RESUMO
Skeletal muscle stem cells (MuSCs) are recognised as functionally heterogeneous. Cranial MuSCs are reported to have greater proliferative and regenerative capacity when compared with those in the limb. A comprehensive understanding of the mechanisms underlying this functional heterogeneity is lacking. Here, we have used clonal analysis, live imaging and single cell transcriptomic analysis to identify crucial features that distinguish extraocular muscle (EOM) from limb muscle stem cell populations. A MyogeninntdTom reporter showed that the increased proliferation capacity of EOM MuSCs correlates with deferred differentiation and lower expression of the myogenic commitment gene Myod. Unexpectedly, EOM MuSCs activated in vitro expressed a large array of extracellular matrix components typical of mesenchymal non-muscle cells. Computational analysis underscored a distinct co-regulatory module, which is absent in limb MuSCs, as driver of these features. The EOM transcription factor network, with Foxc1 as key player, appears to be hardwired to EOM identity as it persists during growth, disease and in vitro after several passages. Our findings shed light on how high-performing MuSCs regulate myogenic commitment by remodelling their local environment and adopting properties not generally associated with myogenic cells.
Assuntos
Músculo Esquelético , Músculos Oculomotores , Camundongos , Animais , Músculo Esquelético/metabolismo , Músculos Oculomotores/metabolismo , Camundongos Endogâmicos C57BL , Proliferação de Células , Células-TroncoRESUMO
Accurately counting and localising cellular events from movies is an important bottleneck of high-content tissue/embryo live imaging. Here, we propose a new methodology based on deep learning that allows automatic detection of cellular events and their precise xyt localisation on live fluorescent imaging movies without segmentation. We focused on the detection of cell extrusion, the expulsion of dying cells from the epithelial layer, and devised DeXtrusion: a pipeline based on recurrent neural networks for automatic detection of cell extrusion/cell death events in large movies of epithelia marked with cell contour. The pipeline, initially trained on movies of the Drosophila pupal notum marked with fluorescent E-cadherin, is easily trainable, provides fast and accurate extrusion predictions in a large range of imaging conditions, and can also detect other cellular events, such as cell division or cell differentiation. It also performs well on other epithelial tissues with reasonable re-training. Our methodology could easily be applied for other cellular events detected by live fluorescent microscopy and could help to democratise the use of deep learning for automatic event detections in developing tissues.
Assuntos
Aprendizado de Máquina , Redes Neurais de Computação , Células Epiteliais , Morte Celular , MicroscopiaRESUMO
Meiotic maturation is a crucial step of oocyte formation, allowing its potential fertilization and embryo development. Elucidating this process is important for both fundamental research and assisted reproductive technology. However, few computational tools based on non-invasive measurements are available to characterize oocyte meiotic maturation. Here, we develop a computational framework to phenotype oocytes based on images acquired in transmitted light. We trained neural networks to segment the contour of oocytes and their zona pellucida using oocytes from diverse species. We defined a comprehensive set of morphological features to describe an oocyte. These steps were implemented in an open-source Fiji plugin. We present a feature-based machine learning pipeline to recognize oocyte populations and determine morphological differences between them. We first demonstrate its potential to screen oocytes from different strains and automatically identify their morphological characteristics. Its second application is to predict and characterize the maturation potential of oocytes. We identify the texture of the zona pellucida and cytoplasmic particle size as features to assess mouse oocyte maturation potential and tested whether these features were applicable to the developmental potential of human oocytes. This article has an associated First Person interview with the first author of the paper.
Assuntos
Células do Cúmulo , Oócitos , Animais , Feminino , Humanos , Aprendizado de Máquina , Camundongos , Oogênese/genética , Zona PelúcidaRESUMO
MOTIVATION: Due to the complexity and heterogeneity of multicellular biological systems, mathematical models that take into account cell signalling, cell population behaviour and the extracellular environment are particularly helpful. We present PhysiBoSS, an open source software which combines intracellular signalling using Boolean modelling (MaBoSS) and multicellular behaviour using agent-based modelling (PhysiCell). RESULTS: PhysiBoSS provides a flexible and computationally efficient framework to explore the effect of environmental and genetic alterations of individual cells at the population level, bridging the critical gap from single-cell genotype to single-cell phenotype and emergent multicellular behaviour. PhysiBoSS thus becomes very useful when studying heterogeneous population response to treatment, mutation effects, different modes of invasion or isomorphic morphogenesis events. To concretely illustrate a potential use of PhysiBoSS, we studied heterogeneous cell fate decisions in response to TNF treatment. We explored the effect of different treatments and the behaviour of several resistant mutants. We highlighted the importance of spatial information on the population dynamics by considering the effect of competition for resources like oxygen. AVAILABILITY AND IMPLEMENTATION: PhysiBoSS is freely available on GitHub (https://github.com/sysbio-curie/PhysiBoSS), with a Docker image (https://hub.docker.com/r/gletort/physiboss/). It is distributed as open source under the BSD 3-clause license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Modelos Genéticos , Transdução de Sinais , Software , Genótipo , Humanos , Transdução de Sinais/genética , Análise de SistemasRESUMO
The different actin structures governing eukaryotic cell shape and movement are not only determined by the properties of the actin filaments and associated proteins, but also by geometrical constraints. We recently demonstrated that limiting nucleation to specific regions was sufficient to obtain actin networks with different organization. To further investigate how spatially constrained actin nucleation determines the emergent actin organization, we performed detailed simulations of the actin filament system using Cytosim. We first calibrated the steric interaction between filaments, by matching, in simulations and experiments, the bundled actin organization observed with a rectangular bar of nucleating factor. We then studied the overall organization of actin filaments generated by more complex pattern geometries used experimentally. We found that the fraction of parallel versus antiparallel bundles is determined by the mechanical properties of actin filament or bundles and the efficiency of nucleation. Thus nucleation geometry, actin filaments local interactions, bundle rigidity, and nucleation efficiency are the key parameters controlling the emergent actin architecture. We finally simulated more complex nucleation patterns and performed the corresponding experiments to confirm the predictive capabilities of the model.
Assuntos
Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Modelos Moleculares , Multimerização Proteica , Citoesqueleto de Actina/ultraestrutura , Animais , Fenômenos Biomecânicos , Biologia Computacional , Simulação por Computador , Humanos , Técnicas In VitroRESUMO
A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named oocytes. Their development is marked by cell growth and subsequent divisions, two critical phases that prepare the oocyte for fusion with sperm to initiate embryogenesis. During growth, oocytes reorganize their cytoplasm to position the nucleus at the cell center, an event predictive of successful oocyte development in mice and humans and, thus, their embryogenic potential. In mouse oocytes, this cytoplasmic reorganization was shown to be driven by the cytoskeleton, the activity of which generates mechanical forces that agitate, reposition, and penetrate the nucleus. Consequently, this cytoplasmic-to-nucleoplasmic force transmission tunes the dynamics of nuclear RNA-processing organelles known as biomolecular condensates. This protocol provides an experimental framework to document, with high temporal resolution, the impact of the cytoskeleton on the nucleus across spatial scales in mouse oocytes. It details the imaging and image analysis steps and tools necessary to evaluate i) cytoskeletal activity in the oocyte cytoplasm, ii) cytoskeleton-based agitation of the oocyte nucleus, and iii) its effects on biomolecular condensate dynamics in the oocyte nucleoplasm. Beyond oocyte biology, the methods elaborated here can be adapted for use in somatic cells to similarly address cytoskeleton-based tuning of nuclear dynamics across scales.
Assuntos
Citoesqueleto , Sêmen , Humanos , Masculino , Feminino , Camundongos , Animais , Oócitos , Citoplasma , Núcleo CelularRESUMO
The cortex controls cell shape. In mouse oocytes, the cortex thickens in an Arp2/3-complex-dependent manner, ensuring chromosome positioning and segregation. Surprisingly, we identify that mouse oocytes lacking the Arp2/3 complex undergo cortical actin remodeling upon division, followed by cortical contractions that are unprecedented in mammalian oocytes. Using genetics, imaging, and machine learning, we show that these contractions stir the cytoplasm, resulting in impaired organelle organization and activity. Oocyte capacity to avoid polyspermy is impacted, leading to a reduced female fertility. We could diminish contractions and rescue cytoplasmic anomalies. Similar contractions were observed in human oocytes collected as byproducts during IVF (in vitro fertilization) procedures. These contractions correlate with increased cytoplasmic motion, but not with defects in spindle assembly or aneuploidy in mice or humans. Our study highlights a multiscale effect connecting cortical F-actin, contractions, and cytoplasmic organization and affecting oocyte quality, with implications for female fertility.
Assuntos
Oócitos , Fuso Acromático , Humanos , Feminino , Animais , Camundongos , Citoplasma , Citoesqueleto de Actina , Complexo 2-3 de Proteínas Relacionadas à Actina , Actinas , Meiose , MamíferosRESUMO
The mechanical properties of living cells reflect their physiological and pathological state. In particular, cancer cells undergo cytoskeletal modifications that typically make them softer than healthy cells, a property that could be used as a diagnostic tool. However, this is challenging because cells are complex structures displaying a broad range of morphologies when cultured in standard 2D culture dishes. Here, we use adhesive micropatterns to impose the cell geometry and thus standardize the mechanics and morphologies of cancer cells, which we measure by atomic force microscopy (AFM), mechanical nanomapping, and membrane nanotube pulling. We show that micropatterning cancer cells leads to distinct morphological and mechanical changes for different cell lines. Micropatterns did not systematically lower the variability in cell elastic modulus distribution. These effects emerge from a variable cell spreading rate associated with differences in the organization of the cytoskeleton, thus providing detailed insights into the structure-mechanics relationship of cancer cells cultured on micropatterns. Combining AFM with micropatterns reveals new mechanical and morphological observables applicable to cancer cells and possibly other cell types.
Assuntos
Citoesqueleto , Neoplasias , Humanos , Microscopia de Força Atômica , Linhagem Celular , Módulo de ElasticidadeRESUMO
The oocyte must grow and mature before fertilization, thanks to a close dialogue with the somatic cells that surround it. Part of this communication is through filopodia-like protrusions, called transzonal projections (TZPs), sent by the somatic cells to the oocyte membrane. To investigate the contribution of TZPs to oocyte quality, we impaired their structure by generating a full knockout mouse of the TZP structural component myosin-X (MYO10). Using spinning disk and super-resolution microscopy combined with a machine-learning approach to phenotype oocyte morphology, we show that the lack of Myo10 decreases TZP density during oocyte growth. Reduction in TZPs does not prevent oocyte growth but impairs oocyte-matrix integrity. Importantly, we reveal by transcriptomic analysis that gene expression is altered in TZP-deprived oocytes and that oocyte maturation and subsequent early embryonic development are partially affected, effectively reducing mouse fertility. We propose that TZPs play a role in the structural integrity of the germline-somatic complex, which is essential for regulating gene expression in the oocyte and thus its developmental potential.
Assuntos
Folículo Ovariano , Pseudópodes , Feminino , Animais , Camundongos , Folículo Ovariano/metabolismo , Oócitos/metabolismo , Oogênese/fisiologia , Células Germinativas , MiosinasRESUMO
Although great efforts to characterize the embryonic phase of brain microvascular system development have been made, its postnatal maturation has barely been described. Here, we compared the molecular and functional properties of brain vascular cells on postnatal day (P)5 vs. P15, via a transcriptomic analysis of purified mouse cortical microvessels (MVs) and the identification of vascular-cell-type-specific or -preferentially expressed transcripts. We found that endothelial cells (EC), vascular smooth muscle cells (VSMC) and fibroblasts (FB) follow specific molecular maturation programs over this time period. Focusing on VSMCs, we showed that the arteriolar VSMC network expands and becomes contractile resulting in a greater cerebral blood flow (CBF), with heterogenous developmental trajectories within cortical regions. Samples of the human brain cortex showed the same postnatal maturation process. Thus, the postnatal phase is a critical period during which arteriolar VSMC contractility required for vessel tone and brain perfusion is acquired and mature.
Assuntos
Células Endoteliais , Músculo Liso Vascular , Humanos , Camundongos , Animais , Músculo Liso Vascular/fisiologia , Encéfalo/irrigação sanguínea , Contração MuscularRESUMO
Cells remodel their cytoplasm with force-generating cytoskeletal motors. Their activity generates random forces that stir the cytoplasm, agitating and displacing membrane-bound organelles like the nucleus in somatic and germ cells. These forces are transmitted inside the nucleus, yet their consequences on liquid-like biomolecular condensates residing in the nucleus remain unexplored. Here, we probe experimentally and computationally diverse nuclear condensates, that include nuclear speckles, Cajal bodies, and nucleoli, during cytoplasmic remodeling of female germ cells named oocytes. We discover that growing mammalian oocytes deploy cytoplasmic forces to timely impose multiscale reorganization of nuclear condensates for the success of meiotic divisions. These cytoplasmic forces accelerate nuclear condensate collision-coalescence and molecular kinetics within condensates. Disrupting the forces decelerates nuclear condensate reorganization on both scales, which correlates with compromised condensate-associated mRNA processing and hindered oocyte divisions that drive female fertility. We establish that cytoplasmic forces can reorganize nuclear condensates in an evolutionary conserved fashion in insects. Our work implies that cells evolved a mechanism, based on cytoplasmic force tuning, to functionally regulate a broad range of nuclear condensates across scales. This finding opens new perspectives when studying condensate-associated pathologies like cancer, neurodegeneration and viral infections.
Assuntos
Nucléolo Celular , Núcleo Celular , Animais , Corpos Enovelados , Citoplasma , Feminino , Mamíferos , OócitosRESUMO
In this work, we analyzed the reliability of alginate-gelatin microcapsules as artificial tumor model. These tumor-like scaffolds are characterized by their composition and stiffness (â¼25 kPa), and their capability to restrict -but not hinder- cell migration, proliferation and release from confinement. Hydrogel-based microcapsules were initially utilized to detect differences in mechano-sensitivity between MCF7 and MDA-MB-231 breast cancer cells, and the endothelial cell line EA.hy926. Additionally, we used RNA-seq and transcriptomic methods to determine how the culture strategy (i.e. 2D v/s 3D) may pre-set the expression of genes involved in multidrug resistance, being then validated by performing cytotoxicological tests and assays of cell morphology. Our results show that both breast cancer cells can generate elongated multicellular spheroids inside the microcapsules, prior being released (mimicking intravasation stages), a behavior which was not observed in endothelial cells. Further, we demonstrate that cells isolated from 3D scaffolds show resistance to cisplatin, a process which seems to be strongly influenced by mechanical stress, instead of hypoxia. We finally discuss the role played by aneuploidy in malignancy and resistance to anticancer drugs, based on the increased number of polynucleated cells found within these microcapsules. Overall, our outcomes demonstrate that alginate-gelatin microcapsules represent a simple, yet very accurate tumor-like model, enabling us to mimic the most relevant malignant hints described in vivo, suggesting that confinement and mechanical stress need to be considered when studying pathogenicity and drug resistance of cancer cells in vitro. STATEMENT OF SIGNIFICANCE: In this work, we analyzed the reliability of alginate-gelatin microcapsules as an artificial tumor model. These scaffolds are characterized by their composition, elastic properties, and their ability to restrict cell migration, proliferation, and release from confinement. Our results demonstrate four novel outcomes: (i) studying cell migration and proliferation in 3D enabled discrimination between malignant and non-pathogenic cells, (ii) studying the cell morphology of cancer aggregates entrapped in alginate-gelatin microcapsules enabled determination of malignancy degree in vitro, (iii) determination that confinement and mechanical stress, instead of hypoxia, are required to generate clones resistant to anticancer drugs (i.e. cisplatin), and (iv) evidence that resistance to anticancer drugs could be due to the presence of polynucleated cells localized inside polymer-based artificial tumors.
Assuntos
Antineoplásicos , Neoplasias da Mama , Alginatos/farmacologia , Antineoplásicos/farmacologia , Cápsulas , Movimento Celular , Cisplatino/farmacologia , Resistência a Medicamentos , Células Endoteliais , Feminino , Gelatina/farmacologia , Humanos , Hidrogéis/farmacologia , Hipóxia , Reprodutibilidade dos TestesRESUMO
The centrosome is the main organizer of microtubules and as such, its position is a key determinant of polarized cell functions. As the name says, the default position of the centrosome is considered to be the cell geometrical center. However, the mechanism regulating centrosome positioning is still unclear and often confused with the mechanism regulating the position of the nucleus to which it is linked. Here, we used enucleated cells plated on adhesive micropatterns to impose regular and precise geometrical conditions to centrosome-microtubule networks. Although frequently observed there, the equilibrium position of the centrosome is not systematically at the cell geometrical center and can be close to cell edge. Centrosome positioning appears to respond accurately to the architecture and anisotropy of the actin network, which constitutes, rather than cell shape, the actual spatial boundary conditions the microtubule network is sensitive to. We found that the contraction of the actin network defines a peripheral margin in which microtubules appear bent by compressive forces. The progressive disassembly of the actin network at distance from the cell edges defines an inner zone where actin bundles were absent, where microtubules were more radially organized and where dynein concentration was higher. We further showed that the production of dynein-based forces on microtubules places the centrosome at the center of this zone. In conclusion, the spatial distribution of cell adhesion and the production of contractile forces define the architecture of the actin network with respect to which the centrosome-microtubule network is centered.
Assuntos
Actinas , Centrossomo , Dineínas , Miosinas , Actinas/metabolismo , Centrossomo/metabolismo , Dineínas/metabolismo , Microtúbulos/metabolismo , Miosinas/metabolismoRESUMO
As 2D surfaces fail to resemble the tumoral milieu, current discussions are focused on which 3D cell culture strategy may better lead the cells to express in vitro most of the malignant hints described in vivo. In this study, this question is assessed by analyzing the full genetic profile of MCF7 cells cultured either as 3D spheroids-considered as "gold standard" for in vitro cancer research- or immobilized in 3D tumor-like microcapsules, by RNA-Seq and transcriptomic methods, allowing to discriminate at big-data scale, which in vitro strategy can better resemble most of the malignant features described in neoplastic diseases. The results clearly show that mechanical stress, rather than 3D morphology only, stimulates most of the biological processes involved in cancer pathogenicity, such as cytoskeletal organization, migration, and stemness. Furthermore, cells entrapped in hydrogel-based scaffolds are likely expressing other physiological hints described in malignancy, such as the upregulated expression of metalloproteinases or the resistance to anticancer drugs, among others. According to the knowledge, this study represents the first attempt to answer which 3D experimental system can better mimic the neoplastic architecture in vitro, emphasizing the relevance of confinement in cancer pathogenicity, which can be easily achieved by using hydrogel-based matrices.
Assuntos
Neoplasias , Esferoides Celulares , Cápsulas , Técnicas de Cultura de Células , Humanos , Hidrogéis , Neoplasias/genética , Estresse MecânicoRESUMO
Ploidy variations such as genome doubling are frequent in human tumors and have been associated with genetic instability favoring tumor progression. How polyploid cells deal with increased centrosome numbers and DNA content remains unknown. Using Drosophila neuroblasts and human cancer cells to study mitotic spindle assembly in polyploid cells, we found that most polyploid cells divide in a multipolar manner. We show that even if an initial centrosome clustering step can occur at mitotic entry, the establishment of kinetochore-microtubule attachments leads to spatial chromosome configurations, whereby the final coalescence of supernumerary poles into a bipolar array is inhibited. Using in silico approaches and various spindle and DNA perturbations, we show that chromosomes act as a physical barrier blocking spindle pole coalescence and bipolarity. Importantly, microtubule stabilization suppressed multipolarity by improving both centrosome clustering and pole coalescence. This work identifies inhibitors of bipolar division in polyploid cells and provides a rationale to understand chromosome instability typical of polyploid cancer cells.
Assuntos
Centrossomo/metabolismo , Poliploidia , Fuso Acromático/metabolismo , Animais , Células Cultivadas , Drosophila , Feminino , Células HEK293 , Humanos , Fuso Acromático/genéticaRESUMO
Human and mouse oocytes' developmental potential can be predicted by their mechanical properties. Their development into blastocysts requires a specific stiffness window. In this study, we combine live-cell and computational imaging, laser ablation, and biophysical measurements to investigate how deregulation of cortex tension in the oocyte contributes to early developmental failure. We focus on extra-soft cells, the most common defect in a natural population. Using two independent tools to artificially decrease cortical tension, we show that chromosome alignment is impaired in extra-soft mouse oocytes, despite normal spindle morphogenesis and dynamics, inducing aneuploidy. The main cause is a cytoplasmic increase in myosin-II activity that could sterically hinder chromosome capture. We describe here an original mode of generation of aneuploidies that could be very common in oocytes and could contribute to the high aneuploidy rate observed during female meiosis, a leading cause of infertility and congenital disorders.
Assuntos
Aneuploidia , Proteínas do Citoesqueleto/metabolismo , Miosina Tipo II/metabolismo , Oócitos/patologia , Animais , Segregação de Cromossomos , Feminino , Infertilidade/etiologia , Meiose , Camundongos , OogêneseRESUMO
Nucleus centering in mouse oocytes results from a gradient of actin-positive vesicle activity and is essential for developmental success. Here, we analyze 3D model simulations to demonstrate how a gradient in the persistence of actin-positive vesicles can center objects of different sizes. We test model predictions by tracking the transport of exogenous passive tracers. The gradient of activity induces a centering force, akin to an effective pressure gradient, leading to the centering of oil droplets with velocities comparable to nuclear ones. Simulations and experimental measurements show that passive particles subjected to the gradient exhibit biased diffusion toward the center. Strikingly, we observe that the centering mechanism is maintained in meiosis I despite chromosome movement in the opposite direction; thus, it can counteract a process that specifically off-centers the spindle. In conclusion, our findings reconcile how common molecular players can participate in the two opposing functions of chromosome centering versus off-centering.
Assuntos
Núcleo Celular/metabolismo , Meiose , Prófase Meiótica I , Modelos Biológicos , Oócitos/metabolismo , Vesículas Transportadoras/metabolismo , Actinas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/genética , Células Cultivadas , Simulação por Computador , Difusão , Feminino , Gotículas Lipídicas/metabolismo , Camundongos , Análise Numérica Assistida por Computador , Tamanho das Organelas , Tamanho da Partícula , Fatores de Tempo , Vesículas Transportadoras/genéticaRESUMO
The mitotic spindle is an ensemble of microtubules responsible for the repartition of the chromosomal content between the two daughter cells during division. In metazoans, spindle assembly is a gradual process involving dynamic microtubules and recruitment of numerous associated proteins and motors. During mitosis, centrosomes organize and nucleate the majority of spindle microtubules. In contrast, oocytes lack canonical centrosomes but are still able to form bipolar spindles, starting from an initial ball that self-organizes in several hours. Interfering with early steps of meiotic spindle assembly can lead to erroneous chromosome segregation. Although not fully elucidated, this process is known to rely on antagonistic activities of plus end- and minus end-directed motors. We developed a model of early meiotic spindle assembly in mouse oocytes, including key factors such as microtubule dynamics and chromosome movement. We explored how the balance between plus end- and minus end-directed motors, as well as the influence of microtubule nucleation, impacts spindle morphology. In a refined model, we added spatial regulation of microtubule stability and minus-end clustering. We could reproduce the features of early stages of spindle assembly from 12 different experimental perturbations and predict eight additional perturbations. With its ability to characterize and predict chromosome individualization, this model can help deepen our understanding of spindle assembly.
Assuntos
Biologia Computacional/métodos , Fuso Acromático/metabolismo , Fuso Acromático/fisiologia , Animais , Divisão do Núcleo Celular , Centrossomo/metabolismo , Segregação de Cromossomos , Cromossomos/metabolismo , Simulação por Computador , Feminino , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose/fisiologia , Modelos Biológicos , Oócitos/metabolismoRESUMO
Successive cell divisions during embryonic cleavage create increasingly smaller cells, so intracellular structures must adapt accordingly. Mitotic spindle size correlates with cell size, but the mechanisms for this scaling remain unclear. Using live cell imaging, we analyzed spindle scaling during embryo cleavage in the nematode Caenorhabditis elegans and sea urchin Paracentrotus lividus. We reveal a common scaling mechanism, where the growth rate of spindle microtubules scales with cell volume, which explains spindle shortening. Spindle assembly timing is, however, constant throughout successive divisions. Analyses in silico suggest that controlling the microtubule growth rate is sufficient to scale spindle length and maintain a constant assembly timing. We tested our in silico predictions to demonstrate that modulating cell volume or microtubule growth rate in vivo induces a proportional spindle size change. Our results suggest that scalability of the microtubule growth rate when cell size varies adapts spindle length to cell volume.
Assuntos
Caenorhabditis elegans/embriologia , Tamanho Celular , Embrião não Mamífero/fisiologia , Microtúbulos/fisiologia , Paracentrotus/embriologia , Fuso Acromático/fisiologia , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Embrião não Mamífero/citologia , Paracentrotus/fisiologiaRESUMO
Tumor development progresses through a complex path of biomechanical changes leading first to cell growth and contraction and then cell deadhesion, scattering, and invasion. Tumorigenic factors may act specifically on one of these steps or have a wider spectrum of actions, leading to a variety of effects and thus sometimes to apparent contradictory outcomes. Here we used micropatterned lines of collagen type I/fibronectin on deformable surfaces to standardize cell behavior and measure simultaneously cell size, speed of motion and magnitude of the associated traction forces at the level of a single cell. We analyzed and compared the normal human breast cell line MCF10A in control conditions and in response to various tumorigenic factors. In all conditions, a wide range of biomechanical properties was identified. Despite this heterogeneity, normal and transformed motile cells followed a common trend whereby size and contractile forces were negatively correlated with cell speed. Some tumorigenic factors, such as activation of ErbB2 or loss of the ßsubunit of casein kinase 2, shifted the whole population toward a faster speed and lower contractility state. Treatment with transforming growth factor ß induced some cells to adopt opposing behaviors such as extremely high versus extremely low contractility. Thus tumor transformation amplified preexisting population heterogeneity and led some cells to exhibit biomechanical properties that were more extreme than those observed with normal cells.