RESUMO
During wound healing, different pools of stem cells (SCs) contribute to skin repair. However, how SCs become activated and drive the tissue remodeling essential for skin repair is still poorly understood. Here, by developing a mouse model allowing lineage tracing and basal cell lineage ablation, we monitor SC fate and tissue dynamics during regeneration using confocal and intravital imaging. Analysis of basal cell rearrangements shows dynamic transitions from a solid-like homeostatic state to a fluid-like state allowing tissue remodeling during repair, as predicted by a minimal mathematical modeling of the spatiotemporal dynamics and fate behavior of basal cells. The basal cell layer progressively returns to a solid-like state with re-epithelialization. Bulk, single-cell RNA, and epigenetic profiling of SCs, together with functional experiments, uncover a common regenerative state regulated by the EGFR/AP1 axis activated during tissue fluidization that is essential for skin SC activation and tissue repair.
RESUMO
During embryonic and postnatal development, organs and tissues grow steadily to achieve their final size at the end of puberty. However, little is known about the cellular dynamics that mediate postnatal growth. By combining in vivo clonal lineage tracing, proliferation kinetics, single-cell transcriptomics, and in vitro micro-pattern experiments, we resolved the cellular dynamics taking place during postnatal skin epidermis expansion. Our data revealed that harmonious growth is engineered by a single population of developmental progenitors presenting a fixed fate imbalance of self-renewing divisions with an ever-decreasing proliferation rate. Single-cell RNA sequencing revealed that epidermal developmental progenitors form a more uniform population compared with adult stem and progenitor cells. Finally, we found that the spatial pattern of cell division orientation is dictated locally by the underlying collagen fiber orientation. Our results uncover a simple design principle of organ growth where progenitors and differentiated cells expand in harmony with their surrounding tissues.
Assuntos
Células Epidérmicas/metabolismo , Epiderme/crescimento & desenvolvimento , Pele/crescimento & desenvolvimento , Animais , Animais não Endogâmicos , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Linhagem da Célula/genética , Proliferação de Células/fisiologia , Células Cultivadas , Células Epidérmicas/patologia , Epiderme/metabolismo , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco/citologiaRESUMO
Key cellular decisions, such as proliferation or growth arrest, typically occur at spatially defined locations within tissues. Loss of this spatial control is a hallmark of many diseases, including cancer. Yet, how these patterns are established is incompletely understood. Here, we report that physical and architectural features of a multicellular sheet inform cells about their proliferative capacity through mechanical regulation of YAP and TAZ, known mediators of Hippo signaling and organ growth. YAP/TAZ activity is confined to cells exposed to mechanical stresses, such as stretching, location at edges/curvatures contouring an epithelial sheet, or stiffness of the surrounding extracellular matrix. We identify the F-actin-capping/severing proteins Cofilin, CapZ, and Gelsolin as essential gatekeepers that limit YAP/TAZ activity in cells experiencing low mechanical stresses, including contact inhibition of proliferation. We propose that mechanical forces are overarching regulators of YAP/TAZ in multicellular contexts, setting responsiveness to Hippo, WNT, and GPCR signaling.
Assuntos
Proteínas de Capeamento de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/metabolismo , Proliferação de Células , Fosfoproteínas/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Actinas/metabolismo , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Humanos , Fenômenos Mecânicos , Fosfoproteínas/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Proteínas de Sinalização YAPRESUMO
The ability of the skin to grow in response to stretching has been exploited in reconstructive surgery1. Although the response of epidermal cells to stretching has been studied in vitro2,3, it remains unclear how mechanical forces affect their behaviour in vivo. Here we develop a mouse model in which the consequences of stretching on skin epidermis can be studied at single-cell resolution. Using a multidisciplinary approach that combines clonal analysis with quantitative modelling and single-cell RNA sequencing, we show that stretching induces skin expansion by creating a transient bias in the renewal activity of epidermal stem cells, while a second subpopulation of basal progenitors remains committed to differentiation. Transcriptional and chromatin profiling identifies how cell states and gene-regulatory networks are modulated by stretching. Using pharmacological inhibitors and mouse mutants, we define the step-by-step mechanisms that control stretch-mediated tissue expansion at single-cell resolution in vivo.
Assuntos
Mecanotransdução Celular/fisiologia , Análise de Célula Única , Pele/citologia , Pele/crescimento & desenvolvimento , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Junções Aderentes/metabolismo , Animais , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Redes Reguladoras de Genes/efeitos dos fármacos , Hidrogéis/administração & dosagem , Hidrogéis/farmacologia , Mecanotransdução Celular/efeitos dos fármacos , Mecanotransdução Celular/genética , Camundongos , Camundongos Transgênicos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , RNA Mensageiro/genética , RNA-Seq , Pele/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Transativadores/antagonistas & inibidores , Transativadores/metabolismo , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteínas de Sinalização YAPRESUMO
Stratified epithelia are made up of several layers of cells, which act as a protective barrier for the organ they cover. To ensure their shielding effect, epithelia are naturally able to cope with constant environmental insults. This ability is enabled by their morphology and architecture, as well as the continuous turnover of stem and progenitor cells that constitute their building blocks. Stem cell fate decisions and dynamics are fundamental key biological processes that allow epithelia to exert their functions. By focusing on the skin epidermis, this review discusses how tissue architecture is generated during development, maintained through adult life, and re-established during regeneration.
Assuntos
Células Epidérmicas , Epiderme , Homeostase , Pele , Células-TroncoRESUMO
Inactivation of ARID1A and other components of the nuclear SWI/SNF protein complex occurs at very high frequencies in a variety of human malignancies, suggesting a widespread role for the SWI/SNF complex in tumour suppression1. However, the underlying mechanisms remain poorly understood. Here we show that ARID1A-containing SWI/SNF complex (ARID1A-SWI/SNF) operates as an inhibitor of the pro-oncogenic transcriptional coactivators YAP and TAZ2. Using a combination of gain- and loss-of-function approaches in several cellular contexts, we show that YAP/TAZ are necessary to induce the effects of the inactivation of the SWI/SNF complex, such as cell proliferation, acquisition of stem cell-like traits and liver tumorigenesis. We found that YAP/TAZ form a complex with SWI/SNF; this interaction is mediated by ARID1A and is alternative to the association of YAP/TAZ with the DNA-binding platform TEAD. Cellular mechanotransduction regulates the association between ARID1A-SWI/SNF and YAP/TAZ. The inhibitory interaction of ARID1A-SWI/SNF and YAP/TAZ is predominant in cells that experience low mechanical signalling, in which loss of ARID1A rescues the association between YAP/TAZ and TEAD. At high mechanical stress, nuclear F-actin binds to ARID1A-SWI/SNF, thereby preventing the formation of the ARID1A-SWI/SNF-YAP/TAZ complex, in favour of an association between TEAD and YAP/TAZ. We propose that a dual requirement must be met to fully enable the YAP/TAZ responses: promotion of nuclear accumulation of YAP/TAZ, for example, by loss of Hippo signalling, and inhibition of ARID1A-SWI/SNF, which can occur either through genetic inactivation or because of increased cell mechanics. This study offers a molecular framework in which mechanical signals that emerge at the tissue level together with genetic lesions activate YAP/TAZ to induce cell plasticity and tumorigenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mecanotransdução Celular , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Carcinogênese/genética , Proteínas de Ciclo Celular , Linhagem Celular , Núcleo Celular/metabolismo , Proliferação de Células , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Via de Sinalização Hippo , Humanos , Masculino , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/deficiência , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Mecânico , Fatores de Transcrição de Domínio TEA , Transativadores , Fatores de Transcrição/metabolismo , Via de Sinalização WntRESUMO
BACKGROUND & AIMS: In vitro, cell function can be potently regulated by the mechanical properties of cells and of their microenvironment. Cells measure these features by developing forces via their actomyosin cytoskeleton, and respond accordingly by regulating intracellular pathways, including the transcriptional coactivators YAP/TAZ. Whether mechanical cues are relevant for in vivo regulation of adult organ homeostasis, and whether this occurs through YAP/TAZ, remains largely unaddressed. METHODS: We developed Capzb conditional knockout mice and obtained primary fibroblasts to characterize the role of CAPZ in vitro. In vivo functional analyses were carried out by inducing Capzb inactivation in adult hepatocytes, manipulating YAP/Hippo activity by hydrodynamic tail vein injections, and treating mice with the ROCK inhibitor, fasudil. RESULTS: We found that the F-actin capping protein CAPZ restrains actomyosin contractility: Capzb inactivation alters stress fiber and focal adhesion dynamics leading to enhanced myosin activity, increased traction forces, and increased liver stiffness. In vitro, this rescues YAP from inhibition by a small cellular geometry; in vivo, it induces YAP activation in parallel to the Hippo pathway, causing extensive hepatocyte proliferation and leading to striking organ overgrowth. Moreover, Capzb is required for the maintenance of the differentiated hepatocyte state, for metabolic zonation, and for gluconeogenesis. In keeping with changes in tissue mechanics, inhibition of the contractility regulator ROCK, or deletion of the Yap1 mechanotransducer, reverse the phenotypes emerging in Capzb-null livers. CONCLUSIONS: These results indicate a previously unsuspected role for CAPZ in tuning the mechanical properties of cells and tissues, which is required in hepatocytes for the maintenance of the differentiated state and to regulate organ size. More generally, it indicates for the first time that mechanotransduction has a physiological role in maintaining liver homeostasis in mammals. LAY SUMMARY: The mechanical properties of cells and tissues (i.e. whether they are soft or stiff) are thought to be important regulators of cell behavior. Herein, we found that inactivation of the protein CAPZ alters the mechanical properties of cells and liver tissues, leading to YAP hyperactivation. In turn, this profoundly alters liver physiology, causing organ overgrowth, defects in liver cell differentiation and metabolism. These results reveal a previously uncharacterized role for mechanical signals in the maintenance of adult liver homeostasis.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína de Capeamento de Actina CapZ/metabolismo , Proteínas de Ciclo Celular/metabolismo , Hepatócitos/fisiologia , Fígado , Mecanotransdução Celular/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Células Cultivadas , Elasticidade , Via de Sinalização Hippo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Fígado/fisiopatologia , Camundongos , Camundongos Knockout , Transdução de Sinais , Proteínas de Sinalização YAPRESUMO
Increased glucose metabolism and reprogramming toward aerobic glycolysis are a hallmark of cancer cells, meeting their metabolic needs for sustained cell proliferation. Metabolic reprogramming is usually considered as a downstream consequence of tumor development and oncogene activation; growing evidence indicates, however, that metabolism on its turn can support oncogenic signaling to foster tumor malignancy. Here, we explored how glucose metabolism regulates gene transcription and found an unexpected link with YAP/TAZ, key transcription factors regulating organ growth, tumor cell proliferation and aggressiveness. When cells actively incorporate glucose and route it through glycolysis, YAP/TAZ are fully active; when glucose metabolism is blocked, or glycolysis is reduced, YAP/TAZ transcriptional activity is decreased. Accordingly, glycolysis is required to sustain YAP/TAZ pro-tumorigenic functions, and YAP/TAZ are required for the full deployment of glucose growth-promoting activity. Mechanistically we found that phosphofructokinase (PFK1), the enzyme regulating the first committed step of glycolysis, binds the YAP/TAZ transcriptional cofactors TEADs and promotes their functional and biochemical cooperation with YAP/TAZ. Strikingly, this regulation is conserved in Drosophila, where phosphofructokinase is required for tissue overgrowth promoted by Yki, the fly homologue of YAP. Moreover, gene expression regulated by glucose metabolism in breast cancer cells is strongly associated in a large dataset of primary human mammary tumors with YAP/TAZ activation and with the progression toward more advanced and malignant stages. These findings suggest that aerobic glycolysis endows cancer cells with particular metabolic properties and at the same time sustains transcription factors with potent pro-tumorigenic activities such as YAP/TAZ.
Assuntos
Bactérias Aeróbias/metabolismo , Fatores de Transcrição/metabolismo , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Drosophila , Glicólise/genética , Glicólise/fisiologia , Humanos , Imunoprecipitação , Fosfoproteínas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas de Sinalização YAPRESUMO
Cells perceive their microenvironment not only through soluble signals but also through physical and mechanical cues, such as extracellular matrix (ECM) stiffness or confined adhesiveness. By mechanotransduction systems, cells translate these stimuli into biochemical signals controlling multiple aspects of cell behaviour, including growth, differentiation and cancer malignant progression, but how rigidity mechanosensing is ultimately linked to activity of nuclear transcription factors remains poorly understood. Here we report the identification of the Yorkie-homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1) as nuclear relays of mechanical signals exerted by ECM rigidity and cell shape. This regulation requires Rho GTPase activity and tension of the actomyosin cytoskeleton, but is independent of the Hippo/LATS cascade. Crucially, YAP/TAZ are functionally required for differentiation of mesenchymal stem cells induced by ECM stiffness and for survival of endothelial cells regulated by cell geometry; conversely, expression of activated YAP overrules physical constraints in dictating cell behaviour. These findings identify YAP/TAZ as sensors and mediators of mechanical cues instructed by the cellular microenvironment.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mecanotransdução Celular/fisiologia , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Proteínas de Ciclo Celular , Diferenciação Celular , Linhagem Celular , Forma Celular , Sobrevivência Celular , Sinais (Psicologia) , Citoesqueleto/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Transativadores , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAPRESUMO
The Spemann organizer stands out from other signaling centers of the embryo because of its broad patterning effects. It defines development along the anteroposterior and dorsoventral axes of the vertebrate body, mainly by secreting antagonists of growth factors. Qualitative models proposed more than a decade ago explain the organizer's region-specific inductions (i.e., head and trunk) as the result of different combinations of antagonists. For example, head induction is mediated by extracellular inhibition of Wnt, BMP, and Nodal ligands. However, little is known about how the levels of these antagonists become harmonized with those of their targets and with the factors initially responsible for germ layers and organizer formation, including Nodal itself. Here we show that key ingredients of the head-organizer development, namely Nodal ligands, Nodal antagonists, and ADMP ligands reciprocally adjust each other's strength and range of activity by a self-regulating network of interlocked feedback and feedforward loops. A key element in this cross-talk is the limited availability of ACVR2a, for which Nodal and ADMP must compete. By trapping Nodal extracellularly, the Nodal antagonists Cerberus and Lefty are permissive for ADMP activity. The system self-regulates because ADMP/ACVR2a/Smad1 signaling in turn represses the expression of the Nodal antagonists, reestablishing the equilibrium. In sum, this work reveals an unprecedented set of interactions operating within the organizer that is critical for embryonic patterning.
Assuntos
Organizadores Embrionários/metabolismo , Receptores de Ativinas Tipo I/metabolismo , Receptores de Activinas Tipo II/metabolismo , Animais , Padronização Corporal , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Galinhas , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização In Situ , Ligantes , Modelos Biológicos , Transdução de Sinais , Fatores de Tempo , Transfecção , Xenopus laevis/metabolismoRESUMO
The definition of embryonic potency and induction of specific cell fates are intimately linked to the tight control over TGFbeta signaling. Although extracellular regulation of ligand availability has received considerable attention in recent years, surprisingly little is known about the intracellular factors that negatively control Smad activity in mammalian tissues. By means of genetic ablation, we show that the Smad4 inhibitor ectodermin (Ecto, also known as Trim33 or Tif1gamma) is required to limit Nodal responsiveness in vivo. New phenotypes, which are linked to excessive Nodal activity, emerge from such a modified landscape of Smad responsiveness in both embryonic and extra-embryonic territories. In extra-embryonic endoderm, Ecto is required to confine expression of Nodal antagonists to the anterior visceral endoderm. In trophoblast cells, Ecto precisely doses Nodal activity, balancing stem cell self-renewal and differentiation. Epiblast-specific Ecto deficiency shifts mesoderm fates towards node/organizer fates, revealing the requirement of Smad inhibition for the precise allocation of cells along the primitive streak. This study unveils that intracellular negative control of Smad function by ectodermin/Tif1gamma is a crucial element in the cellular response to TGFbeta signals in mammalian tissues.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas Smad/metabolismo , Fatores de Transcrição/metabolismo , Alelos , Animais , Padronização Corporal , Diferenciação Celular , Cruzamentos Genéticos , Ectoderma/metabolismo , Mesoderma/metabolismo , Camundongos , Modelos Biológicos , Fenótipo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
In this issue of Cell Stem Cell,Larsen et al. (2021) reveal the molecular machinery that empowers epidermal stem cells to remember. They find that STAT3 and AP1 family establish memory, enabling JUN to remain on the open chromatin and allowing fast recruitment of FOS in case of a second attack.
Assuntos
Proteínas Proto-Oncogênicas c-jun , Células-TroncoRESUMO
COVID-19 has unfortunately halted lab work, conferences, and in-person networking, which is especially detrimental to researchers just starting their labs. Through social media and our reviewer networks, we met some early-career stem cell investigators impacted by the closures. Here, they introduce themselves and their research to our readers.
Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Pesquisadores , Animais , COVID-19 , Humanos , Pandemias , SARS-CoV-2 , Células-Tronco/citologiaRESUMO
Wound healing is essential to repair the skin after injury. In the epidermis, distinct stem cells (SCs) populations contribute to wound healing. However, how SCs balance proliferation, differentiation and migration to repair a wound remains poorly understood. Here, we show the cellular and molecular mechanisms that regulate wound healing in mouse tail epidermis. Using a combination of proliferation kinetics experiments and molecular profiling, we identify the gene signatures associated with proliferation, differentiation and migration in different regions surrounding the wound. Functional experiments show that SC proliferation, migration and differentiation can be uncoupled during wound healing. Lineage tracing and quantitative clonal analysis reveal that, following wounding, progenitors divide more rapidly, but conserve their homoeostatic mode of division, leading to their rapid depletion, whereas SCs become active, giving rise to new progenitors that expand and repair the wound. These results have important implications for tissue regeneration, acute and chronic wound disorders.