We have continued analysis of changes in T lymphocyte cytoskeleton which are essential to T cell migration and recognition; we have found novel and conceptually important remodeling of intermediate filaments and associated proteins. Two model systems of acute activation are being investigated: a) T cell interaction with anti-CD3-coated beads, as a simplified model in which to understand in detail what happens when T cells encounter antigen-positive cells; and b) T cell stimulation by chemokines which regulate their adhesion and migration. In response to chemokine, there is a quick multiphasic cytoskeletal response. First there is a dramatic polymerization of cortical actin (5-30 seconds). This is followed by a rapid actin depolymerization and by cell polarization involving condensation of vimentin into a uropod. During this response there is a transient increase in vimentin solubility followed by a marked decrease, consistent with the concept that existing intermediate filaments are being partially disassembled and then assembled in the uropod. Several other large cytoskeletal proteins become localized to the uropod and cleared from the remainder of the cell periphery demonstrating the extent of cytoskeletal reorganization. Functional implications of this reorganization are currently under investigation. Studies with a range of inhibitors of serine/theonine phosphorylation (or dephosphorylation) profoundly change the process, indicating a critical role of such phosphorylation in the process; they suggest a role for members of the protein kinase C family (PKC). Details of these phosphorylation changes are being studied. Many of the same processes of intermediate filament reorganization are observed in the model system with CD3 stimulation suggesting a broad relevance of intermediate filament remodeling in T cell activation. Because of the putative importance in hematopoietic cells of members of the PKC family, we have begun a detailed structural and functional analysis, emphasizing the novel PKCs (nPKCs) delta and theta. We have investigated PKC translocation both by microscopic analysis of distribution in whole cells, and by biochemical characterization of PKC transfer between cytosolic and membrane fractions. Similarities and differences are being studied in two model systems of receptor-driven activation of hematopoietic cells: CD3-mediated activation of JurkatT cell line and FcERI-mediated activation of RBL mast cell line. In contrast to the published literature, both CD3-delta and theta translocate rapidly (microscopically and biochemically) in response to CD3 activation without requiring a costimulatory signal. In contrast to the nPKCs, the classical PKCs (alpha, beta) undergo much slower and more limited translocation. Furthermore, translocation following PMA treatment is much slower than by T cell receptor activation. The pattern of isoform translocation in response to CD3 in Jurkat is more restricted than the pattern for FcERI in RBL. These findings do not easily fit into the paradigms established for regulated translocation of classical PKCs. Therefore we are undertaking a systematic analysis of the structural features of novel PKCs which regulate translocation; our analysis has started with domain swap and domain deletion constructs and will continue with targeted sites within domains. Our continuing studies of lymph node architecture indicate that the T cell-dependent area of lymph node is a much more highly structured micro-environment than had previously been proposed We have established that there is a spatial boundary, provided by sinus lining cells, which is a barrier to entry both of cells, and of molecules into the T cell area. Penetration of molecules is influenced by their molecular weight: there is a cutoff of approximately 80kd. which allows entry of low molecular weight molecules, including chemokines. Once they have penetrated the barrier, tracers do not appear to diffuse freely, but instead move preferentially via structures which we call ?conduits? which are collagen fibers bounded by fibroblastic reticular cells. Those conduits, connect to the basement membrane of high endothelial venules, which is an abluminal potential space called the ?perivenular channel?. Although it has been more technically difficult than expected, we have now been able to directly demonstrate that the chemokines given intralymphatically follows this route and thereby rapidly reach high endothelial venules, through which lymphocytes migrate. This has important implications regarding regulation of lymphocyte trafficking.
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