Membrane protrusion powers clathrin-independent endocytosis of interleukin-2 receptor.

Endocytosis controls many functions including nutrient uptake, cell division, migration and signal transduction. A clathrin- and caveolin-independent endocytosis pathway is used by important physiological cargos, including interleukin-2 receptors (IL-2R). However, this process lacks morphological and dynamic data. Our electron microscopy (EM) and tomography studies reveal that IL-2R-pits and vesicles are initiated at the base of protrusions. We identify the WAVE complex as a specific endocytic actor. The WAVE complex interacts with IL-2R, via a WAVE-interacting receptor sequence (WIRS) present in the receptor polypeptide, and allows for receptor clustering close to membrane protrusions. In addition, using total internal reflection fluorescent microscopy (TIRF) and automated analysis we demonstrate that two timely distinct bursts of actin polymerization are required during IL-2R uptake, promoted first by the WAVE complex and then by N-WASP. Finally, our data reveal that dynamin acts as a transition controller for the recruitment of Arp2/3 activators required for IL-2R endocytosis. Altogether, our work identifies the spatio-temporal specific role of factors initiating clathrin-independent endocytosis by a unique mechanism that does not depend on the deformation of a flat membrane, but rather on that of membrane protrusions.

AP-1/KIF13A Blocking Peptides Impair Melanosome Maturation and Melanin Synthesis.

Melanocytes are specialized cells that generate unique organelles called melanosomes in which melanin is synthesized and stored. Melanosome biogenesis and melanocyte pigmentation require the transport and delivery of melanin synthesizing enzymes, such as tyrosinase and related proteins (e.g., TYRP1), from endosomes to maturing melanosomes. Among the proteins controlling endosome-melanosome transport, AP-1 together with KIF13A coordinates the endosomal sorting and trafficking of TYRP1 to melanosomes. We identify here ß1-adaptin AP-1 subunit-derived peptides of 5 amino acids that block the interaction of KIF13A with AP-1 in cells. Incubating these peptides with human MNT-1 cells or 3D-reconstructed pigmented epidermis decreases pigmentation by impacting the maturation of melanosomes in fully pigmented organelles. This study highlights that peptides targeting the intracellular trafficking of melanocytes are candidate molecules to tune pigmentation in health and disease.

Spatial organization of adenylyl cyclase and its impact on dopamine signaling in neurons.

The cAMP cascade is widely recognized to transduce its physiological effects locally through spatially limited cAMP gradients. However, little is known about how the adenylyl cyclase enzymes, which initiate cAMP gradients, are localized. Here we answer this question in physiologically relevant striatal neurons and delineate how AC localization impacts downstream signaling functions. We show that the major striatal AC isoforms are differentially sorted between ciliary and extraciliary domains of the plasma membrane, and that AC9 is uniquely targeted to endosomes. We identify key sorting determinants in the N-terminal cytoplasmic domain responsible for isoform-specific localization. We also show that AC9-containing endosomes accumulate activated dopamine receptors and form an elaborately intertwined network with juxtanuclear PKA stores bound to Golgi membranes. Finally, we show that endosomal localization is critical for AC9 to selectively elevate PKA activity in the nucleus relative to the cytoplasm. These results reveal a precise spatial landscape of the cAMP cascade in neurons and a key role of AC localization in directing downstream signal transduction to the nucleus.

Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers.

Vesicular and tubular transport intermediates regulate organellar cargo dynamics. Transport carrier release involves local and profound membrane remodeling before fission. Pinching the neck of a budding tubule or vesicle requires mechanical forces, likely exerted by the action of molecular motors on the cytoskeleton. Here, we show that myosin VI, together with branched actin filaments, constricts the membrane of tubular carriers that are then released from melanosomes, the pigment containing lysosome-related organelles of melanocytes. By combining superresolution fluorescence microscopy, correlative light and electron microscopy, and biochemical analyses, we find that myosin VI motor activity mediates severing by constricting the neck of the tubule at specific melanosomal subdomains. Pinching of the tubules involves the cooperation of the myosin adaptor optineurin and the activity of actin nucleation machineries, including the WASH and Arp2/3 complexes. The fission and release of these tubules allows for the export of components from melanosomes, such as the SNARE VAMP7, and promotes melanosome maturation and transfer to keratinocytes. Our data reveal a new myosin VI- and actin-dependent membrane fission mechanism required for organelle function.

BLOC-1 Brings Together the Actin and Microtubule Cytoskeletons to Generate Recycling Endosomes.

Recycling endosomes consist of a tubular network that emerges from vacuolar sorting endosomes and diverts cargoes toward the cell surface, the Golgi, or lysosome-related organelles. How recycling tubules are formed remains unknown. We show that recycling endosome biogenesis requires the protein complex BLOC-1. Mutations in BLOC-1 subunits underlie an inherited disorder characterized by albinism, the Hermansky-Pudlak Syndrome, and are associated with schizophrenia risk. We show here that BLOC-1 coordinates the kinesin KIF13A-dependent pulling of endosomal tubules along microtubules to the Annexin A2/actin-dependent stabilization and detachment of recycling tubules. These components cooperate to extend, stabilize and form tubular endosomal carriers that function in cargo recycling and in the biogenesis of pigment granules in melanocytic cells. By shaping recycling endosomal tubules, our data reveal that dysfunction of the BLOC-1-KIF13A-Annexin A2 molecular network underlies the pathophysiology of neurological and pigmentary disorders.