PROJECT SUMMARY/ABSTRACT The remarkable functional versatility of the actin cytoskeleton stems from its ability to assemble into a variety of diverse structures – branched networks, meshes, and bundles. This architectural complexity is orchestrated by actin-binding proteins, whose activity is delicately regulated in response to internal and external signals. Our long-term goal is to contribute to human health and well-being by advancing the understanding of the actin cytoskeleton organization by actin-bundling proteins and their contribution to pathologies (e.g., congenital diseases and metastatic cancers). Plastin/fimbrin family of cytoskeleton organizers are conserved proteins that promote assembly of actin filaments into bundles involved in cell migration, adhesion, cytokinesis, and formation of stereocilia and microvilli structures of the inner ear, intestinal and kidney epithelia. Of three human plastin (PLS) isoforms, PLS1 deletion results in deafness, PLS2 contributes to pathologies of the immune system and the development of aggressive metastatic cancers, while mutations in PLS3 lead to severe osteoporosis with bone fragility and other connective tissue disorders. Despite the importance and a long-lasting interest of the research community to these proteins, understanding of their interaction with actin and their regulation is superficial, whereas published structural and biochemical data are incomplete, scattered, and sometimes contradictory. The overall objective of the current proposal is to fill these major gaps by providing a thorough characterization of the molecular and cellular mechanisms governing the function of plastins and to demonstrate how this improved understanding can contribute to explaining the pathology of plastin-related diseases. We propose that the unique domain organization of plastins enables several regulation modes interconnected via a central allosteric mechanism that confers multifaceted contribution to various actin-governed cellular processes. Biochemical characterization of plastin isoforms will reveal mechanisms of their regulation and function at the molecular level (Aim 1a,b); high-resolution cryo-electron microscopy (EM)/cryo-electron tomography (ET) reconstruction will provide structural details of plastin interaction with actin (Aim 1c); structural analysis and atomistic molecular dynamics (MD) simulations will generate a model of the auto-inhibition allowing to predict functional outcomes of congenital mutations (Aim 2); while Aim 3 will focus on understanding functional significance and implications of the allosteric auto-inhibition of plastins and its role in cooperation with other actin- binding proteins. These approaches, supported by single-molecule speckle (SiMS), total internal reflection fluorescence (TIRF), and bulk epi-fluorescence microscopy, will unveil plastin dynamics, cooperation with protein partners, and contribution to actin-dependent processes in living cells. The proposal will result in a breakthrough in the understanding of the actin-dependent cellular events controlled by the plastin/fimbrin family of cytoskeleton organizers, uncover molecular mechanisms behind plastin-linked congenital (deafness, osteoporosis, and diaphragmatic hernia) and acquired (cancer) diseases, opening opportunities for their specific therapeutics.

PROJECT NARRATIVE Plastin family of the actin cytoskeleton organizers contributes to the activation of immune cells, bone formation, cell division, migration, and adhesion as well as to shaping biological membranes essential for the absorbance of nutrients by the intestine, blood dialysis by kidneys, and audial perception by the inner ear. When disrupted or expressed ectopically, plastins contribute to the metastatic activity of cancer cells, autoimmune processes, congenital osteoporosis, syndrome of diaphragmatic hernia, and deafness. Our investigation will reveal fundamental molecular mechanisms underlying the activity of human plastins and will generate a strong and lasting impact on the fundamental understanding of the actin cytoskeleton leading to potential therapeutic interventions for cancers, familial osteoporosis, and autoimmune disorders.