Mechanisms of regulation of lymphocyte migration by actin cytoskeletal effectors

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Lymphocytes must correctly localize to mount effective immune responses. To do this, lymphocytes migrate through tissue environments with very different biophysical characteristics, including extravasation from blood vessels and crawling through cell-packed or extracellular matrix-rich tissues. To navigate through these diverse environments, lymphocytes squeeze through constrictions and migrate in low- or high-adhesive environments. However, there is a key gap in the understanding of how specific cytoskeletal effectors regulate force generation, shape changes, and cell-cell or cell-matrix interactions during lymphocyte migration in different settings. Given their relevance to immune function, primary T lymphocytes are a highly significant model to investigate cytoskeletal regulation of the varying modes of amoeboid cell motility in three-dimensional (3D) environments. Formin family proteins are cytoskeletal effectors involved in mediating actin network remodeling. Formin-like-1 (FMNL1) and Diaphanous-homologue-1 (mDia1) are the two most highly expressed Formins in T cells. We recently determined that FMNL1 is required for T cell transendothelial migration (TEM) and trafficking to inflamed tissues. Interestingly, our preliminary data support that FMNL1 and mDia1 have distinct functions in T cell migration through confined environments. Our goal is to achieve a more comprehensive understanding of the mechanisms by which the cytoskeleton enables migration through diverse tissue environments by determining the mode of action of Formin proteins in T cell motility. We will investigate the mechanisms by which Formins generate mechanical forces during T cell migration, the contribution of Formins in promoting T cell nucleus passage through constrictions, and how Formins regulate T cell motility in vivo. We will also determine if FMNL1 and mDia1 act independently or in concert with the Arp2/3 complex and/or Myosin-IIA. Our hypothesis is that to promote migration through complex environments FMNL1 mediates force generation from the back of the T cell while mDia1 creates force and membrane protrusions at the cell front. To test this hypothesis, we will use a multi-faceted approach, including genetic/mutational approaches and advanced imaging techniques in complementary model systems in vitro and physiological environments in vivo. Aim 1: Determine the mechanisms by which FMNL1 and mDia1 promote T cell transendothelial migration. Aim 2: Determine how FMNL1 and mDia1 regulate T cell motility within 3D environments with diverse biophysical characteristics. Aim 3: Define how Formins regulate T cell extravasation and interstitial motility in vivo. Overall, our studies are significant in that they will advance our knowledge of T cell migration by providing new data to determine the mechanism of action of Formins in T cell motility and if they cooperate with Myosin-IIA to promote migration through environments with varied biophysical characteristics. Thus, this work has the potential to provide important insight into novel ways to therapeutically modulate lymphocyte migration, such as in autoimmune settings and inflammatory diseases.