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© The Author(s) 2020., Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that the motor protein myosin-Va works with dynamic actin tracks to drive long-range organelle dispersion in opposition to microtubules. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here, we show that the SPIRE-type actin nucleators (predominantly SPIRE1) are Rab27a effectors that co-operate with formin-1 to generate actin tracks required for myosin-Va-dependent transport in melanocytes. Thus, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIREs to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this, we suggest a model in which organelles and force generators (motors and track assemblers) are linked, forming an organelle-based, cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm., This work was supported by a Medical Research Council New Investigator Award to A.N.H. (grant reference G1100063), Wellcome Trust Grant Awards (grant reference 108429/Z/15/Z to E.V.S. and 204843/Z/16/Z to A.N.H.), a Biotechnology and Biological Sciences Research Council (grant reference BB/F016956/1) and University of Nottingham funded PhD studentship awarded to C.L.R. D.A.B. was funded by Biochemical Society Vacation Studentship. L.M. is funded through the Spanish Ministry of Economy. Industry and Competitiveness (MINECO) [BIO2015-70978-R]. E.K. and T.W. are funded by DFG SPP1464, KE 447/10-2” and DFG KE 447/18-1. F.S. is funded by DFG Research Training Group, GRK 2174 Award. N.A. is supported by a PhD studentship from Taibah University, Medina, Kingdom of Saudi Arabia. W.W.K. and E.W.T. are funded by The Royal Society (Newton International Fellowship grant NF161582 to W.W.K.), European Commission (Marie Sklodowska Curie Individual Fellowship grant 752165 to W.W.K.), and Cancer Research UK (C29637/A20183 to E.W.T.). A.K.M. acknowledges financial support of the John and Lucille van Geest Foundation.

Dennis et al. analyze cycling of the v-SNARE VAMP7 during melanosome biogenesis in melanocytes. VAMP7 is targeted to and retrieved from maturing melanosomes in separate tubular carriers whose formation requires distinct BLOCs, each defective in variants of Hermansky–Pudlak syndrome., Endomembrane organelle maturation requires cargo delivery via fusion with membrane transport intermediates and recycling of fusion factors to their sites of origin. Melanosomes and other lysosome-related organelles obtain cargoes from early endosomes, but the fusion machinery involved and its recycling pathway are unknown. Here, we show that the v-SNARE VAMP7 mediates fusion of melanosomes with tubular transport carriers that also carry the cargo protein TYRP1 and that require BLOC-1 for their formation. Using live-cell imaging, we identify a pathway for VAMP7 recycling from melanosomes that employs distinct tubular carriers. The recycling carriers also harbor the VAMP7-binding scaffold protein VARP and the tissue-restricted Rab GTPase RAB38. Recycling carrier formation is dependent on the RAB38 exchange factor BLOC-3. Our data suggest that VAMP7 mediates fusion of BLOC-1–dependent transport carriers with melanosomes, illuminate SNARE recycling from melanosomes as a critical BLOC-3–dependent step, and likely explain the distinct hypopigmentation phenotypes associated with BLOC-1 and BLOC-3 deficiency in Hermansky–Pudlak syndrome variants.

Dynamic mapping of extracellular pH (pHe) at the single-cell level is critical for understanding the role of H+ in cellular and subcellular processes, with particular importance in cancer. While several pHe sensing techniques have been developed, accessing this information at the single-cell level requires improvement in sensitivity, spatial and temporal resolution. We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensitivity > 0.01 units, 2 ms response time, and 50 nm spatial resolution. The platform was integrated into a double-barrel nanoprobe combining pH sensing with feedback-controlled distance dependance via Scanning Ion Conductance Microscopy. This allows for the simultaneous 3D topographical imaging and pHe monitoring of living cancer cells. These classes of nanoprobes were used for real-time high spatiotemporal resolution pHe mapping at the subcellular level and revealed tumour heterogeneity of the peri-cellular environments of melanoma and breast cancer cells., Current methods to measure extracellular pH are often limited in resolution and response times. Here the authors present a label-free nanoprobe, consisting of a zwitterionic nanomembrane at the tip of a nanopipette, which enables high spatiotemporal resolution pH measurements and topography-pH 3D mapping in live cancer cells.