RESUMEN
Extracellular vesicles (EVs) facilitate the transfer of proteins, lipids, and genetic material between cells and are recognized as an additional mechanism for sustaining intercellular communication. In the epidermis, the communication between melanocytes and keratinocytes is tightly regulated to warrant skin pigmentation. Melanocytes synthesize the melanin pigment in melanosomes that are transported along the dendrites prior to the transfer of melanin pigment to keratinocytes. EVs secreted by keratinocytes modulate pigmentation in melanocytes [(A. Lo Cicero et al., Nat. Commun. 6, 7506 (2015)]. However, whether EVs secreted by keratinocytes contribute to additional processes essential for melanocyte functions remains elusive. Here, we show that keratinocyte EVs enhance the ability of melanocytes to generate dendrites and mature melanosomes and promote their efficient transfer. Further, keratinocyte EVs carrying Rac1 induce important morphological changes, promote dendrite outgrowth, and potentiate melanin transfer to keratinocytes. Hence, in addition to modulating pigmentation, keratinocytes exploit EVs to control melanocyte plasticity and transfer capacity. These data demonstrate that keratinocyte-derived EVs, by regulating melanocyte functions, are major contributors to cutaneous pigmentation and expand our understanding of the mechanism underlying skin pigmentation via a paracrine EV-mediated communication.
Asunto(s)
Vesículas Extracelulares , Melanosomas , Melaninas , Melanocitos , QueratinocitosRESUMEN
Pigment organelles of vertebrates belong to the lysosome-related organelle (LRO) family, of which melanin-producing melanosomes are the prototypes. While their anabolism has been extensively unraveled through the study of melanosomes in skin melanocytes, their catabolism remains poorly known. Here, we tap into the unique ability of crab spiders to reversibly change body coloration to examine the catabolism of their pigment organelles. By combining ultrastructural and metal analyses on high-pressure frozen integuments, we first assess whether pigment organelles of crab spiders belong to the LRO family and second, how their catabolism is intracellularly processed. Using scanning transmission electron microscopy, electron tomography, and nanoscale Synchrotron-based scanning X-ray fluorescence, we show that pigment organelles possess ultrastructural and chemical hallmarks of LROs, including intraluminal vesicles and metal deposits, similar to melanosomes. Monitoring ultrastructural changes during bleaching suggests that the catabolism of pigment organelles involves the degradation and removal of their intraluminal content, possibly through lysosomal mechanisms. In contrast to skin melanosomes, anabolism and catabolism of pigments proceed within the same cell without requiring either cell death or secretion/phagocytosis. Our work hence provides support for the hypothesis that the endolysosomal system is fully functionalized for within-cell turnover of pigments, leading to functional maintenance under adverse conditions and phenotypic plasticity. First formulated for eye melanosomes in the context of human vision, the hypothesis of intracellular turnover of pigments gets unprecedented strong support from pigment organelles of spiders.
Asunto(s)
Color , Lisosomas/metabolismo , Melanosomas/fisiología , Orgánulos/fisiología , Pigmentos Biológicos/fisiología , Piel/metabolismo , Arañas/fisiología , Animales , Endosomas/metabolismoRESUMEN
HIV-2 is less pathogenic for humans than HIV-1 and might provide partial cross-protection from HIV-1-induced pathology. Although both viruses replicate in the T cells of infected patients, only HIV-2 replicates efficiently in dendritic cells (DCs) and activates innate immune pathways. How HIV is sensed in DC is unknown. Capsid-mutated HIV-2 revealed that sensing by the host requires viral cDNA synthesis, but not nuclear entry or genome integration. The HIV-1 capsid prevented viral cDNA sensing up to integration, allowing the virus to escape innate recognition. In contrast, DCs sensed capsid-mutated HIV-1 and enhanced stimulation of T cells in the absence of productive infection. Finally, we found that DC sensing of HIV-1 and HIV-2 required the DNA sensor cGAS. Thus, the HIV capsid is a determinant of innate sensing of the viral cDNA by cGAS in dendritic cells. This pathway might potentially be harnessed to develop effective vaccines against HIV-1.
Asunto(s)
Cápside/inmunología , ADN Complementario/metabolismo , Células Dendríticas , Infecciones por VIH/inmunología , VIH-1/inmunología , VIH-2/inmunología , Nucleotidiltransferasas/metabolismo , Células Cultivadas , ADN Viral/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/virología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/metabolismo , VIH-2/genética , VIH-2/metabolismo , Humanos , Inmunidad Innata/fisiología , Modelos BiológicosRESUMEN
The metabolism of PI(3,5)P2 is regulated by the PIKfyve, VAC14 and FIG4 complex, mutations in which are associated with hypopigmentation in mice. These pigmentation defects indicate a key, but as yet unexplored, physiological relevance of this complex in the biogenesis of melanosomes. Here, we show that PIKfyve activity regulates formation of amyloid matrix composed of PMEL protein within the early endosomes in melanocytes, called stage I melanosomes. PIKfyve activity controls the membrane remodeling of stage I melanosomes, which regulates PMEL abundance, sorting and processing. PIKfyve activity also affects stage I melanosome kiss-and-run interactions with lysosomes, which are required for PMEL amyloidogenesis and the establishment of melanosome identity. Mechanistically, PIKfyve activity promotes both the formation of membrane tubules from stage I melanosomes and their release by modulating endosomal actin branching. Taken together, our data indicate that PIKfyve activity is a key regulator of the melanosomal import-export machinery that fine tunes the formation of functional amyloid fibrils in melanosomes and the maintenance of melanosome identity.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Flavoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisosomas/metabolismo , Melanocitos/metabolismo , Melanosomas/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoinosítido Fosfatasas/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Amiloide/metabolismo , Animales , Células Cultivadas , Flavoproteínas/genética , Homeostasis , Péptidos y Proteínas de Señalización Intracelular/genética , Melanocitos/patología , Melanosomas/ultraestructura , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Fosfoinosítido Fosfatasas/genética , Transporte de Proteínas , Epitelio Pigmentado de la Retina/patología , Antígeno gp100 del Melanoma/metabolismoRESUMEN
The protein complex composed of the kinase PIKfyve, the phosphatase FIG4 and the scaffolding protein VAC14 regulates the metabolism of phosphatidylinositol 3,5-bisphosphate, which serves as both a signaling lipid and the major precursor for phosphatidylinositol 5-phosphate. This complex is involved in the homeostasis of late endocytic compartments, but its precise role in maintaining the dynamic equilibrium of late endosomes, endolysosomes and lysosomes remains to be determined. Here, we report that inhibition of PIKfyve activity impairs terminal lysosome reformation from acidic and hydrolase-active, but enlarged endolysosomes. Our live-cell imaging and electron tomography data show that PIKfyve activity regulates extensive membrane remodeling that initiates reformation of lysosomes from endolysosomes. Altogether, our findings show that PIKfyve activity is required to maintain the dynamic equilibrium of late endocytic compartments by regulating the reformation of terminal storage lysosomes.
Asunto(s)
Endosomas/metabolismo , Lisosomas/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Técnicas de Cultivo de Célula , Endosomas/ultraestructura , Flavoproteínas/metabolismo , Células HeLa , Homeostasis , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lisosomas/ultraestructura , Microscopía Electrónica , Microscopía Fluorescente , Monoéster Fosfórico Hidrolasas/metabolismo , Transporte de ProteínasRESUMEN
Correlating complementary multiple scale images of the same object is a straightforward means to decipher biological processes. Light microscopy and electron microscopy are the most commonly used imaging techniques, yet despite their complementarity, the experimental procedures available to correlate them are technically complex. We designed and manufactured a new device adapted to many biological specimens, the CryoCapsule, that simplifies the multiple sample preparation steps, which at present separate live cell fluorescence imaging from contextual high-resolution electron microscopy, thus opening new strategies for full correlative light to electron microscopy. We tested the biological application of this highly optimized tool on three different specimens: the in vitro Xenopus laevis mitotic spindle, melanoma cells over-expressing YFP-langerin sequestered in organized membranous subcellular organelles and a pigmented melanocytic cell in which the endosomal system was labeled with internalized fluorescent transferrin.
Asunto(s)
Microscopía por Crioelectrón/métodos , Crioultramicrotomía/instrumentación , Animales , Línea Celular , Microscopía por Crioelectrón/instrumentación , Crioultramicrotomía/métodos , Perros , Endosomas/metabolismo , Endosomas/ultraestructura , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Imagen Óptica/instrumentación , Imagen Óptica/métodos , Huso Acromático/metabolismo , Huso Acromático/ultraestructura , XenopusRESUMEN
BACKGROUND INFORMATION: Rab6 is one of the most conserved Rab GTPaes throughout evolution and the most abundant Rab protein associated with the Golgi complex. The two ubiquitous Rab isoforms, Rab6A and Rab6A', that are generated by alternative splicing of the RAB6A gene, regulate several transport steps at the Golgi level, including retrograde transport between endosomes and Golgi, anterograde transport between Golgi and the plasma membrane, and intra-Golgi and Golgi to endoplasmic reticulum transport. RESULTS: We have generated mice with a conditional null allele of RAB6A. Mice homozygous for the RAB6A null allele died at an early stage of embryonic development. Mouse embryonic fibroblasts (MEFs) were isolated from RAB6A(loxP/loxP) Rosa26-CreERT2 and incubated with 4-hydroxy tamoxifen, resulting in the efficient depletion of Rab6A and Rab6A'. We show that Rab6 depletion affects cell growth, alters Golgi morphology and decreases the Golgi-associated levels of some known Rab6 effectors such as Bicaudal-D and myosin II. We also show that Rab6 depletion protects MEFs against ricin toxin and delays VSV-G secretion. CONCLUSIONS: Our study shows that RAB6 is an essential gene required for normal embryonic development. We confirm in MEF cells most of the functions previously attributed to the two ubiquitous Rab6 isoforms.
Asunto(s)
Empalme Alternativo/genética , Desarrollo Embrionario/genética , Retículo Endoplásmico/metabolismo , Proteínas de Unión al GTP rab/genética , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/genética , Retículo Endoplásmico/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Humanos , Ratones , Ratones Noqueados , Ricina/toxicidad , Tamoxifeno/administración & dosificación , Proteínas de Unión al GTP rab/biosíntesisRESUMEN
Amyloids are often associated with pathologic processes such as in Alzheimer's disease (AD), but can also underlie physiological processes such as pigmentation. Formation of pathological and functional amyloidogenic substrates can require precursor processing by proteases, as exemplified by the generation of Aß peptide from amyloid precursor protein (APP) by beta-site APP cleaving enzyme (BACE)1 and γ-secretase. Proteolytic processing of the pigment cell-specific Melanocyte Protein (PMEL) is also required to form functional amyloid fibrils during melanogenesis, but the enzymes involved are incompletely characterized. Here we show that the BACE1 homologue BACE2 processes PMEL to generate functional amyloids. BACE2 is highly expressed in pigment cells and Bace2(-/-) but not Bace1(-/-) mice display coat color defects, implying a specific role for BACE2 during melanogenesis. By using biochemical and morphological analyses, combined with RNA silencing, pharmacologic inhibition, and BACE2 overexpression in a human melanocytic cell line, we show that BACE2 cleaves the integral membrane form of PMEL within the juxtamembrane domain, releasing the PMEL luminal domain into endosomal precursors for the formation of amyloid fibrils and downstream melanosome morphogenesis. These studies identify an amyloidogenic substrate of BACE2, reveal an important physiological role for BACE2 in pigmentation, and highlight analogies in the generation of PMEL-derived functional amyloids and APP-derived pathological amyloids.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Amiloide/biosíntesis , Ácido Aspártico Endopeptidasas/metabolismo , Melanosomas/metabolismo , Antígeno gp100 del Melanoma/metabolismo , Secretasas de la Proteína Precursora del Amiloide/deficiencia , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Ácido Aspártico Endopeptidasas/deficiencia , Ácido Aspártico Endopeptidasas/genética , Línea Celular , Células HeLa , Humanos , Melaninas/biosíntesis , Melanocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Epitelio Pigmentado Ocular/metabolismo , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Pigmentación de la Piel/genética , Pigmentación de la Piel/fisiologíaRESUMEN
Melanocytes are essential for skin homeostasis and protection, and their defects in humans lead to a wide array of diseases that are potentially extremely severe. To date, the analysis of molecular mechanisms and the function of human melanocytes have been limited because of the difficulties in accessing large numbers of cells with the specific phenotypes. This issue can now be addressed via a differentiation protocol that allows melanocytes to be obtained from pluripotent stem cell lines, either induced or of embryonic origin, based on the use of moderate concentrations of a single cytokine, bone morphogenic protein 4. Human melanocytes derived from pluripotent stem cells exhibit all the characteristic features of their adult counterparts. This includes the enzymatic machinery required for the production and functional delivery of melanin to keratinocytes. Melanocytes also integrate appropriately into organotypic epidermis reconstructed in vitro. The availability of human cells committed to the melanocytic lineage in vitro will enable the investigation of those mechanisms that guide the developmental processes and will facilitate analysis of the molecular mechanisms responsible for genetic diseases. Access to an unlimited resource may also prove a vital tool for the treatment of hypopigmentation disorders when donors with matching haplotypes become available in clinically relevant banks of pluripotent stem cell lines.
Asunto(s)
Células Madre Adultas/citología , Proteína Morfogenética Ósea 4/farmacología , Diferenciación Celular/efectos de los fármacos , Células Epidérmicas , Melanocitos/citología , Células Madre Pluripotentes/citología , Células Madre Adultas/metabolismo , Línea Celular , Epidermis/metabolismo , Humanos , Hipopigmentación/metabolismo , Hipopigmentación/terapia , Melanocitos/metabolismo , Células Madre Pluripotentes/metabolismo , Trasplante de Células MadreRESUMEN
Transmission electron microscopy (TEM) is currently the only method that enables the observation of extracellular vesicles (EVs) at a nanometer scale. Direct visualization of the whole content of EV preparation provides not only crucial insights on the morphology of EVs but also an objective evaluation of the content and purity of the preparation. Coupled to immunogold labeling, TEM allows the detection and association of proteins at the surface of EVs. In these techniques, EVs are deposited on grids and are chemically immobilized and contrasted to withstand a high-voltage electron beam. Under high vacuum, the electron beam hits the sample and the electrons that scatter forward are collected to form an image. Here, we describe the steps needed to observe EVs by classical TEM and the extra steps required to label proteins through immunolabeling electron microscopy (IEM).
Asunto(s)
Vesículas Extracelulares , Microscopía Electrónica de Transmisión , Microscopía Electrónica , Vesículas Extracelulares/metabolismo , Proteínas/metabolismoRESUMEN
Our knowledge of the organization of the cell is linked, to a great extent, to light and electron microscopy. Choosing either photons or electrons for imaging has many consequences on the image obtained, as well as on the experiment required in order to generate the image. One apparent effect on the experimental side is in the sample preparation, which can be quite elaborate for electron microscopy. In recent years, rapid freezing, cryo-preparation and cryo-electron microscopy have been more widely used because they introduce fewer artefacts during preparation when compared with chemical fixation and room temperature processing. In addition, cryo-electron microscopy allows the visualization of the hydrated specimens. In the present review, we give an introduction to the rapid freezing of biological samples and describe the preparation steps. We focus on bulk samples that are too big to be directly viewed under the electron microscope. Furthermore, we discuss the advantages and limitations of freeze substitution and cryo-electron microscopy of vitreous sections and compare their application to the study of bacteria and mammalian cells and to tomography.
Asunto(s)
Microscopía por Crioelectrón/métodos , Criopreservación/métodos , Animales , Tomografía con Microscopio Electrónico , Humanos , Melanocitos/ultraestructura , Microtomía , Adhesión en Plástico , Shigella flexneri/ultraestructura , Fijación del Tejido , VitrificaciónRESUMEN
Morphogens are secreted molecules that regulate and coordinate major developmental processes, such as cell differentiation and tissue morphogenesis. Depending on the mechanisms of secretion and the nature of their carriers, morphogens act at short and long range. We investigated the paradigmatic long-range activity of Hedgehog (Hh), a well-known morphogen, and its contribution to the growth and patterning of the Drosophila wing imaginal disc. Extracellular vesicles (EVs) contribute to Hh long-range activity; however, the nature, the site, and the mechanisms underlying the biogenesis of these vesicular carriers remain unknown. Here, through the analysis of mutants and a series of Drosophila RNAi-depleted wing imaginal discs using fluorescence and live-imaging electron microscopy, including tomography and 3D reconstruction, we demonstrate that microvilli of the wing imaginal disc epithelium are the site of generation of small EVs that transport Hh across the tissue. Further, we show that the Prominin-like (PromL) protein is critical for microvilli integrity. Together with actin cytoskeleton and membrane phospholipids, PromL maintains microvilli architecture that is essential to promote its secretory function. Importantly, the distribution of Hh to microvilli and its release via these EVs contribute to the proper morphogenesis of the wing imaginal disc. Our results demonstrate that microvilli-derived EVs are carriers for Hh long-range signaling in vivo. By establishing that members of the Prominin protein family are key determinants of microvilli formation and integrity, our findings support the view that microvilli-derived EVs conveying Hh may provide a means for exchanging signaling cues of high significance in tissue development and cancer.
Asunto(s)
Proteínas de Drosophila , Vesículas Extracelulares , Antígeno AC133/metabolismo , Animales , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Discos Imaginales , Microvellosidades/metabolismo , Morfogénesis , Alas de AnimalesRESUMEN
Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pregnancy, little is known about their role during viral congenital infection, especially at the beginning of pregnancy. In this study, we examined the consequences of human cytomegalovirus (hCMV) infection on sEVs production, composition, and function using an immortalized human cytotrophoblast cell line derived from first trimester placenta. By combining complementary approaches of biochemistry, electron microscopy, and quantitative proteomic analysis, we showed that hCMV infection increases the yield of sEVs produced by cytotrophoblasts and modifies their protein content towards a potential proviral phenotype. We further demonstrate that sEVs secreted by hCMV-infected cytotrophoblasts potentiate infection in naive recipient cells of fetal origin, including human neural stem cells. Importantly, these functional consequences are also observed with sEVs prepared from an ex vivo model of infected histocultures from early placenta. Based on these findings, we propose that placental sEVs could be important actors favoring viral dissemination to the fetal brain during hCMV congenital infection.
Asunto(s)
Infecciones por Citomegalovirus , Vesículas Extracelulares , Citomegalovirus/genética , Vesículas Extracelulares/metabolismo , Femenino , Humanos , Placenta , Embarazo , ProteómicaRESUMEN
Intracellular trafficking is mediated by transport carriers that originate by membrane remodeling from donor organelles. Tubular carriers contribute to the flux of membrane lipids and proteins to acceptor organelles, but how lipids and proteins impose a tubular geometry on the carriers is incompletely understood. Using imaging approaches on cells and in vitro membrane systems, we show that phosphatidylinositol-4-phosphate (PI4P) and biogenesis of lysosome-related organelles complex 1 (BLOC-1) govern the formation, stability, and functions of recycling endosomal tubules. In vitro, BLOC-1 binds and tubulates negatively charged membranes, including those containing PI4P. In cells, endosomal PI4P production by type II PI4-kinases is needed to form and stabilize BLOC-1-dependent recycling endosomal tubules. Decreased PI4KIIs expression impairs the recycling of endosomal cargoes and the life cycles of intracellular pathogens such as Chlamydia bacteria and influenza virus that exploit the membrane dynamics of recycling endosomes. This study demonstrates how a phospholipid and a protein complex coordinate the remodeling of cellular membranes into functional tubules.
Asunto(s)
Endosomas , Membranas Intracelulares , Péptidos y Proteínas de Señalización Intracelular , Fosfatos de Fosfatidilinositol , Membrana Celular/metabolismo , Endosomas/metabolismo , Membranas Intracelulares/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisosomas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transporte de ProteínasRESUMEN
Cargo partitioning into intralumenal vesicles (ILVs) of multivesicular endosomes underlies such cellular processes as receptor downregulation, viral budding, and biogenesis of lysosome-related organelles such as melanosomes. We show that the melanosomal protein Pmel17 is sorted into ILVs by a mechanism that is dependent upon lumenal determinants and conserved in non-pigment cells. Pmel17 targeting to ILVs does not require its native cytoplasmic domain or cytoplasmic residues targeted by ubiquitylation and, unlike sorting of ubiquitylated cargo, is insensitive to functional inhibition of Hrs and ESCRT complexes. Chimeric protein and deletion analyses indicate that two N-terminal lumenal subdomains are necessary and sufficient for ILV targeting. Pmel17 fibril formation, which occurs during melanosome maturation in melanocytes, requires a third lumenal subdomain and proteolytic processing that itself requires ILV localization. These results establish an Hrs- and perhaps ESCRT-independent pathway of ILV sorting by lumenal determinants and a requirement for ILV sorting in fibril formation.
Asunto(s)
Endosomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Orgánulos/metabolismo , Transporte de Proteínas , Vesículas Transportadoras/metabolismo , Antígenos de Neoplasias , Biomarcadores/metabolismo , Línea Celular , Humanos , Antígeno MART-1 , Melanosomas/metabolismo , Glicoproteínas de Membrana/genética , Morfogénesis , Proteínas de Neoplasias/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Antígeno gp100 del MelanomaRESUMEN
Melanosomes are lysosome-related organelles (LROs) in which melanins are synthesized and stored. Early stage melanosomes are characterized morphologically by intralumenal fibrils upon which melanins are deposited in later stages. The integral membrane protein Pmel17 is a component of the fibrils, can nucleate fibril formation in the absence of other pigment cell-specific proteins, and forms amyloid-like fibrils in vitro. Before fibril formation Pmel17 traffics through multivesicular endosomal compartments, but how these compartments participate in downstream events leading to fibril formation is not fully known. By using high-pressure freezing of MNT-1 melanoma cells and freeze substitution to optimize ultrastructural preservation followed by double tilt 3D electron tomography, we show that the amyloid-like fibrils begin to form in multivesicular compartments, where they radiate from the luminal side of intralumenal membrane vesicles. The fibrils in fully formed stage II premelanosomes organize into sheet-like arrays and exclude the remaining intralumenal vesicles, which are smaller and often in continuity with the limiting membrane. These observations indicate that premelanosome fibrils form in association with intralumenal endosomal membranes. We suggest that similar processes regulate amyloid formation in pathological models.
Asunto(s)
Amiloide/biosíntesis , Melaninas/biosíntesis , Melanocitos/ultraestructura , Melanosomas/ultraestructura , Amiloide/química , Animales , Línea Celular Tumoral , Microscopía por Crioelectrón , Citocalasina D/química , Citocalasina D/metabolismo , Endosomas/química , Endosomas/metabolismo , Endosomas/ultraestructura , Congelación , Humanos , Melanocitos/metabolismo , Melanosomas/química , Melanosomas/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Ratones , Miosina Tipo I/química , Miosina Tipo I/metabolismo , Presión , Estructura Secundaria de Proteína , Antígeno gp100 del MelanomaRESUMEN
Skin pigmentation is dependent on cellular processes including melanosome biogenesis, transport, maturation and transfer to keratinocytes. However, how the cells finely control these processes in space and time to ensure proper pigmentation remains unclear. Here, we show that a component of the cytoplasmic dynein complex, Dynlt3, is required for efficient melanosome transport, acidity and transfer. In Mus musculus melanocytes with decreased levels of Dynlt3, pigmented melanosomes undergo a more directional motion, leading to their peripheral location in the cell. Stage IV melanosomes are more acidic, but still heavily pigmented, resulting in a less efficient melanosome transfer. Finally, the level of Dynlt3 is dependent on ß-catenin activity, revealing a function of the Wnt/ß-catenin signalling pathway during melanocyte and skin pigmentation, by coupling the transport, positioning and acidity of melanosomes required for their transfer.
Asunto(s)
Dineínas/genética , Melanocitos/metabolismo , Melanosomas/fisiología , Animales , Dineínas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Pigmentación de la PielRESUMEN
Extracellular vesicles (EVs) have increasingly been recognized as key players in a wide variety of physiological and pathological contexts, including during pregnancy. Notably, EVs appear both as possible biomarkers and as mediators involved in the communication of the placenta with the maternal and fetal sides. A better understanding of the physiological and pathological roles of EVs strongly depends on the development of adequate and reliable study models, specifically at the beginning of pregnancy where many adverse pregnancy outcomes have their origin. In this study, we describe the isolation of small EVs from a histoculture model of first trimester placental explants in normal conditions as well as upon infection by human cytomegalovirus. Using bead-based multiplex cytometry and electron microscopy combined with biochemical approaches, we characterized these small EVs and defined their associated markers and ultrastructure. We observed that infection led to changes in the expression level of several surface markers, without affecting the secretion and integrity of small EVs. Our findings lay the foundation for studying the functional role of EVs during early pregnancy, along with the identification of new predictive biomarkers for the severity and outcome of this congenital infection, which are still sorely lacking.
RESUMEN
Important progress has been made during the past decade in the identification of molecular motors required in the distribution of early and late endosomes and the proper trafficking along the endocytic pathway. There is little direct evidence, however, that these motors drive movement of the endosomes. To evaluate the contributions of kinesin-1, dynein and kinesin-2 to the movement of early and late endosomes along microtubules, we made use of a cytosol-free motility assay using magnetically isolated early and late endosomes as well as biochemical analyses and live-cell imaging. By making use of specific antibodies, we confirmed that kinesin-1 and dynein move early endosomes and we found that kinesin-2 moves both early and late endosomes in the cell-free assay. Unexpectedly, dynein did not move late endosomes in the cell-free assay. We provide evidence from disruption of dynein function and latrunculin A treatment, suggesting that dynein regulates late endosome movement indirectly, possibly through a mechanism involving the actin cytoskeleton. These data provide new insights into the complex regulation of endosomes' motility and suggest that dynein is not the major motor required to move late endosomes toward the minus end of microtubules.