RESUMEN
Understanding how complex tissues are formed, maintained, and regenerated through local growth, differentiation, and remodeling requires knowledge on how single-cell behaviors are coordinated on the population level. The self-renewing hair follicle, maintained by a distinct stem cell population, represents an excellent paradigm to address this question. A major obstacle in mechanistic understanding of hair follicle stem cell (HFSC) regulation has been the lack of a culture system that recapitulates HFSC behavior while allowing their precise monitoring and manipulation. Here, we establish an in vitro culture system based on a 3D extracellular matrix environment and defined soluble factors, which for the first time allows expansion and long-term maintenance of murine multipotent HFSCs in the absence of heterologous cell types. Strikingly, this scheme promotes de novo generation of HFSCs from non-HFSCs and vice versa in a dynamic self-organizing process. This bidirectional interconversion of HFSCs and their progeny drives the system into a population equilibrium state. Our study uncovers regulatory dynamics by which phenotypic plasticity of cells drives population-level homeostasis within a niche, and provides a discovery tool for studies on adult stem cell fate.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Folículo Piloso/citología , Técnicas de Cultivo de Órganos/métodos , Células Madre/fisiología , Animales , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Stem cells have the ability to self-renew and differentiate along multiple lineages, driving tissue homeostasis and regeneration. Paradigms of unidirectional, hierarchical differentiation trajectories observed in embryonic and hematopoietic stem cells have traditionally been applied to tissue-resident stem cells. However, accumulating evidence implicates stemness as a bidirectional, dynamic state that is largely governed by the niche, which facilitates plasticity and adaptability to changing conditions. In this Review, we discuss mechanisms of cell fate regulation through niche-derived cues, with a particular focus on epithelial stem cells of the mammalian skin, intestine and lung. We discuss a spectrum of niche-derived biochemical, mechanical and architectural inputs that define stem cell states during morphogenesis, homeostasis and regeneration, and highlight how these diverse inputs influence stem cell plasticity.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Plasticidad de la Célula , Transducción de Señal/fisiología , Nicho de Células Madre/fisiología , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Fenómenos Fisiológicos Celulares , Plasticidad de la Célula/genética , Humanos , Transducción de Señal/genética , Nicho de Células Madre/genéticaRESUMEN
Coordinated assembly and disassembly of actin into filaments and higher order structures such as stress fibers and lamellipodia are fundamental for cell migration and adhesion. However, the precise spatiotemporal regulation of F-actin structures is not completely understood. SWAP-70, a phosphatidylinositol 3,4,5-trisphosphate-interacting, F-actin-binding protein, participates in actin rearrangements through yet unknown mechanisms. Here, we show that SWAP-70 is an F-actin-bundling protein that oligomerizes through a Gln/Glu-rich stretch within a coiled-coil region. SWAP-70 bundles filaments in parallel and anti-parallel fashion through its C-terminal F-actin binding domain and delays dilution-induced F-actin depolymerization. We further demonstrate that SWAP-70 co-localizes and directly interacts with cofilin, an F-actin severing and depolymerization factor, and contributes to the regulation of cofilin activity in vivo. In line with these activities, upon stem cell factor stimulation, murine bone marrow-derived mast cells lacking SWAP-70 display aberrant regulation of F-actin and actin free barbed ends dynamics. Moreover, proper stem cell factor-dependent cofilin activation via dephosphorylation and subcellular redistribution into a detergent-resistant cytoskeletal compartment also require SWAP-70. Together, these findings reveal an important role of SWAP-70 in the dynamic spatiotemporal regulation of F-actin networks.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Factores Despolimerizantes de la Actina/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/deficiencia , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/deficiencia , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratones , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/deficiencia , Antígenos de Histocompatibilidad Menor , Células 3T3 NIH , Proteínas Nucleares/química , Proteínas Nucleares/deficiencia , Polimerizacion/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Factor de Células Madre/farmacología , Sus scrofaRESUMEN
The generation of plasma cells (PCs) is key for proper humoral immune responses. The transcription factors IRF-4 and BLIMP-1 (B-lymphocyte induce maturation protein-1) control PC commitment, but the underlying regulatory mechanisms are incompletely understood. Here we have identified SWAP-70 as being critically involved in Toll-like receptor (TLR)-triggered PC differentiation. Upon activation through various TLRs, Swap-70(-/-) B cells were activated and proliferated normally. However, expression of BLIMP-1 was markedly reduced and PC differentiation was impaired. Four hours of LPS stimulation were sufficient to drive PC differentiation, and SWAP-70 was required during this initial period. Swap-70(-/-) B cells pre-activated in vitro failed to efficiently differentiate into PCs upon adoptive transfer into recipient mice. Re-introduction of SWAP-70 into Swap-70(-/-) B cells rescued their development into PCs, and SWAP-70 over-expression in wild-type (WT) B cells increased PC generation. In the absence of SWAP-70, IRF-4 protein levels were reduced and the IRF-4(high) B220(+) CD138(-) compartment, including PC precursors, was strongly diminished. Ectopic expression of SWAP-70 increases IRF-4 protein levels and PC differentiation in WT and Swap-70(-/-) B cells, and IRF-4 over-expression in Swap-70(-/-) B cells elevates PC differentiation to WT levels. Thus, in a dose-dependent manner, SWAP-70 controls IRF-4 protein expression and thereby regulates the initiation of PC differentiation.
Asunto(s)
Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores Reguladores del Interferón/biosíntesis , Proteínas Nucleares/metabolismo , Células Plasmáticas/metabolismo , Factores de Transcripción/metabolismo , Animales , Anticuerpos Monoclonales , Linfocitos B/metabolismo , Inmunidad Humoral , Immunoblotting , Lipopolisacáridos/farmacología , Activación de Linfocitos , Ratones , Ratones Noqueados , Antígenos de Histocompatibilidad Menor , Células Plasmáticas/citología , Células Plasmáticas/inmunología , Reacción en Cadena de la Polimerasa , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismo , Factores de Transcripción/biosíntesisRESUMEN
Tissue turnover requires activation and lineage commitment of tissue-resident stem cells (SCs). These processes are impacted by ageing, but the mechanisms remain unclear. Here, we addressed the mechanisms of ageing in murine hair follicle SCs (HFSCs) and observed a widespread reduction in chromatin accessibility in aged HFSCs, particularly at key self-renewal and differentiation genes, characterized by bivalent promoters occupied by active and repressive chromatin marks. Consistent with this, aged HFSCs showed reduced ability to activate bivalent genes for efficient self-renewal and differentiation. These defects were niche dependent as the transplantation of aged HFSCs into young recipients or synthetic niches restored SC functions. Mechanistically, the aged HFSC niche displayed widespread alterations in extracellular matrix composition and mechanics, resulting in mechanical stress and concomitant transcriptional repression to silence promoters. As a consequence, increasing basement membrane stiffness recapitulated age-related SC changes. These data identify niche mechanics as a central regulator of chromatin state, which, when altered, leads to age-dependent SC exhaustion.
Asunto(s)
Diferenciación Celular , Autorrenovación de las Células , Senescencia Celular , Ensamble y Desensamble de Cromatina , Folículo Piloso/fisiología , Regiones Promotoras Genéticas , Nicho de Células Madre , Células Madre/fisiología , Animales , Diferenciación Celular/genética , Linaje de la Célula , Autorrenovación de las Células/genética , Células Cultivadas , Senescencia Celular/genética , Matriz Extracelular/fisiología , Silenciador del Gen , Folículo Piloso/citología , Folículo Piloso/metabolismo , Masculino , Mecanotransducción Celular , Ratones Endogámicos C57BL , Ratones Noqueados , Envejecimiento de la Piel , Células Madre/metabolismo , Estrés Mecánico , Transcripción GenéticaRESUMEN
Stem cells reside in specialized niches that are critical for their function. Upon activation, hair follicle stem cells (HFSCs) exit their niche to generate the outer root sheath (ORS), but a subset of ORS progeny returns to the niche to resume an SC state. Mechanisms of this fate reversibility are unclear. We show that the ability of ORS cells to return to the SC state requires suppression of a metabolic switch from glycolysis to oxidative phosphorylation and glutamine metabolism that occurs during early HFSC lineage progression. HFSC fate reversibility and glutamine metabolism are regulated by the mammalian target of rapamycin complex 2 (mTORC2)-Akt signaling axis within the niche. Deletion of mTORC2 results in a failure to re-establish the HFSC niche, defective hair follicle regeneration, and compromised long-term maintenance of HFSCs. These findings highlight the importance of spatiotemporal control of SC metabolic states in organ homeostasis.
Asunto(s)
Glutamina/metabolismo , Folículo Piloso/metabolismo , Células Madre/metabolismo , Animales , Células Cultivadas , Folículo Piloso/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Imagen Óptica , Células Madre/citologíaRESUMEN
SWAP-70 and DEF6, two proteins that feature similar domain and motif arrangements, are mainly known for their functions in differentiated hematopoietic cells. Both proteins interact with and regulate RhoGTPases and F-actin dynamics, yet their role in hematopoietic stem and precursor cells (HSPCs) remained unexplored. Here, the role of the SWEF proteins SWAP-70 and DEF6 in HSPCs was examined. Both SWEF proteins are expressed in HSCs. HSCs and different precursor populations were analyzed in mice deficient for SWAP-70, DEF6, SWAP-70 and DEF6 (double knockout, DKO), and wild-type controls. HSPCs isolated from these strains were used for competitive adoptive transfer into irradiated wild-type mice. Reconstitution of the myeloid and lymphoid lineages in the recipient mice was determined. The numbers of HSPCs in the bone marrow of Swap-70-/- and Swap-70-/-Def6-/- mice were >3-fold increased. When transplanted into lethally irradiated wild-type recipients, the reconstitution potential of Swap-70-/- HSPCs was intrinsically impaired in competing with wild-type HSPCs for contribution to hematopoiesis. Def6-/- HSPCs show wild type-like reconstitution potential under the same transplantation conditions. DKO HSPCs reconstituted to only 25% of wild-type levels, indicating a partial rescue by DEF6 deficiency in the Swap-70-/- background. Our study reveals the two SWEF proteins as important contributors to HSPC biology. Despite their similarity these two proteins regulate HSC/progenitor homeostasis, self-renewal, lineage contributions and repopulation in a distinct and mostly antagonistic manner.