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.

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.

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 longrange transport. Here, we show that the SPIRE-type actin nucleators (predominantly SPIRE1) are Rab27a effectors that cooperate 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.

Located in the basal epidermis and hair follicles, melanocytes of the integument are responsible for its coloration through production of melanin pigments. Melanin is produced in lysosomal‐like organelles called melanosomes. In humans, this skin pigmentation acts as an ultraviolet radiation filter. Abnormalities in the division of melanocytes are quite common, with potentially oncogenic growth usually followed by cell senescence producing benign naevi (moles), or occasionally melanoma. Therefore, melanocytes are a useful model for studying melanoma, as well as pigmentation and organelle transport and the diseases affecting these mechanisms. This chapter focuses on the isolation, culture, and transfection of human and murine melanocytes. The first basic protocol describes the primary culture of melanocytes from human skin and the maintenance of growing cultures. The second basic protocol details the subculture and preparation of mouse keratinocyte feeder cells. The primary culture of melanocytes from mouse skin is described in the third basic protocol, and, lastly, the fourth basic protocol outlines a technique for transfecting melanocytes and melanoma cells. Curr. Protoc. Cell Biol. 63:1.8.1‐1.8.20. © 2014 by John Wiley & Sons, Inc.

This 'highways and local roads' model suggests that MTs are tracks for long-range transport (highways) between the cell centre and periphery, driven by kinesin and dynein motors. Meanwhile AFs (local roads) and myosin motors work down-stream picking up cargo at the periphery and transporting it for the 'last m' to its final destination. This model makes intuitive sense as MTs in animal cells in culture typically form a polarised radial network of tracks spanning >10 m from the centrally located centrosome to the periphery and appear ideally distributed for long-distance transport. Meanwhile, with some exceptions in which AFs form uniformly polarised arrays, e.g. lamellipodia, filopodia and dendritic spines, AF architecture appears much more complex. In many fixed cells AF appear to comprise populations of short (1-2 m length), with random or anti-parallel filament polarity, and not an obvious system of tracks for directed transport 5,6 . This view is exemplified by the co-operative capture (CC) model of melanosome transport in melanocytes 7,8 . Skin melanocytes make pigmented melanosomes and then distribute them, via dendrites, to adjacent keratinocytes, thus providing pigmentation and photo-protection (reviewed in 9 ). The CC model proposes that transport of melanosomes into dendrites occurs by sequential longdistance transport from the cell body into dendrites along MTs (propelled by kinesin/dynein motors), followed by AF/myosin-Va dependent tethering in the dendrites. Consistent with this, in myosin-Va-null cells melanosomes move bi-directionally along MTs into dendrites, but do not accumulate therein, and instead cluster in the cell body 7,10 . This defect results in partial albinism in mammals due to uneven pigment transfer from melanocytes to keratinocytes (e.g. dilute mutant mouse and human Griscelli syndrome (GS) type I patients; Figure 1A) 11,12 . Subsequent studies revealed similar defects in mutant mice (and human GS types II and III patients) lacking the small