RESUMEN
Adaptor protein 2 (AP2) is a major constituent of clathrin-coated pits (CCPs). Whether it is essential for all forms of clathrin-mediated endocytosis (CME) in mammalian cells is an open issue. Here, we demonstrate, by live TIRF microscopy, the existence of a subclass of relatively short-lived CCPs lacking AP2 under physiological, unperturbed conditions. This subclass is retained in AP2-knockout cells and is able to support the internalization of epidermal growth factor receptor (EGFR) but not of transferrin receptor (TfR). The AP2-independent internalization mechanism relies on the endocytic adaptors eps15, eps15L1, and epsin1. The absence of AP2 impairs the recycling of the EGFR to the cell surface, thereby augmenting its degradation. Accordingly, under conditions of AP2 ablation, we detected dampening of EGFR-dependent AKT signaling and cell migration, arguing that distinct classes of CCPs could provide specialized functions in regulating EGFR recycling and signaling.
Asunto(s)
Vesículas Cubiertas por Clatrina/fisiología , Transducción de Señal , Complejo 2 de Proteína Adaptadora/antagonistas & inhibidores , Complejo 2 de Proteína Adaptadora/genética , Complejo 2 de Proteína Adaptadora/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/antagonistas & inhibidores , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Línea Celular , Movimiento Celular , Endocitosis , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Edición Génica , Células HeLa , Humanos , Ratones , Microscopía Fluorescente , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Activación TranscripcionalRESUMEN
Membrane remodeling is an essential part of transferring components to and from the cell surface and membrane-bound organelles and for changes in cell shape, which are particularly critical during cell division. Earlier analyses, based on classical optical live-cell imaging and mostly restricted by technical necessity to the attached bottom surface, showed persistent formation of endocytic clathrin pits and vesicles during mitosis. Taking advantage of the resolution, speed, and noninvasive illumination of the newly developed lattice light-sheet fluorescence microscope, we reexamined their assembly dynamics over the entire cell surface and found that clathrin pits form at a lower rate during late mitosis. Full-cell imaging measurements of cell surface area and volume throughout the cell cycle of single cells in culture and in zebrafish embryos showed that the total surface increased rapidly during the transition from telophase to cytokinesis, whereas cell volume increased slightly in metaphase and was relatively constant during cytokinesis. These applications demonstrate the advantage of lattice light-sheet microscopy and enable a new standard for imaging membrane dynamics in single cells and multicellular assemblies.
Asunto(s)
Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Animales , Ciclo Celular , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Clatrina/metabolismo , Citocinesis/fisiología , Endosomas/metabolismo , Metafase , Microscopía/métodos , Mitosis/fisiología , Pez Cebra/embriologíaRESUMEN
Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.
Asunto(s)
Citoesqueleto/ultraestructura , Endocitosis , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Orgánulos/ultraestructura , Actinina/análisis , Actinas/análisis , Animales , Línea Celular , Clatrina/análisis , Vesículas Cubiertas por Clatrina/química , Vesículas Cubiertas por Clatrina/ultraestructura , Invaginaciones Cubiertas de la Membrana Celular/química , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Citoesqueleto/química , Citoesqueleto/metabolismo , Endosomas/química , Endosomas/ultraestructura , Aparato de Golgi/ultraestructura , Procesamiento de Imagen Asistido por Computador , Imagenología Tridimensional/instrumentación , Microscopía Fluorescente/instrumentación , Mitocondrias/química , Mitocondrias/ultraestructura , Orgánulos/química , Orgánulos/metabolismo , Proteínas de Unión al GTP rab5/análisisRESUMEN
Batrachochytrium dendrobatidis (Bd), a chytrid fungus, has increasingly been implicated as a major factor in the worldwide decline of amphibian populations. The fungus causes chytridiomycosis in susceptible species leading to massive die-offs of adult amphibians. Although Bd infects the keratinized mouthparts of tadpoles and negatively affects foraging behavior, these infections are non-lethal. An important morphogen controlling amphibian metamorphosis is thyroid hormone (T3). Tadpoles may be infected with Bd and the fungus may be exposed to T3 during metamorphosis. We hypothesize that exposure of Bd to T3 may induce the expression of factors associated with host colonization and pathogenicity. We utilized a proteomics approach to better understand the dynamics of the Bd-T3 interaction. Using liquid chromatography-mass spectrometry (LC-MS), we generated a data set of a large number of cytoplasmic and membrane proteins following exposure of Bd to T3. From these data, we identified a total of 263 proteins whose expression was significantly changed following T3 exposure. We provide evidence for expression of an array of proteins that may play key roles in both genomic and non-genomic actions of T3 in Bd. Additionally, our proteomics study shows an increase in several proteins including proteases and a class of uncommon crinkler and crinkler-like effector proteins suggesting their importance in Bd pathogenicity as well as those involved in metabolism and energy transfer, protein fate, transport and stress responses. This approach provides insights into the mechanistic basis of the Bd-amphibian interaction following T3 exposure.
Asunto(s)
Quitridiomicetos/efectos de los fármacos , Proteómica , Triyodotironina/farmacología , Animales , Anuros/microbiología , Transporte Biológico/efectos de los fármacos , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Quitridiomicetos/aislamiento & purificación , Quitridiomicetos/metabolismo , Electroforesis , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Péptidos/análisis , Espectrometría de Masas en Tándem , Tripsina/metabolismoRESUMEN
Clathrin-dependent endocytosis is an essential cellular process shared by all cell types. Despite this, precisely how endocytosis is regulated in a cell-type-specific manner and how this key pathway functions physiologically or pathophysiologically remain largely unknown. PICALM, which encodes the clathrin adaptor protein PICALM, was originally identified as a component of the CALM/AF10 leukemia oncogene. Here we show, by employing a series of conditional Picalm knockout mice, that PICALM critically regulates transferrin uptake in erythroid cells by functioning as a cell-type-specific regulator of transferrin receptor endocytosis. While transferrin receptor is essential for the development of all hematopoietic lineages, Picalm was dispensable for myeloid and B-lymphoid development. Furthermore, global Picalm inactivation in adult mice did not cause gross defects in mouse fitness, except for anemia and a coat color change. Freeze-etch electron microscopy of primary erythroblasts and live-cell imaging of murine embryonic fibroblasts revealed that Picalm function is required for efficient clathrin coat maturation. We showed that the PICALM PIP2 binding domain is necessary for transferrin receptor endocytosis in erythroblasts and absolutely essential for erythroid development from mouse hematopoietic stem/progenitor cells in an erythroid culture system. We further showed that Picalm deletion entirely abrogated the disease phenotype in a Jak2(V617F) knock-in murine model of polycythemia vera. Our findings provide new insights into the regulation of cell-type-specific transferrin receptor endocytosis in vivo. They also suggest a new strategy to block cellular uptake of transferrin-bound iron, with therapeutic potential for disorders characterized by inappropriate red blood cell production, such as polycythemia vera.
Asunto(s)
Hematopoyesis/genética , Proteínas de Ensamble de Clatrina Monoméricas/genética , Policitemia Vera/genética , Anemia Hipocrómica/genética , Animales , Diferenciación Celular , Vesículas Cubiertas por Clatrina/metabolismo , Vesículas Cubiertas por Clatrina/ultraestructura , Modelos Animales de Enfermedad , Endocitosis , Eritroblastos/metabolismo , Eritroblastos/ultraestructura , Eritropoyesis/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Inmunofenotipificación , Linfopoyesis/genética , Ratones , Ratones Noqueados , Proteínas de Ensamble de Clatrina Monoméricas/química , Proteínas de Ensamble de Clatrina Monoméricas/deficiencia , Proteínas de Ensamble de Clatrina Monoméricas/metabolismo , Mielopoyesis/genética , Fenotipo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Policitemia Vera/mortalidad , Dominios y Motivos de Interacción de Proteínas , Receptores de Transferrina/metabolismoRESUMEN
Cell entry by non-enveloped viruses requires translocation into the cytosol of a macromolecular complex--for double-strand RNA viruses, a complete subviral particle. We have used live-cell fluorescence imaging to follow rotavirus entry and penetration into the cytosol of its â¼ 700 Å inner capsid particle ("double-layered particle", DLP). We label with distinct fluorescent tags the DLP and each of the two outer-layer proteins and track the fates of each species as the particles bind and enter BSC-1 cells. Virions attach to their glycolipid receptors in the host cell membrane and rapidly become inaccessible to externally added agents; most particles that release their DLP into the cytosol have done so by â¼ 10 minutes, as detected by rapid diffusional motion of the DLP away from residual outer-layer proteins. Electron microscopy shows images of particles at various stages of engulfment into tightly fitting membrane invaginations, consistent with the interpretation that rotavirus particles drive their own uptake. Electron cryotomography of membrane-bound virions also shows closely wrapped membrane. Combined with high resolution structural information about the viral components, these observations suggest a molecular model for membrane disruption and DLP penetration.
Asunto(s)
Antígenos Virales/metabolismo , Proteínas de la Cápside/metabolismo , Membrana Celular/química , Rotavirus/química , Virión , Ensamble de Virus/fisiología , Internalización del Virus , Animales , Antígenos Virales/química , Antígenos Virales/genética , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Membrana Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops/virología , Procesamiento de Imagen Asistido por Computador , Riñón/virología , Microscopía Electrónica , Mutación/genética , Rotavirus/fisiologíaRESUMEN
Immune-privileged Sertoli cells (SCs) exhibit long-term survival after allotransplantation or xenotransplantation, suggesting they can be used as a vehicle for cell-based gene therapy. Previously, we demonstrated that SCs engineered to secrete insulin by using an adenoviral vector normalized blood glucose levels in diabetic mice. However, the expression of insulin was transient, and the use of immunocompromised mice did not address the question of whether SCs can stably express insulin in immunocompetent animals. Thus, the objective of the current study was to use a lentiviral vector to achieve stable expression of insulin in SCs and test the ability of these cells to survive after allotransplantation. A mouse SC line transduced with a recombinant lentiviral vector containing furin-modified human proinsulin cDNA (MSC-EhI-Zs) maintained stable insulin expression in vitro. Allotransplantation of MSC-EhI-Zs cells into diabetic BALB/c mice demonstrated 88% and 75% graft survival rates at 20 and 50 days post-transplantation, respectively. Transplanted MSC-EhI-Zs cells continued to produce insulin mRNA throughout the study (i.e., 50 days); however, insulin protein was detected only in patches of cells within the grafts. Consistent with low insulin protein detection, there was no significant change in blood glucose levels in the transplant recipients. Nevertheless, MSC-EhI-Zs cells isolated from the grafts continued to express insulin protein in culture. Collectively, this demonstrates that MSC-EhI-Zs cells stably expressed insulin and survived allotransplantation without immunosuppression. This further strengthens the use of SCs as targets for cell-based gene therapy for the treatment of numerous chronic diseases, especially those that require basal protein expression.
Asunto(s)
Diabetes Mellitus Experimental/terapia , Proinsulina/metabolismo , Células de Sertoli/trasplante , Animales , Línea Celular , Supervivencia Celular/fisiología , Ingeniería Genética/métodos , Terapia Genética/métodos , Inmunohistoquímica , Masculino , Ratones Endogámicos BALB C , Proinsulina/genética , ARN/química , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células de Sertoli/metabolismoRESUMEN
Primary Sertoli cells isolated from mouse testes survive when transplanted across immunological barriers and protect cotransplanted allogeneic and xenogeneic cells from rejection in rodent models. In contrast, the mouse Sertoli cell line (MSC-1) lacks immunoprotective properties associated with primary Sertoli cells. In this study, enriched primary Sertoli cells or MSC-1 cells were transplanted as allografts into the renal subcapsular area of naive BALB/c mice, and their survival in graft sites was compared. While Sertoli cells were detected within the grafts with 100% graft survival throughout the 20-day study, MSC-1 cells were rejected between 11 and 14 days, with 0% graft survival at 20 days posttransplantation. Nonetheless, the mechanism for primary Sertoli cell survival and immunoprotection remains unresolved. To identify immune factors or functional pathways potentially responsible for immune privilege, gene expression profiles of enriched primary Sertoli cells were compared with those of MSC-1 cells. Microarray analysis identified 2369 genes in enriched primary Sertoli cells that were differentially expressed at ±4-fold or higher levels than in MSC-1 cells. Ontological analyses identified multiple immune pathways, which were used to generate a list of 340 immune-related genes. Three functions were identified in primary Sertoli cells as potentially important for establishing immune privilege: suppression of inflammation by specific cytokines and prostanoid molecules, slowing of leukocyte migration by controlled cell junctions and actin polymerization, and inhibition of complement activation and membrane-associated cell lysis. These results increase our understanding of testicular immune privilege and, in the long-term, could lead to improvements in transplantation success.