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Neuronal and non-neuronal tissues show distinctly different intracellular localization of synaptotagmin (Syt) homologues. Therefore, cell type-specific mechanisms are likely to direct Syt homologues to their final cellular destinations. Syt IX localizes to dense core vesicles in PC12 cells. However, in the rat basophilic leukemia (RBL-2H3) mast cell line, as well as in CHO cells, Syt IX is localized at the endocytic recycling compartment (ERC). We show that targeting of Syt IX to the ERC involves constitutive trafficking to the plasma membrane followed by internalization and transport to the ERC. We further show that internalization from the plasma membrane and delivery to the ERC are dependent on phosphorylation by Ca2+-dependent protein kinase Cα or β. As such, correct targeting of Syt IX is facilitated by the phorbol ester TPA but prevented by the cPKC inhibitor Go 6976.

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Background: Actin remodeling is a key regulator of mast cell (MC) migration and secretion. However, the precise mechanism underlying the coordination of these processes has remained obscure. Objective: We sought to characterize the actin rearrangements that occur during MC secretion or chemotactic migration and identify the underlying mechanism of their coordination. Methods: Using high-resolution microscopy, we analyzed the dynamics of actin rearrangements in MCs triggered to migration by IL-8 or prostaglandin E 2 or to FcεRI-stimulated secretion. Results: We show that a major feature of the actin skeleton in MCs stimulated to migration is the buildup of pericentral actin clusters that prevent cell flattening and converge the secretory granules (SGs) in the cell center. This migratory phenotype is replaced on encounter of an IgE cross-linking antigen that stimulates secretion through a secretory phenotype characterized by cell flattening, reduction of actin mesh density, ruffling of cortical actin, and mobilization of SGs. Furthermore, we show that knockdown of mammalian diaphanous-related formin 1 (mDia1) inhibits chemotactic migration and its typical actin rearrangements, whereas expression of an active mDia1 mutant recapitulates the migratory actin phenotype and enhances cell migration while inhibiting FcεRI-triggered secretion. However, mice deficient in mDia1 appear to have normal numbers of MCs in various organs at baseline. Conclusion: Our results demonstrate a unique role of actin rearrangements in clustering the SGs and inhibiting their

The secretory granules (SGs) of secretory cells of the hematopoietic lineage, such as the mast cells, are lysosomerelated organelles whose membrane proteins travel through the plasma membrane and the endocytic system. Therefore, a mechanism must exist to prevent proteins destined to recycling or to the trans-Golgi network (TGN) from reaching the SGs. We now show that synaptotagmin (