RESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Interacciones Huésped-Patógeno/fisiología , Fosfatos de Fosfatidilinositol/metabolismo , Salmonella typhimurium/metabolismo , Factores de Virulencia/metabolismo , Proteínas Bacterianas/genética , Membrana Celular/metabolismo , Membrana Celular/microbiología , Células HeLa/microbiología , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Infecciones por Salmonella/metabolismo , Infecciones por Salmonella/microbiología , Salmonella typhimurium/patogenicidad , Vacuolas/metabolismo , Factores de Virulencia/genéticaRESUMEN
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. Typhimurium, cells infected by ΔsteA mutant bacteria displayed fewer Salmonella-induced filaments (SIFs), an increased clustering of SCVs, and morphologically abnormal vacuoles containing more than one bacterium. The increased clustering of SCVs and the appearance of vacuoles containing more than one bacterium were suppressed by inhibition of the activity of the microtubule motor dynein or kinesin-1. Clustering and positioning of SCVs are controlled by the effectors SseF and SseG, possibly by helping to maintain a balanced activity of microtubule motors on the bacterial vacuoles. Deletion of steA in S. Typhimurium ΔsseF or ΔsseG mutants revealed that SteA contributes to the characteristic scattered distribution of ΔsseF or ΔsseG mutant SCVs in infected cells. Overall, this shows that SteA participates in the control of SCV membrane dynamics. Moreover, it indicates that SteA is functionally linked to SseF and SseG and suggests that it might contribute directly or indirectly to the regulation of microtubule motors on the bacterial vacuoles.
Asunto(s)
Proteínas Bacterianas/metabolismo , Salmonella typhimurium/metabolismo , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Línea Celular , Dineínas , Regulación de la Expresión Génica/inmunología , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Ratones , Mutación , Factores de Virulencia/genéticaRESUMEN
Endospores 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.
Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas/fisiología , Proteínas de la Membrana/fisiología , Sustitución de Aminoácidos , Bacillus subtilis/citología , Bacillus subtilis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia Conservada/genética , ADN Bacteriano/análisis , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Morfogénesis/genética , Mutación , Biblioteca de Péptidos , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Esporas Bacterianas/química , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
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.
Asunto(s)
Caveolas/metabolismo , Queratinocitos/metabolismo , Melanocitos/metabolismo , Pigmentación de la Piel/fisiología , Piel/metabolismo , Caveolina 1/metabolismo , Comunicación Celular/fisiología , Comunicación Celular/efectos de la radiación , Células Cultivadas , Técnicas de Cocultivo , Células Epidérmicas/metabolismo , Epidermis/metabolismo , Epidermis/ultraestructura , Células HeLa , Humanos , Queratinocitos/citología , Melanocitos/citología , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Transducción de Señal/fisiología , Transducción de Señal/efectos de la radiación , Piel/citología , Piel/ultraestructura , Rayos UltravioletaRESUMEN
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.