Melanosomes are a type of lysosome-related organelle that is commonly defective in Hermansky–Pudlak syndrome. Biogenesis of melanosomes is regulated by BLOC-1, -2, -3, or AP-1, -3 complexes, which mediate cargo transport from recycling endosomes to melanosomes. Although several Rab GTPases have been shown to regulate these trafficking steps, the precise role of Rab9A remains unknown. Here, we found that a cohort of Rab9A associates with the melanosomes and its knockdown in melanocytes results in hypopigmented melanosomes due to mistargeting of melanosomal proteins to lysosomes. In addition, the Rab9A-depletion phenotype resembles Rab38/32-inactivated or BLOC-3-deficient melanocytes, suggesting that Rab9A works in line with BLOC-3 and Rab38/32 during melanosome cargo transport. Furthermore, silencing of Rab9A, Rab38/32 or its effector VARP, or BLOC-3-deficiency in melanocytes decreased the length of STX13-positive recycling endosomal tubules and targeted the SNARE to lysosomes. This result indicates a defect in directing recycling endosomal tubules to melanosomes. Thus, Rab9A and its co-regulatory GTPases control STX13-mediated cargo delivery to maturing melanosomes.

CTLs are serial killers that kill multiple target cells via exocytosis of cytotoxic granules (CGs). CG exocytosis is tightly regulated and has been investigated in great detail; however, whether CG proteins are endocytosed following exocytosis and contribute to serial killing remains unknown. By using primary CTLs derived from a knock-in mouse of the CG membrane protein Synaptobrevin2, we show that CGs are endocytosed in a clathrin- and dynamin-dependent manner. Following acidification, endocytosed CGs are recycled through early and late, but not recycling endosomes. CGs are refilled with granzyme B at the late endosome stage and polarize to subsequent synapses formed between the CTL and new target cells. Importantly, inhibiting CG endocytosis in CTLs results in a significant reduction of their cytotoxic activity. Thus, our data demonstrate that continuous endocytosis of CG membrane proteins is a prerequisite for efficient serial killing of CTLs and identify key events in this process.

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Cytotoxic T lymphocytes kill infected or malignant cells through the directed release of cytotoxic substances at the site of target cell contact, the immunological synapse. While genetic association studies of genes predisposing to early-onset life-threatening hemophagocytic lymphohistiocytosis has identified components of the plasma membrane fusion machinery, the identity of the vesicular components remain enigmatic. Here, we identify VAMP7 as an essential component of the vesicular fusion machinery of primary, human T cells. VAMP7 co-localizes with granule markers throughout all stages of T cell maturation and simultaneously fuses with granule markers at the IS. Knock-down of VAMP7 expression significantly decreased the killing efficiency of T cells, without diminishing early T cell receptor signaling. VAMP7 exerts its function in a SNARE complex with Syntaxin11 and SNAP-23 on the plasma membrane. The identification of the minimal fusion machinery in T cells provides a starting point for the development of potential drugs in immunotherapy.

Cytotoxic T lymphocytes patrol our body in search for infected cells which they kill through the release of cytotoxic substances contained in cytotoxic granules. The fusion of cytotoxic granules occurs at a specially formed contact site, the immunological synapse, and is tightly controlled to ensure specificity. In this review, we discuss the contribution of two intracellular compartments, endosomes and cytotoxic granules, to the formation, function and disassembly of the immunological synapse. We highlight a recently proposed sequential process of fusion events at the IS upon target cell recognition. First, recycling endosomes fuse with the plasma membrane to deliver cargo required for the docking of cytotoxic granules. Second, cytotoxic granules arrive and fuse upon docking in a SNARE-dependent manner. Following fusion, membrane components of the cytotoxic granule are retrieved through endocytosis to ensure the fast, efficient serial killing of target cells that is characteristic of cytotoxic T lymphocytes.