SummaryEndospores formed by Bacillus subtilis are encased in a tough protein shell known as the coat, which consists of at least 70 different proteins. We investigated the process of spore coat morphogenesis using a library of 40 coat proteins fused to green fluorescent protein and demonstrate that two successive steps can be distinguished in coat assembly. The first step, initial localization of proteins to the spore surface, is dependent on the coat morphogenetic proteins SpoIVA and SpoVM. The second step, spore encasement, requires a third protein, SpoVID. We show that in spoVID mutant cells, most coat proteins assembled into a cap at one side of the developing spore but failed to migrate around and encase it. We also found that SpoIVA directly interacts with SpoVID. A domain analysis revealed that the N-terminus of SpoVID is required for encasement and is a structural homologue of a virion protein, whereas the C-terminus is necessary for the interaction with SpoIVA. Thus, SpoVM, SpoIVA and SpoVID are recruited to the spore surface in a concerted manner and form a tripartite machine that drives coat formation and spore encasement.

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.

Many bacterial pathogens use specialized secretion systems to deliver virulence effector proteins into eukaryotic host cells. The function of these effectors depends on their localization within infected cells, but the mechanisms determining subcellular targeting of each effector are mostly elusive. Here, we show that the Salmonella type III secretion effector SteA binds specifically to phosphatidylinositol 4-phosphate [PI(4)P]. Ectopically expressed SteA localized at the plasma membrane (PM) of eukaryotic cells. However, SteA was displaced from the PM of Saccharomyces cerevisiae in mutants unable to synthesize the local pool of PI(4)P and from the PM of HeLa cells after localized depletion of PI(4)P. Moreover, in infected cells, bacterially translocated or ectopically expressed SteA localized at the membrane of the Salmonella-containing vacuole (SCV) and to Salmonella-induced tubules; using the PI(4)P-binding domain of the Legionella type IV secretion effector SidC as probe, we found PI(4)P at the SCV membrane and associated tubules throughout Salmonella infection of HeLa cells. Both binding of SteA to PI(4)P and the subcellular localization of ectopically expressed or bacterially translocated SteA were dependent on a lysine residue near the N-terminus of the protein. Overall, this indicates that binding of SteA to PI(4)P is necessary for its localization within host cells.

Salmonella enterica serovar Typhimurium is a bacterial pathogen causing gastroenteritis in humans and a typhoid-like systemic disease in mice. S. Typhimurium virulence is related to its capacity to multiply intracellularly within a membrane-bound compartment, the Salmonella-containing vacuole (SCV), and depends on type III secretion systems that deliver bacterial effector proteins into host cells. Here, we analyzed the cellular function of the Salmonella effector SteA. We show that, compared to cells infected by wild-type S.

Tissue homeostasis requires regulation of cell-cell communication, which relies on signaling molecules and cell contacts. In skin epidermis, keratinocytes secrete factors transduced by melanocytes into signaling cues promoting their pigmentation and dendrite outgrowth, while melanocytes transfer melanin pigments to keratinocytes to convey skin photoprotection. How epidermal cells integrate these functions remains poorly characterized. Here, we show that caveolae are asymmetrically distributed in melanocytes and particularly abundant at the melanocyte-keratinocyte interface in epidermis. Caveolae in melanocytes are modulated by ultraviolet radiations and keratinocytes-released factors, like miRNAs. Preventing caveolae formation in melanocytes increases melanin pigment synthesis through upregulation of cAMP signaling and decreases cell protrusions, cell-cell contacts, pigment transfer and epidermis pigmentation. Altogether, we identify that caveolae serve as molecular hubs that couple signaling outputs from keratinocytes to mechanical plasticity of pigment cells. The coordination of intercellular communication and contacts by caveolae is thus crucial to skin pigmentation and tissue homeostasis.

22Tissue homeostasis requires regulation of cell-cell communication, which relies on signaling 23 molecules and cell contacts. In skin epidermis, keratinocytes secrete specific factors transduced 24 by melanocytes into signaling cues to promote their pigmentation and dendrite outgrowth, while 25 melanocytes transfer melanin pigments to keratinocytes to convey skin photoprotection. How 26 epidermal cells integrate these functions remains poorly characterized. Here, we found that 27 caveolae polarize in melanocytes and are particularly abundant at melanocyte-keratinocyte 28 interface. Caveolae in melanocytes are sensitive to ultra-violet radiations and miRNAs released 29 by keratinocytes. Preventing caveolae formation in melanocytes results in increased production 30 of intracellular cAMP and melanin pigments, but decreases cell protrusions, cell-cell contacts, 31 pigment transfer and epidermis pigmentation. Altogether, our data establish that, in 32 melanocytes, caveolae serve as key molecular hubs that couple signaling outputs from 33 keratinocytes to mechanical plasticity. This process is crucial to maintain cell-cell contacts and 34 intercellular communication, skin pigmentation and tissue homeostasis. 84Epidermal melanocytes and keratinocytes are in constant communication, not only via secreted 85 factors and exosomes that modulate cellular responses, but also by the physical contacts they 86 establish to maintain the tissue homeostasis and pigmentation. Here, we report a new function 87 for caveolae, which, by integrating the biochemical and mechanical behavior of melanocytes, 88 control melanin transfer to keratinocytes and epidermis pigmentation. Altogether, this study 89 provides the first evidence for a physiologic role of caveolae as a molecular sensing platform 90 required for the homeostasis of the largest human tissue, the skin epidermis. 91 5 Results 92 Caveolae polarize in melanocytes and are positively-regulated by keratinocytes-secreted 93 factors 94Melanocytes and keratinocytes establish a complex intercellular dialogue required for skin 95 photoprotection. 2D-co-culture systems, where these two cell types share the same medium, 96have been widely used to study intercellular communication and pigment transfer between 97 epidermal cells (Hirobe, 2005; Lei et al., 2002). To evaluate the distribution of caveolae within 98 the epidermal unit in 2D, normal human melanocytes and keratinocytes were co-cultured and 99 labelled for the two constituents of caveolae, Cav1 or Cavin1. Immunofluorescence microscopy 100 revealed that both Cav1 and Cavin1, and therefore caveolae, were asymmetrically distributed in 101 melanocytes (Figures 1A and B), which were identified by the abundant staining of the 102 premelanosome protein PMEL [hereafter referred as melanin, see Experimental Procedures; 103 (Raposo et al., 2001)]. This polarization was not observed in keratinocytes.104 Cells can break their symmetry in response to local external chemical and/or mechanical cues 105 such as signaling molecules and/or cell-ce...