| Summary: | The cell cortex is a thin network of actin, myosin motors, and associated proteins that underlies the plasma membrane in most eukaryotic cells. It enables cells to resist extracellular stresses, perform mechanical work, and change shape. The actin network undergoes constant reorganisation due to molecular turnover. Hence, cortical structural and mechanical properties depend strongly on the relative turnover rates of its constituents and the actin filament length-distribution, but quantitative data on these dynamics remains elusive. I combined single molecule speckle microscopy and photobleaching experiments with microscopic computer simulations to analyse how molecular binding dynamics of G-actin to filaments sets network turnover and consequently the mechanical properties of the cellular actin cortex in living cells. Using photobleaching experiments, I found that two filament families with very different turnover rates composed the actin cortex: one with fast turnover dynamics and polymerisation resulting from addition of monomers to free barbed-ends and one with slow turnover dynamics with polymerisation resulting from formin-mediated filament growth. I show that filaments in the second subpopulation are on average longer than those in the first and that cofilin-mediated severing of formin-capped filaments contributes to replenishing the filament subpopulation with free barbed-ends. Additionally, I measured the molecular association rates and the distribution of travel-distances of single actin monomers and formin dimers in speckle experiments and showed that this travel-distance distribution is consistent with the actin filament length-distribution found from photobleaching experiments and molecular simulations. Furthermore, I compare the steady state cortex of different cell lines and newly formed cortices in cellular blebs and discuss the role of cross-linkers like α-actinin and myosin mini-filaments in the actin cortex. Together, my results provide a quantitative characterisation of essential mechanisms underlying actin cortex homeostasis.
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