RESUMEN
Chemical synapses between axons and dendrites mediate neuronal intercellular communication. Here, we describe a synapse between axons and primary cilia: the axo-ciliary synapse. Using enhanced focused ion beam-scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between brainstem serotonergic axons and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, the 5-hydroxytryptamine receptor 6 (5-HTR6). Using a cilia-targeted serotonin sensor, we show that opto- and chemogenetic stimulation of serotonergic axons releases serotonin onto cilia. Ciliary 5-HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway, which modulates nuclear actin and increases histone acetylation and chromatin accessibility. Ablation of this pathway reduces chromatin accessibility in CA1 pyramidal neurons. As a signaling apparatus with proximity to the nucleus, axo-ciliary synapses short circuit neurotransmission to alter the postsynaptic neuron's epigenetic state.
Asunto(s)
Axones/fisiología , Cromatina/química , Cilios , Sinapsis , Núcleo Celular/metabolismo , Cromatina/metabolismo , Cilios/metabolismo , Hipocampo/citología , Hipocampo/fisiología , Serotonina/metabolismo , Transducción de Señal , Sinapsis/fisiologíaRESUMEN
Piloerection (goosebumps) requires concerted actions of the hair follicle, the arrector pili muscle (APM), and the sympathetic nerve, providing a model to study interactions across epithelium, mesenchyme, and nerves. Here, we show that APMs and sympathetic nerves form a dual-component niche to modulate hair follicle stem cell (HFSC) activity. Sympathetic nerves form synapse-like structures with HFSCs and regulate HFSCs through norepinephrine, whereas APMs maintain sympathetic innervation to HFSCs. Without norepinephrine signaling, HFSCs enter deep quiescence by down-regulating the cell cycle and metabolism while up-regulating quiescence regulators Foxp1 and Fgf18. During development, HFSC progeny secretes Sonic Hedgehog (SHH) to direct the formation of this APM-sympathetic nerve niche, which in turn controls hair follicle regeneration in adults. Our results reveal a reciprocal interdependence between a regenerative tissue and its niche at different stages and demonstrate sympathetic nerves can modulate stem cells through synapse-like connections and neurotransmitters to couple tissue production with demands.
Asunto(s)
Nervio Accesorio/fisiología , Folículo Piloso/citología , Cabello/crecimiento & desarrollo , Proteínas Hedgehog/metabolismo , Norepinefrina/metabolismo , Transducción de Señal/genética , Células Madre/metabolismo , Células Madre/fisiología , Nervio Accesorio/citología , Animales , Ciclo Celular/genética , Frío , Femenino , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica , Cabello/citología , Cabello/fisiología , Folículo Piloso/crecimiento & desarrollo , Folículo Piloso/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Piloerección , RNA-Seq , Receptores Adrenérgicos beta 2/deficiencia , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Nicho de Células Madre , Células Madre/citología , Sistema Nervioso Simpático/citología , Sistema Nervioso Simpático/fisiología , Sinapsis/fisiologíaRESUMEN
Metabolic coordination between neurons and astrocytes is critical for the health of the brain. However, neuron-astrocyte coupling of lipid metabolism, particularly in response to neural activity, remains largely uncharacterized. Here, we demonstrate that toxic fatty acids (FAs) produced in hyperactive neurons are transferred to astrocytic lipid droplets by ApoE-positive lipid particles. Astrocytes consume the FAs stored in lipid droplets via mitochondrial ß-oxidation in response to neuronal activity and turn on a detoxification gene expression program. Our findings reveal that FA metabolism is coupled in neurons and astrocytes to protect neurons from FA toxicity during periods of enhanced activity. This coordinated mechanism for metabolizing FAs could underlie both homeostasis and a variety of disease states of the brain.
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Astrocitos/metabolismo , Ácidos Grasos/metabolismo , Neuronas/metabolismo , Animales , Apolipoproteínas E/metabolismo , Apolipoproteínas E/fisiología , Astrocitos/fisiología , Encéfalo/metabolismo , Ácidos Grasos/toxicidad , Homeostasis , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Oxidación-Reducción , Ratas , Ratas Sprague-DawleyRESUMEN
Long-distance RNA transport enables local protein synthesis at metabolically-active sites distant from the nucleus. This process ensures an appropriate spatial organization of proteins, vital to polarized cells such as neurons. Here, we present a mechanism for RNA transport in which RNA granules "hitchhike" on moving lysosomes. In vitro biophysical modeling, live-cell microscopy, and unbiased proximity labeling proteomics reveal that annexin A11 (ANXA11), an RNA granule-associated phosphoinositide-binding protein, acts as a molecular tether between RNA granules and lysosomes. ANXA11 possesses an N-terminal low complexity domain, facilitating its phase separation into membraneless RNA granules, and a C-terminal membrane binding domain, enabling interactions with lysosomes. RNA granule transport requires ANXA11, and amyotrophic lateral sclerosis (ALS)-associated mutations in ANXA11 impair RNA granule transport by disrupting their interactions with lysosomes. Thus, ANXA11 mediates neuronal RNA transport by tethering RNA granules to actively-transported lysosomes, performing a critical cellular function that is disrupted in ALS.
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Anexinas/metabolismo , Transporte Axonal/fisiología , Gránulos Citoplasmáticos/metabolismo , Lisosomas/metabolismo , ARN/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Animales Modificados Genéticamente , Anexinas/genética , Axones/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Mutación , Unión Proteica , Ratas/embriología , Ratas Sprague-Dawley , Transfección , Pez CebraRESUMEN
To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites1,2. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites3,4. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle5,6. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation7,8, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.
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Retículo Endoplásmico , Mitocondrias , Membranas Mitocondriales , Movimiento , Proteínas de Transporte Vesicular , Humanos , Esclerosis Amiotrófica Lateral/genética , Retículo Endoplásmico/química , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Mitocondrias/química , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Membranas Mitocondriales/química , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/ultraestructura , Transducción de Señal , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/ultraestructura , Microscopía Electrónica , Imagenología Tridimensional , Sitios de Unión , Difusión , Factores de Tiempo , Mutación , HomeostasisRESUMEN
Establishing and maintaining tolerance to self-antigens or innocuous foreign antigens is vital for the preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (AIRE) have a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (Treg) cell development1-4. Within weeks of birth, a separate wave of Treg cell differentiation occurs in the periphery upon exposure to antigens derived from the diet and commensal microbiota5-8, yet the cell types responsible for the generation of peripheral Treg (pTreg) cells have not been identified. Here we describe the identification of a class of RORγt+ antigen-presenting cells called Thetis cells, with transcriptional features of both mTECs and dendritic cells, comprising four major sub-groups (TC I-TC IV). We uncover a developmental wave of Thetis cells within intestinal lymph nodes during a critical window in early life, coinciding with the wave of pTreg cell differentiation. Whereas TC I and TC III expressed the signature mTEC nuclear factor AIRE, TC IV lacked AIRE expression and was enriched for molecules required for pTreg generation, including the TGF-ß-activating integrin αvß8. Loss of either major histocompatibility complex class II (MHCII) or ITGB8 by Thetis cells led to a profound impairment in intestinal pTreg differentiation, with ensuing colitis. By contrast, MHCII expression by RORγt+ group 3 innate lymphoid cells (ILC3) and classical dendritic cells was neither sufficient nor required for pTreg generation, further implicating TC IV as the tolerogenic RORγt+ antigen-presenting cell with an essential function in early life. Our studies reveal parallel pathways for the establishment of tolerance to self and foreign antigens in the thymus and periphery, respectively, marked by the involvement of shared cellular and transcriptional programmes.
Asunto(s)
Células Presentadoras de Antígenos , Células Dendríticas , Células Epiteliales , Microbioma Gastrointestinal , Tolerancia Inmunológica , Linfocitos T Reguladores , Timo , Diferenciación Celular , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Microbioma Gastrointestinal/inmunología , Inmunidad Innata , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Timo/citología , Timo/inmunología , Factor de Crecimiento Transformador beta/inmunología , Células Presentadoras de Antígenos/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Ganglios Linfáticos/inmunologíaRESUMEN
Through recurrent bouts synchronous with the hair cycle, quiescent melanocyte stem cells (McSCs) become activated to generate proliferative progeny that differentiate into pigment-producing melanocytes. The signaling factors orchestrating these events remain incompletely understood. Here, we use single-cell RNA sequencing with comparative gene expression analysis to elucidate the transcriptional dynamics of McSCs through quiescence, activation, and melanocyte maturation. Unearthing converging signs of increased WNT and BMP signaling along this progression, we endeavored to understand how these pathways are integrated. Employing conditional lineage-specific genetic ablation studies in mice, we found that loss of BMP signaling in the lineage leads to hair graying due to a block in melanocyte maturation. We show that interestingly, BMP signaling functions downstream from activated McSCs and maintains WNT effector, transcription factor LEF1. Employing pseudotime analysis, genetics, and chromatin landscaping, we show that following WNT-mediated activation of McSCs, BMP and WNT pathways collaborate to trigger the commitment of proliferative progeny by fueling LEF1- and MITF-dependent differentiation. Our findings shed light upon the signaling interplay and timing of cues that orchestrate melanocyte lineage progression in the hair follicle and underscore a key role for BMP signaling in driving complete differentiation.
Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Diferenciación Celular/genética , Melanocitos/citología , Transducción de Señal/genética , Células Madre/citología , Animales , Linaje de la Célula/genética , Perfilación de la Expresión Génica , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Ratones , Factor de Transcripción Asociado a Microftalmía/metabolismo , Análisis de la Célula IndividualRESUMEN
Cristae are high-curvature structures in the inner mitochondrial membrane (IMM) that are crucial for ATP production. While cristae-shaping proteins have been defined, analogous lipid-based mechanisms have yet to be elucidated. Here, we combine experimental lipidome dissection with multi-scale modeling to investigate how lipid interactions dictate IMM morphology and ATP generation. When modulating phospholipid (PL) saturation in engineered yeast strains, we observed a surprisingly abrupt breakpoint in IMM topology driven by a continuous loss of ATP synthase organization at cristae ridges. We found that cardiolipin (CL) specifically buffers the inner mitochondrial membrane against curvature loss, an effect that is independent of ATP synthase dimerization. To explain this interaction, we developed a continuum model for cristae tubule formation that integrates both lipid and protein-mediated curvatures. This model highlighted a snapthrough instability, which drives IMM collapse upon small changes in membrane properties. We also showed that cardiolipin is essential in low-oxygen conditions that promote PL saturation. These results demonstrate that the mechanical function of cardiolipin is dependent on the surrounding lipid and protein components of the IMM.
Asunto(s)
Cardiolipinas , Lipidómica , Cardiolipinas/metabolismo , Membranas Mitocondriales/metabolismo , Fosfolípidos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfato/metabolismoRESUMEN
Sweat glands are abundant in the body and essential for thermoregulation. Like mammary glands, they originate from epidermal progenitors. However, they display few signs of cellular turnover, and whether they have stem cells and tissue-regenerative capacity remains largely unexplored. Using lineage tracing, we here identify in sweat ducts multipotent progenitors that transition to unipotency after developing the sweat gland. In characterizing four adult stem cell populations of glandular skin, we show that they display distinct regenerative capabilities and remain unipotent when healing epidermal, myoepithelial-specific, and lumenal-specific injuries. We devise purification schemes and isolate and transcriptionally profile progenitors. Exploiting molecular differences between sweat and mammary glands, we show that only some progenitors regain multipotency to produce de novo ductal and glandular structures, but that these can retain their identity even within certain foreign microenvironments. Our findings provide insight into glandular stem cells and a framework for the further study of sweat gland biology.
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Células Madre Adultas/citología , Células Madre Adultas/fisiología , Homeostasis , Glándulas Sudoríparas/citología , Cicatrización de Heridas , Células Madre Adultas/clasificación , Animales , Células Epidérmicas , Epidermis/fisiología , Femenino , Humanos , Glándulas Mamarias Animales/citología , Ratones , Morfogénesis , Células Madre Multipotentes/fisiología , Análisis de Componente Principal , Trasplante de Células Madre , Glándulas Sudoríparas/embriología , Glándulas Sudoríparas/fisiologíaRESUMEN
Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometre resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations in that they visualize only a single slice or a relatively small volume of the cell, respectively. Focused ion beam-scanning electron microscopy (FIB-SEM) has demonstrated the ability to image small volumes of cellular samples with 4-nm isotropic voxels1. Owing to advances in the precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning, we have increased the volume that can be imaged with 4-nm voxels by two orders of magnitude. Here we present a volume EM atlas at such resolution comprising ten three-dimensional datasets for whole cells and tissues, including cancer cells, immune cells, mouse pancreatic islets and Drosophila neural tissues. These open access data (via OpenOrganelle2) represent the foundation of a field of high-resolution whole-cell volume EM and subsequent analyses, and we invite researchers to explore this atlas and pose questions.
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Conjuntos de Datos como Asunto , Difusión de la Información , Microscopía Electrónica de Rastreo , Orgánulos/ultraestructura , Animales , Línea Celular , Células Cultivadas , Drosophila melanogaster/citología , Drosophila melanogaster/ultraestructura , Femenino , Aparato de Golgi/ultraestructura , Humanos , Interfase , Islotes Pancreáticos/citología , Masculino , Ratones , Microscopía Electrónica de Rastreo/métodos , Microscopía Electrónica de Rastreo/normas , Microtúbulos/ultraestructura , Neuroglía/ultraestructura , Neuronas/ultraestructura , Publicación de Acceso Abierto , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/ultraestructura , Ribosomas/ultraestructura , Vesículas Sinápticas/ultraestructura , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/ultraestructuraRESUMEN
Astrotactin 2 (ASTN2) is a transmembrane neuronal protein highly expressed in the cerebellum that functions in receptor trafficking and modulates cerebellar Purkinje cell (PC) synaptic activity. Individuals with ASTN2 mutations exhibit neurodevelopmental disorders, including autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), learning difficulties, and language delay. To provide a genetic model for the role of the cerebellum in ASD-related behaviors and study the role of ASTN2 in cerebellar circuit function, we generated global and PC-specific conditional Astn2 knockout (KO and cKO, respectively) mouse lines. Astn2 KO mice exhibit strong ASD-related behavioral phenotypes, including a marked decrease in separation-induced pup ultrasonic vocalization calls, hyperactivity, repetitive behaviors, altered behavior in the three-chamber test, and impaired cerebellar-dependent eyeblink conditioning. Hyperactivity and repetitive behaviors are also prominent in Astn2 cKO animals, but they do not show altered behavior in the three-chamber test. By Golgi staining, Astn2 KO PCs have region-specific changes in dendritic spine density and filopodia numbers. Proteomic analysis of Astn2 KO cerebellum reveals a marked upregulation of ASTN2 family member, ASTN1, a neuron-glial adhesion protein. Immunohistochemistry and electron microscopy demonstrate a significant increase in Bergmann glia volume in the molecular layer of Astn2 KO animals. Electrophysiological experiments indicate a reduced frequency of spontaneous excitatory postsynaptic currents (EPSCs), as well as increased amplitudes of both spontaneous EPSCs and inhibitory postsynaptic currents in the Astn2 KO animals, suggesting that pre- and postsynaptic components of synaptic transmission are altered. Thus, ASTN2 regulates ASD-like behaviors and cerebellar circuit properties.
Asunto(s)
Trastorno del Espectro Autista , Cerebelo , Ratones Noqueados , Células de Purkinje , Animales , Ratones , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/fisiopatología , Células de Purkinje/metabolismo , Cerebelo/metabolismo , Conducta Animal/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Modelos Animales de Enfermedad , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , MasculinoRESUMEN
Here, we exploit the hair follicle to define the point at which stem cells (SCs) become irreversibly committed along a differentiation lineage. Employing histone and nucleotide double-pulse-chase and lineage tracing, we show that the early SC descendents en route to becoming transit-amplifying cells retain stemness and slow-cycling properties and home back to the bulge niche when hair growth stops. These become the primary SCs for the next hair cycle, whereas initial bulge SCs become reserves for injury. Proliferating descendents further en route irreversibly lose their stemness, although they retain many SC markers and survive, unlike their transit-amplifying progeny. Remarkably, these progeny also home back to the bulge. Combining purification and gene expression analysis with differential ablation and functional experiments, we define critical functions for these non-SC niche residents and unveil the intriguing concept that an irreversibly committed cell in an SC lineage can become an essential contributor to the niche microenvironment.
Asunto(s)
Folículo Piloso/citología , Folículo Piloso/crecimiento & desarrollo , Nicho de Células Madre/metabolismo , Células Madre/metabolismo , Animales , Antígenos CD34/metabolismo , Diferenciación Celular , Folículo Piloso/metabolismo , Humanos , Ratones , Piel/citologíaRESUMEN
Loss of normal tissue architecture is a hallmark of oncogenic transformation1. In developing organisms, tissues architectures are sculpted by mechanical forces during morphogenesis2. However, the origins and consequences of tissue architecture during tumorigenesis remain elusive. In skin, premalignant basal cell carcinomas form 'buds', while invasive squamous cell carcinomas initiate as 'folds'. Here, using computational modelling, genetic manipulations and biophysical measurements, we identify the biophysical underpinnings and biological consequences of these tumour architectures. Cell proliferation and actomyosin contractility dominate tissue architectures in monolayer, but not multilayer, epithelia. In stratified epidermis, meanwhile, softening and enhanced remodelling of the basement membrane promote tumour budding, while stiffening of the basement membrane promotes folding. Additional key forces stem from the stratification and differentiation of progenitor cells. Tumour-specific suprabasal stiffness gradients are generated as oncogenic lesions progress towards malignancy, which we computationally predict will alter extensile tensions on the tumour basement membrane. The pathophysiologic ramifications of this prediction are profound. Genetically decreasing the stiffness of basement membranes increases membrane tensions in silico and potentiates the progression of invasive squamous cell carcinomas in vivo. Our findings suggest that mechanical forces-exerted from above and below progenitors of multilayered epithelia-function to shape premalignant tumour architectures and influence tumour progression.
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Membrana Basal/metabolismo , Carcinoma Basocelular/metabolismo , Carcinoma Basocelular/patología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Actomiosina/metabolismo , Animales , Carcinogénesis , Proliferación Celular , Simulación por Computador , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Femenino , Humanos , Ratones , Invasividad Neoplásica , DocilidadRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Although in vitro studies of embryonic stem cells have identified polycomb repressor complexes (PRCs) as key regulators of differentiation, it remains unclear as to how PRC-mediated mechanisms control fates of multipotent progenitors in developing tissues. Here, we show that an essential PRC component, Ezh2, is expressed in epidermal progenitors but diminishes concomitant with embryonic differentiation and with postnatal decline in proliferative activity. We show that Ezh2 controls proliferative potential of basal progenitors by repressing the Ink4A-Ink4B locus and tempers the developmental rate of differentiation by preventing premature recruitment of AP1 transcriptional activator to the structural genes that are required for epidermal differentiation. Together, our studies reveal that PRCs control epigenetic modifications temporally and spatially in tissue-restricted stem cells. They maintain their proliferative potential and globally repressing undesirable differentiation programs while selectively establishing a specific terminal differentiation program in a stepwise fashion.
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Diferenciación Celular , Células Epidérmicas , Epidermis/metabolismo , Regulación del Desarrollo de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/metabolismo , Células Madre/metabolismo , Animales , Núcleo Celular/metabolismo , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Histonas/metabolismo , Humanos , Metilación , Ratones , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Proteínas Represoras/metabolismoRESUMEN
Hair follicles (HFs) undergo cyclical periods of growth, which are fueled by stem cells (SCs) at the base of the resting follicle. HF-SC formation occurs during HF development and requires transcription factor SOX9. Whether and how SOX9 functions in HF-SC maintenance remain unknown. By conditionally targeting Sox9 in adult HF-SCs, we show that SOX9 is essential for maintaining them. SOX9-deficient HF-SCs still transition from quiescence to proliferation and launch the subsequent hair cycle. However, once activated, bulge HF-SCs begin to differentiate into epidermal cells, which naturally lack SOX9. In addition, as HF-SC numbers dwindle, outer root sheath production is not sustained, and HF downgrowth arrests prematurely. Probing the mechanism, we used RNA sequencing (RNA-seq) to identify SOX9-dependent transcriptional changes and chromatin immunoprecipitation (ChIP) and deep sequencing (ChIP-seq) to identify SOX9-bound genes in HF-SCs. Intriguingly, a large cohort of SOX9-sensitive targets encode extracellular factors, most notably enhancers of Activin/pSMAD2 signaling. Moreover, compromising Activin signaling recapitulates SOX9-dependent defects, and Activin partially rescues them. Overall, our findings reveal roles for SOX9 in regulating adult HF-SC maintenance and suppressing epidermal differentiation in the niche. In addition, our studies expose a role for SCs in coordinating their own behavior in part through non-cell-autonomous signaling within the niche.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Folículo Piloso/citología , Folículo Piloso/metabolismo , Factor de Transcripción SOX9/metabolismo , Transducción de Señal , Activinas/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Células Epidérmicas , Perfilación de la Expresión Génica , Ratones , Receptores Notch/metabolismo , Factor de Transcripción SOX9/genética , Proteína Smad2/metabolismo , Células Madre/citología , Proteínas Wnt/metabolismoRESUMEN
Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic range of â¼10,000-fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by two orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact zebrafish. We found axon initial segment utilizes a "waterfall" mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially restricted subnuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics in vivo.
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Rastreo Celular/métodos , Neuronas/metabolismo , Vesículas Sinápticas/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Línea Celular Tumoral , Células Cultivadas , Humanos , Cinética , Neuronas/citología , Imagen de Lapso de Tiempo/métodos , Pez CebraRESUMEN
Adult stem cells reside in specialized niches where they receive environmental cues to maintain tissue homeostasis. In mammals, the stem cell niche within hair follicles is home to epithelial hair follicle stem cells and melanocyte stem cells, which sustain cyclical bouts of hair regeneration and pigmentation. To generate pigmented hairs, synchrony is achieved such that upon initiation of a new hair cycle, stem cells of each type activate lineage commitment. Dissecting the inter-stem-cell crosstalk governing this intricate coordination has been difficult, because mutations affecting one lineage often affect the other. Here we identify transcription factor NFIB as an unanticipated coordinator of stem cell behaviour. Hair follicle stem-cell-specific conditional targeting of Nfib in mice uncouples stem cell synchrony. Remarkably, this happens not by perturbing hair cycle and follicle architecture, but rather by promoting melanocyte stem cell proliferation and differentiation. The early production of melanin is restricted to melanocyte stem cells at the niche base. Melanocyte stem cells more distant from the dermal papilla are unscathed, thereby preventing hair greying typical of melanocyte stem cell differentiation mutants. Furthermore, we pinpoint KIT-ligand as a dermal papilla signal promoting melanocyte stem cell differentiation. Additionally, through chromatin-immunoprecipitation with high-throughput-sequencing and transcriptional profiling, we identify endothelin 2 (Edn2) as an NFIB target aberrantly activated in NFIB-deficient hair follicle stem cells. Ectopically induced Edn2 recapitulates NFIB-deficient phenotypes in wild-type mice. Conversely, endothelin receptor antagonists and/or KIT blocking antibodies prevent precocious melanocyte stem cell differentiation in the NFIB-deficient niche. Our findings reveal how melanocyte and hair follicle stem cell behaviours maintain reliance upon cooperative factors within the niche, and how this can be uncoupled in injury, stress and disease states.
Asunto(s)
Folículo Piloso/citología , Melanocitos/citología , Factores de Transcripción NFI/metabolismo , Nicho de Células Madre , Células Madre/citología , Células Madre/metabolismo , Animales , Apoptosis , Diferenciación Celular , Proliferación Celular , Inmunoprecipitación de Cromatina , Endotelina-2/genética , Endotelina-2/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Cabello/citología , Cabello/crecimiento & desarrollo , Color del Cabello , Folículo Piloso/metabolismo , Melanocitos/metabolismo , Ratones , Factores de Transcripción NFI/deficiencia , Factores de Transcripción NFI/genética , Análisis de Secuencia , Factor de Células Madre/metabolismoRESUMEN
Polycomb protein group (PcG)-dependent trimethylation on H3K27 (H3K27me3) regulates identity of embryonic stem cells (ESCs). How H3K27me3 governs adult SCs and tissue development is unclear. Here, we conditionally target H3K27 methyltransferases Ezh2 and Ezh1 to address their roles in mouse skin homeostasis. Postnatal phenotypes appear only in doubly targeted skin, where H3K27me3 is abolished, revealing functional redundancy in EZH1/2 proteins. Surprisingly, while Ezh1/2-null hair follicles (HFs) arrest morphogenesis and degenerate due to defective proliferation and increased apoptosis, epidermis hyperproliferates and survives engraftment. mRNA microarray studies reveal that, despite these striking phenotypic differences, similar genes are up-regulated in HF and epidermal Ezh1/2-null progenitors. Featured prominently are (1) PcG-controlled nonskin lineage genes, whose expression is still significantly lower than in native tissues, and (2) the PcG-regulated Ink4a/Inkb/Arf locus. Interestingly, when EZH1/2 are absent, even though Ink4a/Arf/Ink4b genes are fully activated in HF cells, they are only partially so in epidermal progenitors. Importantly, transduction of Ink4b/Ink4a/Arf shRNAs restores proliferation/survival of Ezh1/2-null HF progenitors in vitro, pointing toward the relevance of this locus to the observed HF phenotypes. Our findings reveal new insights into Polycomb-dependent tissue control, and provide a new twist to how different progenitors within one tissue respond to loss of H3K27me3.