Catch and slip bond dynamics of cytoskeletal cross-linkers in live cells – a new approach to understand principles of cytoskeletal material engineering

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In recent years, several biophysical studies have shown that biological material, such as an animal cell, actively engineers its material properties. Understanding this phenomenon is an important prerequisite for our comprehension of cellular force response, cell shape dynamics and tissue organization. Specifically, the actin cytoskeleton is able to tune its mechanics through active mechanical stress introduced by myosin motors. However, different studies find qualitatively different modes of this mechanical tuning through active stress; both stiffening and softening of actomyosin meshworks have been observed. Here, I suggest to combine experiment and theory to elucidate the influence of active stress on cell mechanical properties. I will focus in my study on the influence of the force-sensing of actin cross-linkers in the actin cytoskeleton. Previous experiments have indicated that two qualitatively different actin cross-linker types exist – catch and slip bond cross-linkers – whose bonds are either enforced or weakened in the presence of tensile stress. Therefore, catch or slip bond cross-linkers are expected to give rise to a stiffening or softening of the actin cytoskeleton at increased active stress, respectively. While the state-of-the-art measurements on the load-dependence of bond lifetimes are done in vitro, I will go an important step ahead and characterize the force-dependence of cross-linker bonds in the cortex of live cells. To this end, I will utilize a new technique for the measurement of active cortical stress and cytoskeletal stiffness that I have recently developed. There, cells are mechanically probed by a parallel plate compression assay using an atomic force microscope. This technique will be combined with photo-bleaching of fluorescently labeled cross-linkers at the actin cortex of cells. The recovery of cross-linker fluorescence will reveal their unbinding dynamics. I will use this assay to characterize the lifetime of cross-linkers at varying levels of active cortical stress and, thus, characterize them as catch or slip bond cross-linkers.Outlook: In a second funding period, I plan to examine how a combination of different catch and slip bond cross-linkers in the actin cytoskeleton can be used to custom-tailor the material properties of the cell. To that end, I intend to develop a mathematical model that characterises the expected influence of catch and slip bond cross-linkers on emergent material properties of the actin cytoskeleton. This model will predict cross-linker concentration regimes in which active stress causes stiffening or softening, solidification or fluidization of a meshwork. In an experimental part, I plan to explore these regimes through targeted knock-down of cross-linker proteins in the cell, thereby, delivering a proof of principle that force-sensing of cross-linkers affects the nonlinear properties of the actin cytoskeleton.