RESUMEN
Ciliogenesis precedes lineage-determining signaling in skin development. To understand why, we performed shRNA-mediated knockdown of seven intraflagellar transport proteins (IFTs) and conditional ablation of Ift-88 and Kif3a during embryogenesis. In both cultured keratinocytes and embryonic epidermis, all of these eliminated cilia, and many (not Kif3a) caused hyperproliferation. Surprisingly and independent of proliferation, ciliary mutants displayed defects in Notch signaling and commitment of progenitors to differentiate. Notch receptors and Notch-processing enzymes colocalized with cilia in wild-type epidermal cells. Moreover, differentiation defects in ciliary mutants were cell autonomous and rescued by activated Notch (NICD). By contrast, Shh signaling was neither operative nor required for epidermal ciliogenesis, Notch signaling, or differentiation. Rather, Shh signaling defects in ciliary mutants occurred later, arresting hair follicle morphogenesis in the skin. These findings unveil temporally and spatially distinct functions for primary cilia at the nexus of signaling, proliferation, and differentiation.
Asunto(s)
Diferenciación Celular , Cilios/metabolismo , Epidermis/embriología , Epidermis/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Animales , Proteínas Portadoras/genética , Polaridad Celular , Proliferación Celular , Proteínas del Citoesqueleto/metabolismo , Células Epidérmicas , Técnicas de Silenciamiento del Gen , Folículo Piloso/citología , Proteínas Hedgehog/metabolismo , Cinesis , Ratones , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Arl/ARF GTPases regulate ciliary trafficking, but their tissue-specific functions are unclear. Here, we demonstrate that ciliary GTPase Arl3 is required for mitotic spindle orientation of mouse basal stem cells during skin development. Arl3 loss diminished cell divisions within the plane of the epithelium, leading to increased perpendicular divisions, expansion of progenitor cells and loss of epithelial integrity. These observations suggest that an Arl3-dependent mechanism maintains cell division polarity along the tissue axis, and disruption of planar spindle orientation has detrimental consequences for epidermal architecture. Defects in planar cell polarity (PCP) can disrupt spindle positioning during tissue morphogenesis. Upon Arl3 loss, the PCP signaling molecules Celsr1 and Vangl2 failed to maintain planar polarized distributions, resulting in defective hair follicle angling, a hallmark of disrupted PCP. In the absence of Celsr1 polarity, frizzled 6 lost its asymmetrical distribution and abnormally segregated to the apical cortex of basal cells. We propose that Arl3 regulates polarized endosomal trafficking of PCP components to compartmentalized membrane domains. Cell-cell communication via ciliary GTPase signaling directs mitotic spindle orientation and PCP signaling, processes that are crucial for the maintenance of epithelial architecture.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Huso Acromático/metabolismo , Factores de Ribosilacion-ADP/genética , Animales , Western Blotting , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Polaridad Celular/genética , Polaridad Celular/fisiología , Células Cultivadas , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Técnicas In Vitro , Queratinocitos/metabolismo , Lentivirus/genética , Ratones , Mitosis/genética , Mitosis/fisiología , Morfogénesis/genética , Morfogénesis/fisiología , Reacción en Cadena de la Polimerasa , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Imaging studies implicate microtubule targeting of focal adhesions in focal adhesion disassembly, although the molecular mechanism is unknown. Here, we develop a model system of focal adhesion disassembly based on the finding that microtubule regrowth after nocodazole washout induces disassembly of focal adhesions, and that this disassembly occurs independently of Rho and Rac, but depends on focal adhesion kinase (FAK) and dynamin. During disassembly, dynamin interacts with FAK and colocalizes with focal adhesions. Inhibition of dynamin prevents migration of cells with a focal adhesion phenotype. Our results show that focal adhesion disassembly involves microtubules, dynamin and FAK, and is not simply the reversal of focal adhesion formation.
Asunto(s)
Adhesión Celular/fisiología , Dinaminas/metabolismo , Adhesiones Focales/metabolismo , Microtúbulos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Antineoplásicos/farmacología , Movimiento Celular/fisiología , Dinaminas/ultraestructura , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Adhesiones Focales/ultraestructura , Proteínas Fluorescentes Verdes , Integrinas/metabolismo , Ratones , Microtúbulos/ultraestructura , Células 3T3 NIH , Nocodazol/farmacología , Proteínas Tirosina Quinasas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Directional cell migration drives embryonic development, cancer metastasis, and tissue repair and regeneration. Here, we examine the role of intraflagellar transport (IFT) 20 (Ift20) during polarized migration of epidermal cells. IFT20 is implicated in regulating cell migration independently of the primary cilium, but how IFT proteins integrate with the cell migration machinery is poorly understood. We show that genetic ablation of IFT20 in vitro slows keratinocyte migration during wound healing. We find that this phenotype is independent of the primary cilium and instead can be attributed to alterations in integrin-mediated mechanotransduction and focal adhesion (FA) dynamics. Loss of Ift20 resulted in smaller and less numerous FAs and reduced the levels of activated FA kinase. Studies of FA dynamics during microtubule-induced FA turnover demonstrated that Ift20 loss specifically impaired the reformation, but not the disassembly, of FAs. In the absence of Ift20 function, ß1 integrins endocytosed during FA disassembly are not transferred out of Rab5 (+) endosomes. This defective transit from the early endosome disrupts eventual recycling of ß1 integrins back to the cell surface, resulting in defective FA reformation. In vivo, conditional ablation of Ift20 in hair follicle stem cells (HF-SCs) similarly impairs their ability to invade and migrate during epidermal wound healing. Using explant studies, lineage tracing, and clonal analysis, we demonstrate that Ift20 is required for HF-SC migration and their contribution to epidermal regeneration. This work identifies a new Ift20 mechanotrafficking mechanism required for polarized cell migration and stem cell-driven tissue repair.
Asunto(s)
Proteínas Portadoras/metabolismo , Movimiento Celular/fisiología , Integrinas/metabolismo , Animales , Proteínas Portadoras/fisiología , Adhesión Celular/fisiología , Membrana Celular/metabolismo , Polaridad Celular/fisiología , Cilios/metabolismo , Endosomas/metabolismo , Femenino , Adhesiones Focales/metabolismo , Integrina beta1/metabolismo , Integrinas/genética , Queratinocitos/metabolismo , Masculino , Mecanotransducción Celular/fisiología , Ratones , Ratones Endogámicos , Microtúbulos/metabolismo , Transporte de Proteínas , Cicatrización de Heridas/fisiologíaRESUMEN
Src kinase is a crucial mediator of adhesion-related signaling and motility. Src binds to focal adhesion kinase (FAK) through its SH2 domain and subsequently activates it for phosphorylation of downstream substrates. In addition to this binding function, data suggested that the SH2 domain might also perform an important role in targeting Src to focal adhesions (FAs) to enable further substrate phosphorylations. To examine this, we engineered an R175L mutation in cSrc to prevent the interaction with FAK pY397. This constitutively open Src kinase mediated up-regulated substrate phosphorylation in SYF cells but was unable to promote malignant transformation. Significantly, SrcR175L cells also had a profound motility defect and an impaired FA generation capacity. Importantly, we were able to recapitulate wild-type motile behavior and FA formation by directing the kinase to FAs, clearly implicating the SH2 domain in recruitment to FAK and indicating that this targeting capacity, and not simply Src-FAK scaffolding, was critical for normal Src function.
Asunto(s)
Movimiento Celular/fisiología , Proteína-Tirosina Quinasas de Adhesión Focal/fisiología , Adhesiones Focales/fisiología , Familia-src Quinasas/química , Familia-src Quinasas/fisiología , Sustitución de Aminoácidos , Animales , Arginina/química , Línea Celular , Pollos , Proteína-Tirosina Quinasas de Adhesión Focal/química , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Genes src , Humanos , Técnicas In Vitro , Mutagénesis Sitio-Dirigida , Fosforilación , Dominios Homologos src , Familia-src Quinasas/genéticaRESUMEN
How primary cilia impact epidermal growth and differentiation during embryogenesis is poorly understood. Here, we show that during skin development, Notch signaling occurs within the ciliated, differentiating cells of the first few suprabasal epidermal layers. Moreover, both Notch signaling and cilia disappear in the upper layers, where key ciliary proteins distribute to cell-cell borders. Extending this correlation, we find that Presenilin-2 localizes to basal bodies/cilia through a conserved VxPx motif. When this motif is mutated, a GFP-tagged Presenilin-2 still localizes to intercellular borders, but basal body localization is lost. Notably, in contrast to wild type, this mutant fails to rescue epidermal differentiation defects seen upon Psen1 and 2 knockdown. Screening components implicated in ciliary targeting and polarized exocytosis, we provide evidence that the small GTPase ARF4 is required for Presenilin basal body localization, Notch signaling, and subsequent epidermal differentiation. Collectively, our findings raise the possibility that ARF4-dependent polarized exocytosis acts through the basal body-ciliary complex to spatially regulate Notch signaling during epidermal differentiation.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Diferenciación Celular , Células Epidérmicas , Presenilina-2/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Secuencias de Aminoácidos , Animales , Cuerpos Basales/metabolismo , Células Cultivadas , Cilios/metabolismo , Cilios/ultraestructura , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Epidermis/embriología , Epidermis/metabolismo , Epidermis/ultraestructura , Genes Reporteros , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Organogénesis , Presenilina-1/metabolismo , Presenilina-2/química , Transporte de ProteínasRESUMEN
Integrin endocytic recycling is critical for cell migration, yet how recycled integrins assemble into new adhesions is unclear. By synchronizing endocytic disassembly of focal adhesions (FAs), we find that recycled integrins reassemble FAs coincident with their return to the cell surface and dependent on Rab5 and Rab11. Unexpectedly, endocytosed integrins remained in an active but unliganded state in endosomes. FAK and Src kinases co-localized with endocytosed integrin and were critical for FA reassembly by regulating integrin activation and recycling, respectively. FAK sustained the active integrin conformation by maintaining talin association with Rab11 endosomes in a type I phosphatidylinositol phosphate kinase (PIPKIγ)-dependent manner. In migrating cells, endocytosed integrins reassembled FAs polarized towards the leading edge, and this polarization required FAK. These studies identify unanticipated roles for FA proteins in maintaining endocytosed integrin in an active conformation. We propose that the conformational memory of endocytosed integrin enhances polarized reassembly of FAs to enable directional cell migration.
Asunto(s)
Polaridad Celular , Endocitosis , Quinasa 1 de Adhesión Focal/metabolismo , Adhesiones Focales/metabolismo , Integrina alfa5/metabolismo , Integrina beta1/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Talina/metabolismo , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Movimiento Celular , Fibronectinas/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Ratones , Microtúbulos/metabolismo , Células 3T3 NIH , Transducción de Señal , Vinculina/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Focal adhesion disassembly is regulated by microtubules (MTs) through an unknown mechanism that involves dynamin. To test whether endocytosis may be involved, we interfered with the function of clathrin or its adaptors autosomal recessive hypercholesteremia (ARH) and Dab2 (Disabled-2) and found that both treatments prevented MT-induced focal adhesion disassembly. Surface labeling experiments showed that integrin was endocytosed in an extracellular matrix-, clathrin-, and ARH- and Dab2-dependent manner before entering Rab5 endosomes. Clathrin colocalized with a subset of focal adhesions in an ARH- and Dab2-dependent fashion. Direct imaging showed that clathrin rapidly accumulated on focal adhesions during MT-stimulated disassembly and departed from focal adhesions with integrin upon their disassembly. In migrating cells, depletion of clathrin or Dab2 and ARH inhibited focal adhesion disassembly and decreased the rate of migration. These results show that focal adhesion disassembly occurs through a targeted mechanism involving MTs, clathrin, and specific clathrin adaptors and that direct endocytosis of integrins from focal adhesions mediates their disassembly in migrating cells.