Actin-based Force Generation in the Vascular Endothelium

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The vascular endothelium is a systemically distributed, highly dynamic, and tightly regulated organ system that plays a central role in the pathogenesis of a wide range of diseases. Contractile actin complexes mediate barrier function, mechano-transduction, directed transcellular transport processes in endothelial cells and are essential for cell migration during angiogenesis. Despite their indispensable contribution to the integrity and function of the vascular endothelium, our knowledge about the isoform-composition and functional properties of the actomyosin-based structures involved is limited. Preliminary experiments using reconstituted actin-tropomyosin-myosin complexes show how the role of cytoskeletal tropomyosin isoforms extends beyond the role of a gatekeeper. Changes in motor activity are not only observed upon exchange of myosin isoforms, but the same myosin shows pronounced variations in strain-sensitivity, processivity, and velocity upon exchange of the associated tropomyosin isoform. We will identify the major contractile complexes present in vascular endothelial cells, to elucidate their function-defining structural features, to deduce first principles that allow an accurate modelling of the impact of allosteric trigger events on their chemo-mechanical properties, thermal stability, protein folding stability and dynamics. We will exploit our leading role in the production of cytoskeletal proteins, reconstitution of contractile complexes, and their functional and structural characterisation, to map the allosteric communication pathways and determine the basis for the observed isoform-dependent differences in chemo-mechanical coupling. We will use serial crystallography approaches to obtain complementary information about conformational and folding dynamics. Based on our characterisation of suitable pharmacological myosin chaperones, we can prevent irreversible denaturation and follow unfolding and refolding processes in these experiments. We expect that our results will provide an unprecedented wealth of structural and dynamic data. Their integration with molecular simulations promises to produce testable models that will lead to the identification of allosteric trigger positions in the different protein isoforms that contribute to actomyosin-dependent force production in vascular endothelial cells and how they contribute to chemo-mechanical coupling and force generation in the context of different combinations of isoforms.