RESUMO
In development, lineage segregation is coordinated in time and space. An important example is the mammalian inner cell mass, in which the primitive endoderm (PrE, founder of the yolk sac) physically segregates from the epiblast (EPI, founder of the fetus). While the molecular requirements have been well studied, the physical mechanisms determining spatial segregation between EPI and PrE remain elusive. Here, we investigate the mechanical basis of EPI and PrE sorting. We find that rather than the differences in static cell surface mechanical parameters as in classical sorting models, it is the differences in surface fluctuations that robustly ensure physical lineage sorting. These differential surface fluctuations systematically correlate with differential cellular fluidity, which we propose together constitute a non-equilibrium sorting mechanism for EPI and PrE lineages. By combining experiments and modeling, we identify cell surface dynamics as a key factor orchestrating the correct spatial segregation of the founder embryonic lineages.
Assuntos
Blastocisto , Embrião de Mamíferos , Endoderma , Animais , Blastocisto/metabolismo , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Membrana Celular/metabolismo , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Endoderma/metabolismo , Mamíferos , Camundongos , Transporte ProteicoRESUMO
Mechanical signalling affects multiple biological processes during development and in adult organisms, including cell fate transitions, cell migration, morphogenesis and immune responses. Here, we review recent insights into the mechanisms and functions of two main routes of mechanical signalling: outside-in mechanical signalling, such as mechanosensing of substrate properties or shear stresses; and mechanical signalling regulated by the physical properties of the cell surface itself. We discuss examples of how these two classes of mechanical signalling regulate stem cell function, as well as developmental processes in vivo. We also discuss how cell surface mechanics affects intracellular signalling and, in turn, how intracellular signalling controls cell surface mechanics, generating feedback into the regulation of mechanosensing. The cooperation between mechanosensing, intracellular signalling and cell surface mechanics has a profound impact on biological processes. We discuss here our understanding of how these three elements interact to regulate stem cell fate and development.
Assuntos
Fenômenos Biológicos , Mecanotransdução Celular , Diferenciação Celular , Mecanotransdução Celular/fisiologia , Morfogênese , Transdução de SinaisRESUMO
Naïve epiblast cells in the embryo and pluripotent stem cells in vitro undergo developmental progression to a formative state competent for lineage specification. During this transition, transcription factors and chromatin are rewired to encode new functional features. Here, we examine the role of mitogen-activated protein kinase (ERK1/2) signalling in pluripotent state transition. We show that a primary consequence of ERK activation in mouse embryonic stem cells is elimination of Nanog, which precipitates breakdown of the naïve state gene regulatory network. Variability in pERK dynamics results in heterogeneous loss of Nanog and metachronous state transition. Knockdown of Nanog allows exit without ERK activation. However, transition to formative pluripotency does not proceed and cells collapse to an indeterminate identity. This outcome is due to failure to maintain expression of the central pluripotency factor Oct4. Thus, during formative transition ERK signalling both dismantles the naïve state and preserves pluripotency. These results illustrate how a single signalling pathway can both initiate and secure transition between cell states.
Assuntos
Sistema de Sinalização das MAP Quinases , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero , Células-Tronco Pluripotentes , Animais , Proteína Homeobox Nanog/metabolismo , Proteína Homeobox Nanog/genética , Camundongos , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/genética , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/metabolismo , Camadas Germinativas/citologia , Redes Reguladoras de Genes , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genéticaRESUMO
The Company of Biologists' 2022 workshop on 'Cell State Transitions: Approaches, Experimental Systems and Models' brought together an international and interdisciplinary team of investigators spanning the fields of cell and developmental biology, stem cell biology, physics, mathematics and engineering to tackle the question of how cells precisely navigate between distinct identities and do so in a dynamic manner. This second edition of the workshop was organized after a successful virtual workshop on the same topic that took place in 2021.
Assuntos
Células-Tronco , Congressos como Assunto , Biologia Celular , Biologia do DesenvolvimentoRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Ageing causes a decline in tissue regeneration owing to a loss of function of adult stem cell and progenitor cell populations1. One example is the deterioration of the regenerative capacity of the widespread and abundant population of central nervous system (CNS) multipotent stem cells known as oligodendrocyte progenitor cells (OPCs)2. A relatively overlooked potential source of this loss of function is the stem cell 'niche'-a set of cell-extrinsic cues that include chemical and mechanical signals3,4. Here we show that the OPC microenvironment stiffens with age, and that this mechanical change is sufficient to cause age-related loss of function of OPCs. Using biological and synthetic scaffolds to mimic the stiffness of young brains, we find that isolated aged OPCs cultured on these scaffolds are molecularly and functionally rejuvenated. When we disrupt mechanical signalling, the proliferation and differentiation rates of OPCs are increased. We identify the mechanoresponsive ion channel PIEZO1 as a key mediator of OPC mechanical signalling. Inhibiting PIEZO1 overrides mechanical signals in vivo and allows OPCs to maintain activity in the ageing CNS. We also show that PIEZO1 is important in regulating cell number during CNS development. Thus we show that tissue stiffness is a crucial regulator of ageing in OPCs, and provide insights into how the function of adult stem and progenitor cells changes with age. Our findings could be important not only for the development of regenerative therapies, but also for understanding the ageing process itself.
Assuntos
Células-Tronco Adultas/patologia , Envelhecimento/patologia , Sistema Nervoso Central/patologia , Células-Tronco Multipotentes/patologia , Nicho de Células-Tronco , Animais , Animais Recém-Nascidos , Contagem de Células , Matriz Extracelular/patologia , Feminino , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Oligodendroglia/patologia , Ratos , Nicho de Células-Tronco/fisiologiaRESUMO
A fundamental challenge when studying biological systems is the description of cell state dynamics. During transitions between cell states, a multitude of parameters may change - from the promoters that are active, to the RNAs and proteins that are expressed and modified. Cells can also adopt different shapes, alter their motility and change their reliance on cell-cell junctions or adhesion. These parameters are integral to how a cell behaves and collectively define the state a cell is in. Yet, technical challenges prevent us from measuring all of these parameters simultaneously and dynamically. How, then, can we comprehend cell state transitions using finite descriptions? The recent virtual workshop organised by The Company of Biologists entitled 'Cell State Transitions: Approaches, Experimental Systems and Models' attempted to address this question. Here, we summarise some of the main points that emerged during the workshop's themed discussions. We also present examples of cell state transitions and describe models and systems that are pushing forward our understanding of how cells rewire their state.
Assuntos
Linhagem da Célula/genética , Regiões Promotoras Genéticas/genética , Proteínas/genética , RNA/genética , Adesão Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Junções Intercelulares/genética , Biologia de SistemasRESUMO
Proper control of division orientation and symmetry, largely determined by spindle positioning, is essential to development and homeostasis. Spindle positioning has been extensively studied in cells dividing in two-dimensional (2D) environments and in epithelial tissues, where proteins such as NuMA (also known as NUMA1) orient division along the interphase long axis of the cell. However, little is known about how cells control spindle positioning in three-dimensional (3D) environments, such as early mammalian embryos and a variety of adult tissues. Here, we use mouse embryonic stem cells (ESCs), which grow in 3D colonies, as a model to investigate division in 3D. We observe that, at the periphery of 3D colonies, ESCs display high spindle mobility and divide asymmetrically. Our data suggest that enhanced spindle movements are due to unequal distribution of the cell-cell junction protein E-cadherin between future daughter cells. Interestingly, when cells progress towards differentiation, division becomes more symmetric, with more elongated shapes in metaphase and enhanced cortical NuMA recruitment in anaphase. Altogether, this study suggests that in 3D contexts, the geometry of the cell and its contacts with neighbors control division orientation and symmetry. This article has an associated First Person interview with the first author of the paper.
Assuntos
Anáfase , Fuso Acromático , Animais , Junções Intercelulares , Metáfase , Camundongos , Mitose , Células-TroncoRESUMO
Cortical stiffness is an important cellular property that changes during migration, adhesion and growth. Previous atomic force microscopy (AFM) indentation measurements of cells cultured on deformable substrates have suggested that cells adapt their stiffness to that of their surroundings. Here we show that the force applied by AFM to a cell results in a significant deformation of the underlying substrate if this substrate is softer than the cell. This 'soft substrate effect' leads to an underestimation of a cell's elastic modulus when analysing data using a standard Hertz model, as confirmed by finite element modelling and AFM measurements of calibrated polyacrylamide beads, microglial cells and fibroblasts. To account for this substrate deformation, we developed a 'composite cell-substrate model'. Correcting for the substrate indentation revealed that cortical cell stiffness is largely independent of substrate mechanics, which has major implications for our interpretation of many physiological and pathological processes.
Assuntos
Córtex Cerebral/citologia , Diferenciação Celular , Módulo de Elasticidade , Microscopia de Força Atômica/métodos , Especificidade por SubstratoRESUMO
It is now well-established that the macrophage and microglial response to CNS demyelination influences remyelination by removing myelin debris and secreting a variety of signaling molecules that influence the behaviour of oligodendrocyte progenitor cells (OPCs). Previous studies have shown that changes in microglia contribute to the age-related decline in the efficiency of remyelination. In this study, we show that microglia increase their expression of the proteoglycan NG2 with age, and that this is associated with an altered micro-niche generated by aged, but not young, microglia that can divert the differentiation OPCs from oligodendrocytes into astrocytes in vitro. We further show that these changes in ageing microglia are generated by exposure to high levels of TGFß. Thus, our findings suggest that the rising levels of circulating TGFß known to occur with ageing contribute to the age-related decline in remyelination by impairing the ability of microglia to promote oligodendrocyte differentiation from OPCs, and therefore could be a potential therapeutic target to promote remyelination.
Assuntos
Senescência Celular/fisiologia , Microglia/metabolismo , Células Precursoras de Oligodendrócitos/metabolismo , Oligodendroglia/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fatores Etários , Animais , Animais Recém-Nascidos , Diferenciação Celular/fisiologia , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Relação Dose-Resposta a Droga , Microglia/efeitos dos fármacos , Células Precursoras de Oligodendrócitos/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Developmental cell biology requires technologies in which the fate of single cells is followed over extended time periods, to monitor and understand the processes of self-renewal, differentiation, and reprogramming. A workflow is presented, in which single cells are encapsulated into droplets (Ø: 80 µm, volume: ≈270 pL) and the droplet compartment is later converted to a hydrogel bead. After on-chip de-emulsification by electrocoalescence, these 3D scaffolds are subsequently arrayed on a chip for long-term perfusion culture to facilitate continuous cell imaging over 68 h. Here, the response of murine embryonic stem cells to different growth media, 2i and N2B27, is studied, showing that the exit from pluripotency can be monitored by fluorescence time-lapse microscopy, by immunostaining and by reverse-transcription and quantitative PCR (RT-qPCR). The defined 3D environment emulates the natural context of cell growth (e.g., in tissue) and enables the study of cell development in various matrices. The large scale of cell cultivation (in 2000 beads in parallel) may reveal infrequent events that remain undetected in lower throughput or ensemble studies. This platform will help to gain qualitative and quantitative mechanistic insight into the role of external factors on cell behavior.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Hidrogéis/farmacologia , Microesferas , Células-Tronco Embrionárias Murinas/citologia , Óptica e Fotônica/métodos , Perfusão , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Fenótipo , Reologia , Fatores de TempoRESUMO
Many biological processes, including tissue morphogenesis, are driven by cell sorting. However, the primary mechanical drivers of sorting in multicellular aggregates (MCAs) remain controversial, in part because there is no appropriate computational model to probe mechanical interactions between cells. To address this important issue, we developed a three-dimensional, local force-based simulation based on the subcellular element method. In our method, cells are modelled as collections of locally interacting force-bearing elements. We use the method to investigate the effects of tension and cell-cell adhesion on MCA sorting. We predict a minimum level of adhesion to produce inside-out sorting of two cell types, which is in excellent agreement with observations in several developmental systems. We also predict the level of tension asymmetry needed for robust sorting. The generality and flexibility of the method make it applicable to tissue self-organization in a myriad of other biological processes, such as tumorigenesis and embryogenesis.
Assuntos
Carcinogênese , Movimento Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Simulação por Computador , Modelos BiológicosRESUMO
Remyelination is a neuroprotective regenerative response to demyelination that restores saltatory conduction and decreases the vulnerability of axons to irreversible degeneration. It is a highly efficient process: however, as with all regenerative processes, its efficiency declines with ageing. Here we argue that this age-related decline in remyelination has a major impact on the natural history of multiple sclerosis (MS), a disease often of several decades' duration. We describe recent work on (1) how ageing changes the function of oligodendrocyte progenitor cells (OPCs), the cells primarily responsible for generating new myelin-forming oligodendrocytes in remyelination, (2) how these changes are induced by age-related changes in the OPC niche and (3) how these changes can be reversed, thereby opening up the possibility of therapeutically maintaining remyelination efficiency throughout the disease, preserving axonal health and treating the progressive phase of MS.
Assuntos
Envelhecimento/fisiologia , Células Precursoras de Oligodendrócitos/fisiologia , Remielinização/fisiologia , Substância Branca/fisiologia , HumanosRESUMO
Auxetic materials, characterised by a negative Poisson's ratio, have properties that are different from most conventional materials. These are a result of the constraints on the kinematics of the material's basic structural components, and have important technological implications. Models of these materials have been studied extensively, but theoretical descriptions have remained largely limited to materials with an ordered microstructure. Here we investigate whether negative Poisson's ratios can arise spontaneously in disordered systems. To this end, we develop a quantitative description of the structure in systems of connected basic elements, which enables us to analyse the local and global responses to small external tensile forces. We find that the Poisson's ratios in these disordered systems are equally likely to be positive or negative on both the element and system scales. Separating the strain into translational, rotational and expansive components, we find that the translational strains of neighbouring basic structural elements are positively correlated, while their rotations are negatively correlated. There is no correlation in this type of system between the local auxeticity and local structural characteristics. Our results suggest that auxeticity is more common in disordered structures than the ubiquity of positive Poisson's ratios in macroscopic materials would suggest.
RESUMO
The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells.
Assuntos
Membrana Celular/metabolismo , Elasticidade , Miosina Tipo II/metabolismo , Células 3T3 , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animais , Adesão Celular , Membrana Celular/ultraestrutura , Células HeLa , Humanos , Camundongos , Microfluídica , Miosina Tipo II/químicaRESUMO
Embryonic stem cells (ESCs) self-renew in a state of naïve pluripotency in which they are competent to generate all somatic cells. It has been hypothesized that, before irreversibly committing, ESCs pass through at least one metastable transition state. This transition would represent a gateway for differentiation and reprogramming of somatic cells. Here, we show that during the transition, the nuclei of ESCs are auxetic: they exhibit a cross-sectional expansion when stretched and a cross-sectional contraction when compressed, and their stiffness increases under compression. We also show that the auxetic phenotype of transition ESC nuclei is driven at least in part by global chromatin decondensation. Through the regulation of molecular turnover in the differentiating nucleus by external forces, auxeticity could be a key element in mechanotransduction. Our findings highlight the importance of nuclear structure in the regulation of differentiation and reprogramming.
Assuntos
Diferenciação Celular , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Células-Tronco Embrionárias/citologia , Camundongos , Células-Tronco Pluripotentes/citologiaRESUMO
A classical question in biology is how different processes are controlled in space and time, with research pointing to different mechanisms as timers. In this collection of Voices, we asked researchers to define their scientific questions related to time-keeping and the approaches they use to answer them.
Assuntos
Fenômenos Cronobiológicos , Animais , Crescimento e DesenvolvimentoRESUMO
Biomechanical cues are instrumental in guiding embryonic development and cell differentiation. Understanding how these physical stimuli translate into transcriptional programs will provide insight into mechanisms underlying mammalian pre-implantation development. Here, we explore this type of regulation by exerting microenvironmental control over mouse embryonic stem cells. Microfluidic encapsulation of mouse embryonic stem cells in agarose microgels stabilizes the naive pluripotency network and specifically induces expression of Plakoglobin (Jup), a vertebrate homolog of ß-catenin. Overexpression of Plakoglobin is sufficient to fully re-establish the naive pluripotency gene regulatory network under metastable pluripotency conditions, as confirmed by single-cell transcriptome profiling. Finally, we find that, in the epiblast, Plakoglobin was exclusively expressed at the blastocyst stage in human and mouse embryos - further strengthening the link between Plakoglobin and naive pluripotency in vivo. Our work reveals Plakoglobin as a mechanosensitive regulator of naive pluripotency and provides a paradigm to interrogate the effects of volumetric confinement on cell-fate transitions.
Assuntos
Desenvolvimento Embrionário , Camadas Germinativas , Animais , Camundongos , Humanos , gama Catenina/genética , gama Catenina/metabolismo , Diferenciação Celular/genética , Camadas Germinativas/metabolismo , Desenvolvimento Embrionário/genética , Perfilação da Expressão Gênica , Blastocisto/metabolismo , Mamíferos/genéticaRESUMO
The interplay between epigenetic modification and chromatin compaction is implicated in the regulation of gene expression, and it comprises one of the most fascinating frontiers in cell biology. Although a complete picture is still lacking, it is generally accepted that the differentiation of embryonic stem (ES) cells is accompanied by a selective condensation into heterochromatin with concomitant gene silencing, leaving access only to lineage-specific genes in the euchromatin. ES cells have been reported to have less condensed chromatin, as they are capable of differentiating into any cell type. However, pluripotency itself-even prior to differentiation-is a split state comprising a naïve state and a state in which ES cells prime for differentiation. Here, we show that naïve ES cells decondense their chromatin in the course of downregulating the pluripotency marker Nanog before they initiate lineage commitment. We used fluorescence recovery after photobleaching, and histone modification analysis paired with a novel, to our knowledge, optical stretching method, to show that ES cells in the naïve state have a significantly stiffer nucleus that is coupled to a globally more condensed chromatin state. We link this biophysical phenotype to coinciding epigenetic differences, including histone methylation, and show a strong correlation of chromatin condensation and nuclear stiffness with the expression of Nanog. Besides having implications for transcriptional regulation and embryonic cell sorting and suggesting a putative mechanosensing mechanism, the physical differences point to a system-level regulatory role of chromatin in maintaining pluripotency in embryonic development.