in clathrin-mediated endocytosis (CME), polymerizing actin pushes against a membrane to generate protrusive force. The dynamics, structure, and force generation of actin are regulated by mechanics and actin binding proteins (ABPs) that bundle, branch, break, soften, stiffen, polymerize, tether, or move actin filaments. Aside from myosin motors, how mechanical force regulates the affinity of ABPs has seldom been investigated. When ABPs are mechanically anchored in the cell, force at the ABP-actin interface regulates the lifetime of the ABP-actin bond, which I refer to hereafter as the force-dependent actin dissociation rate (FDADR). While intuition suggests the lifetimes of molecular bonds should shorten when the molecules are pulled apart (a “slip bond”)1, a surprising majority of recently characterized ABPs involved in cell adhesion form “catch bonds” with actin that increase in lifetime (sometimes >100-fold2) as force increases2–5. These bonds are highly tuned to the direction of force applied relative to the actin filament polarity2,4,6 with the most extreme reported example being the asymmetric catch bond formed by talin's ABS3 domain2. The functional impact of the FDADR of these ABPs is not known. Due to actin's importance in generating and transmitting mechanical force, equilibrium bulk measurements of ABP-actin interactions provide an incomplete picture of how ABPs contribute to actin cytoskeleton structure, function, and regulation. During CME the PM is bent to encapsulate membrane-bound cargoes. When PM tension is high, actin polymerization force is required to bend the membrane and pull the nascent vesicle into the cell7. Actin at the CME pit “adapts” to PM tension by localizing to the surface of the pit preferentially in conditions of elevated PM tension (i.e., precisely only when it is required for CME completion)8. I will test the hypothesis that the THATCH actin binding domains of CME adapter HIP1R forms an asymmetric catch bond like homolog talin ABS3. I will discover mutants with altered binding in a yeast molecular-genetic screen and characterize their FDADR. I will develop a stochastic simulation to uncover the role of the FDADR in actin network structure and function during CME. I hypothesize that HIP1R's FDADR is tuned to selectively bind actin filaments that bear mechanical load, thus supporting endocytosis over a range of membrane tensions amidst dense cortical filamentous actin. Mutant HIP1R THATCH with altered FDADR will be expressed in mammalian cells, and their impact on CME and actin organization will be determined and compared to simulations, thus relating FDADR to actin structure and function.