RESUMEN
Force transmission through adherens junctions (AJs) is crucial for multicellular organization, wound healing and tissue regeneration. Recent studies shed light on the molecular mechanisms of mechanotransduction at the AJs. However, the canonical model fails to explain force transmission when essential proteins of the mechanotransduction module are mutated or missing. Here, we demonstrate that, in absence of α-catenin, ß-catenin can directly and functionally interact with vinculin in its open conformation, bearing physiological forces. Furthermore, we found that ß-catenin can prevent vinculin autoinhibition in the presence of α-catenin by occupying vinculin´s head-tail interaction site, thus preserving force transmission capability. Taken together, our findings suggest a multi-step force transmission process at AJs, where α-catenin and ß-catenin can alternatively and cooperatively interact with vinculin. This can explain the graded responses needed to maintain tissue mechanical homeostasis and, importantly, unveils a force-bearing mechanism involving ß-catenin and extended vinculin that can potentially explain the underlying process enabling collective invasion of metastatic cells lacking α-catenin.
Asunto(s)
Uniones Adherentes , Mecanotransducción Celular , Vinculina , alfa Catenina , beta Catenina , Vinculina/metabolismo , Uniones Adherentes/metabolismo , beta Catenina/metabolismo , alfa Catenina/metabolismo , alfa Catenina/genética , Animales , Humanos , Ratones , Unión ProteicaRESUMEN
Deviations from mirror symmetry in the development of bilateral organisms are common but the mechanisms of initial symmetry breaking are insufficiently understood. The actin cytoskeleton of individual cells self-organises in a chiral manner, but the molecular players involved remain essentially unidentified and the relationship between chirality of an individual cell and cell collectives is unclear. Here, we analysed self-organisation of the chiral actin cytoskeleton in individual cells on circular or elliptical patterns, and collective cell alignment in confined microcultures. Screening based on deep-learning analysis of actin patterns identified actin polymerisation regulators, depletion of which suppresses chirality (mDia1) or reverses chirality direction (profilin1 and CapZß). The reversed chirality is mDia1-independent but requires the function of actin-crosslinker α-actinin1. A robust correlation between the effects of a variety of actin assembly regulators on chirality of individual cells and cell collectives is revealed. Thus, actin-driven cell chirality may underlie tissue and organ asymmetry.
Asunto(s)
Citoesqueleto de Actina , ActinasRESUMEN
Contractile epithelial tubes are found in various organs, such as lung airways and blood capillaries. Their ability to sense luminal pressure and respond with adequate contractility is essential for their physiology, and its mis-regulation results in diseases such as asthma and hypertension. Here, we describe a mechanoresponsive regulatory pathway downstream of tissue stretching that controls contraction of the C. elegans spermatheca, a tubular structure where fertilization occurs. Using live-imaging, we show that ovulation-induced stretching of spermathecal cells leads to recruitment of the RhoGEF RHGF-1 to stress fibers, which activates RHO-1 and myosin II in a positive feedback loop. Through deletion analysis, we identified the PDZ domain of RHGF-1 as responsible for F-actin binding, and genetic epistasis analysis with the RhoGAP spv-1 demonstrated that tension-dependent recruitment of RHGF-1 to F-actin is required for robust spermathecal contractility. Our study illustrates how mechanosensitive regulators of Rho GTPases provide epithelial tubes the ability to tune their contractility in response to internal pressure.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Femenino , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Actinas/metabolismo , Fibras de Estrés/metabolismo , Contracción Muscular , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Proteínas Activadoras de GTPasa/metabolismoRESUMEN
Skeletal myogenesis is dynamic, and it involves cell-shape changes together with cell fusion and rearrangements. However, the final muscle arrangement is highly organized with striated fibers. By combining live imaging with quantitative analyses, we dissected fast-twitch myocyte fusion within the zebrafish myotome in toto. We found a strong mediolateral bias in fusion timing; however, at a cellular scale, there was heterogeneity in cell shape and the relationship between initial position of fast myocytes and resulting fusion partners. We show that the expression of the fusogen myomaker is permissive, but not instructive, in determining the spatiotemporal fusion pattern. Rather, we observed a close coordination between slow muscle rearrangements and fast myocyte fusion. In mutants that lack slow fibers, the spatiotemporal fusion pattern is substantially noisier. We propose a model in which slow muscles guide fast myocytes by funneling them close together, enhancing fusion probability. Thus, despite fusion being highly stochastic, a robust myotome structure emerges at the tissue scale.
Asunto(s)
Células Musculares , Pez Cebra , Animales , Desarrollo de Músculos , Músculo Esquelético/metabolismo , Músculos/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Current imaging approaches limit the ability to perform multi-scale characterization of three-dimensional (3D) organotypic cultures (organoids) in large numbers. Here, we present an automated multi-scale 3D imaging platform synergizing high-density organoid cultures with rapid and live 3D single-objective light-sheet imaging. It is composed of disposable microfabricated organoid culture chips, termed JeWells, with embedded optical components and a laser beam-steering unit coupled to a commercial inverted microscope. It permits streamlining organoid culture and high-content 3D imaging on a single user-friendly instrument with minimal manipulations and a throughput of 300 organoids per hour. We demonstrate that the large number of 3D stacks that can be collected via our platform allows training deep learning-based algorithms to quantify morphogenetic organizations of organoids at multi-scales, ranging from the subcellular scale to the whole organoid level. We validated the versatility and robustness of our approach on intestine, hepatic, neuroectoderm organoids and oncospheres.
Asunto(s)
Imagenología Tridimensional , Organoides , IntestinosRESUMEN
Cells sense a variety of extracellular growth factors and signaling molecules through numerous distinct receptor tyrosine kinases (RTKs) on the cell surface. In many cases, the same intracellular signaling molecules interact with more than one type of RTK. How signals from different RTKs retain the identity of the triggering receptor and how (or if) different receptors may synergize or compete remain largely unknown. Here we utilize an experimental strategy, combining microscale patterning and single-molecule imaging, to measure the competition between ephrin-A1:EphA2 and epidermal growth factor (EGF):EGF receptor (EGFR) ligand-receptor complexes for the shared downstream signaling molecules, Grb2 and SOS. The results reveal a distinct hierarchy, in which newly formed EGF:EGFR complexes outcompete ephrin-A1:EphA2 for Grb2 and SOS, revealing a type of negative crosstalk interaction fundamentally controlled by chemical mass action and protein copy number limitations.
Asunto(s)
Efrina-A1 , Receptor EphA2 , Factor de Crecimiento Epidérmico , Receptores ErbB/metabolismo , Retroalimentación , Receptor EphA2/metabolismo , Transducción de SeñalRESUMEN
Many pathogenic bacteria are encased in a layer of capsular polysaccharide (CPS). This layer is important for virulence by masking surface antigens, preventing opsonophagocytosis, and avoiding mucus entrapment. The bacterial tyrosine kinase (BY-kinase) regulates capsule synthesis and helps bacterial pathogens to survive different host niches. BY-kinases autophosphorylate at the C-terminal tyrosine residues upon external stimuli, but the role of phosphorylation is still unclear. Here, we report that the BY-kinase CpsCD is required for growth in Streptococcus pneumoniae Cells lacking a functional cpsC or cpsD accumulated low molecular weight CPS and lysed because of the lethal sequestration of the lipid carrier undecaprenyl phosphate, resulting in inhibition of peptidoglycan (PG) synthesis. CpsC interacts with CpsD and the polymerase CpsH. CpsD phosphorylation reduces the length of CPS polymers presumably by controlling the activity of CpsC. Finally, pulse-chase experiments reveal the spatiotemporal coordination between CPS and PG synthesis. This coordination is dependent on CpsC and CpsD. Together, our study provides evidence that BY-kinases regulate capsule polymer length by fine-tuning CpsC activity through autophosphorylation.
Asunto(s)
Cápsulas Bacterianas/metabolismo , Proteínas Bacterianas/metabolismo , Galactosiltransferasas/metabolismo , Polisacáridos Bacterianos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Streptococcus pneumoniae/enzimología , Proteínas Bacterianas/genética , Galactosiltransferasas/genética , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/crecimiento & desarrolloRESUMEN
Endothelial Cells (ECs) form cohesive cellular lining of the vasculature and play essential roles in both developmental processes and pathological conditions. Collective migration and proliferation of endothelial cells (ECs) are key processes underlying endothelialization of vessels as well as vascular graft, but the complex interplay of mechanical and biochemical signals regulating these processes are still not fully elucidated. While surface topography and biochemical modifications have been used to enhance endothelialization in vitro, thus far such single-modality modifications have met with limited success. As combination therapy that utilizes multiple modalities has shown improvement in addressing various intractable and complex biomedical conditions, here, we explore a combined strategy that utilizes topographical features in conjunction with pharmacological perturbations. We characterized EC behaviors in response to micrometer-scale grating topography in concert with pharmacological perturbations of endothelial adherens junctions (EAJ) regulators. We found that the protein tyrosine phosphatase, PTP1B, serves as a potent regulator of EAJ stability, with PTP1B inhibition synergizing with grating topographies to modulate EAJ rearrangement, thereby augmenting global EC monolayer sheet orientation, proliferation, connectivity, and collective cell migration. Our data delineates the crosstalk between cell-ECM topography sensing and cell-cell junction integrity maintenance and suggests that the combined use of grating topography and PTP1B inhibitor could be a promising strategy for promoting collective EC migration and proliferation.
Asunto(s)
Uniones Adherentes , Células Endoteliales , Línea Celular , Movimiento Celular , Endotelio VascularRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Microtubules display dynamic turnover during cell migration, leading to cell contractility and focal adhesion maturation regulated by Rho guanosine triphosphatase activity. This interplay between microtubules and actomyosin is mediated by guanine nucleotide exchange factor (GEF)-H1 released after microtubule depolymerization or microtubule disconnection from focal adhesions. However, how GEF-H1 activates Rho upon microtubule disassembly remains elusive. Here, we found that BNIP-2, a BCH domain-containing protein that binds both RhoA and GEF-H1 and traffics with kinesin-1 on microtubules, is important for GEF-H1-driven RhoA activation upon microtubule disassembly. Depletion of BNIP-2 in MDA-MB-231 breast cancer cells decreases RhoA activity and promotes cell migration. Upon nocodazole-induced microtubule disassembly, the interaction between BNIP-2 and GEF-H1 increases, while knockdown of BNIP-2 reduces RhoA activation and cell rounding via uncoupling RhoA-GEF-H1 interaction. Together, these findings revealed that BNIP-2 couples microtubules and focal adhesions via scaffolding GEF-H1 and RhoA, fine-tuning RhoA activity and cell migration.
Asunto(s)
Neoplasias de la Mama , Proteínas Portadoras/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Movimiento Celular , Femenino , Humanos , Microtúbulos/metabolismo , Nocodazol , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The Eph family of receptor tyrosine kinases is crucial for assembly and maintenance of healthy tissues. Dysfunction in Eph signaling is causally associated with cancer progression. In breast cancer cells, dysregulated Eph signaling has been linked to alterations in receptor clustering abilities. Here, we implemented a single-cell assay and a scoring scheme to systematically probe the spatial organization of activated EphA receptors in multiple carcinoma cells. We show that cancer cells retain EphA clustering phenotype over several generations, and the degree of clustering reported for migration potential both at population and single-cell levels. Finally, using patient-derived cancer lines, we probed the evolution of EphA signalling in cell populations that underwent metastatic transformation and acquisition of drug resistance. Taken together, our scalable approach provides a reliable scoring scheme for EphA clustering that is consistent over multiple carcinomas and can assay heterogeneity of cancer cell populations in a cost- and time-effective manner.
Asunto(s)
Carcinoma/genética , Familia de Multigenes/genética , Receptores de la Familia Eph/genética , Análisis de la Célula Individual , Carcinoma/patología , Heterogeneidad Genética , Humanos , Fenotipo , Transducción de Señal/genéticaRESUMEN
Cell migration over heterogeneous substrates during wound healing or morphogenetic processes leads to shape changes driven by different organizations of the actin cytoskeleton and by functional changes including lamellipodial protrusions and contractile actin cables. Cells distinguish between cell-sized positive and negative curvatures in their physical environment by forming protrusions at positive ones and actin cables at negative ones; however, the cellular mechanisms remain unclear. Here, we report that concave edges promote polarized actin structures with actin flow directed towards the cell edge, in contrast to well-documented retrograde flow at convex edges. Anterograde flow and contractility induce a tension anisotropy gradient. A polarized actin network is formed, accompanied by a local polymerization-depolymerization gradient, together with leading-edge contractile actin cables in the front. These cables extend onto non-adherent regions while still maintaining contact with the substrate through focal adhesions. The contraction and dynamic reorganization of this actin structure allows forward movements enabling cell migration over non-adherent regions on the substrate. These versatile functional structures may help cells sense and navigate their environment by adapting to external geometric and mechanical cues.
RESUMEN
Syncytial architecture is an evolutionarily-conserved feature of the germline of many species and plays a crucial role in their fertility. However, the mechanism supporting syncytial organization is largely unknown. Here, we identify a corset-like actomyosin structure within the syncytial germline of Caenorhabditis elegans, surrounding the common rachis. Using laser microsurgery, we demonstrate that actomyosin contractility within this structure generates tension both in the plane of the rachis surface and perpendicular to it, opposing membrane tension. Genetic and pharmacological perturbations, as well as mathematical modeling, reveal a balance of forces within the gonad and show how changing the tension within the actomyosin corset impinges on syncytial germline structure, leading, in extreme cases, to sterility. Thus, our work highlights a unique tissue-level cytoskeletal structure, and explains the critical role of actomyosin contractility in the preservation of a functional germline.
Asunto(s)
Actomiosina/metabolismo , Células Germinativas/metabolismo , Células Gigantes/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Corriente Citoplasmática , Gónadas/metabolismo , Modelos Biológicos , Miosinas/metabolismoRESUMEN
The cell cortex is essential to maintain animal cell shape, and contractile forces generated within it by nonmuscle myosin II (NMY-2) drive cellular morphogenetic processes such as cytokinesis. The role of actin cross-linking proteins in cortical dynamics is still incompletely understood. Here, we show that the evolutionarily conserved actin bundling/cross-linking protein plastin is instrumental for the generation of potent cortical actomyosin contractility in the Caenorhabditis elegans zygote. PLST-1 was enriched in contractile structures and was required for effective coalescence of NMY-2 filaments into large contractile foci and for long-range coordinated contractility in the cortex. In the absence of PLST-1, polarization was compromised, cytokinesis was delayed or failed, and 50% of embryos died during development. Moreover, mathematical modeling showed that an optimal amount of bundling agents enhanced the ability of a network to contract. We propose that by increasing the connectivity of the F-actin meshwork, plastin enables the cortex to generate stronger and more coordinated forces to accomplish cellular morphogenesis.
Asunto(s)
Actinas/metabolismo , Caenorhabditis elegans/citología , Polaridad Celular , Citocinesis , Glicoproteínas de Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Actomiosina/metabolismo , Animales , Caenorhabditis elegans/embriología , Forma de la Célula , Morfogénesis , Factores de Tiempo , Cigoto/citologíaRESUMEN
Classical cadherins are well known for their essential function in mediating cell-cell adhesion via their extra-cellular cadherin domains and intra-cellular connections to the actin cytoskeleton [1-3]. There is evidence, however, of adhesion-independent cadherin clusters existing outside of cell-cell junctions [4-6]. What function, if any, these clusters have is not known. HMR-1, the sole classical cadherin in Caenorhabditis elegans, plays essential roles during gastrulation, blastomere polarity establishment, and epidermal morphogenesis [7-11]. To elucidate the physiological roles of non-junctional cadherin, we analyzed HMR-1 in the C. elegans zygote, which is devoid of neighbors. We show that non-junctional clusters of HMR-1 form during the one-cell polarization stage and associate with F-actin at the cortex during episodes of cortical flow. Non-junctional HMR-1 clusters downregulate RHO-1 activity and inhibit accumulation of non-muscle myosin II (NMY-2) at the anterior cortex. We found that HMR-1 clusters impede cortical flows and play a role in preserving the integrity of the actomyosin cortex, preventing it from splitting in two. Importantly, we uncovered an inverse relationship between the amount of HMR-1 at the cell surface and the rate of cytokinesis. The effect of HMR-1 clusters on cytokinesis is independent of their effect on NMY-2 levels, and is also independent of their extra-cellular domains. Thus, in addition to their canonical role in inter-cellular adhesion, HMR-1 clusters regulate RHO-1 activity and NMY-2 level at the cell surface, reinforce the stability of the actomyosin cortex, and resist its movement to influence cell-shape dynamics.
Asunto(s)
Actomiosina/metabolismo , Cadherinas/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Cigoto/metabolismo , Actinas/metabolismo , Actomiosina/genética , Animales , Cadherinas/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Adhesión Celular , Citocinesis , Regulación de la Expresión Génica , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Collective migration of cells is of fundamental importance for a number of biological functions such as tissue development and regeneration, wound healing and cancer metastasis. The movement of cell groups consisting of multiple cells connected by cell-cell junctions depends on both extracellular and intercellular contacts. Epithelial cell assemblies are thus regulated by a cross-talk between cell-substrate and cell-cell interactions. Here, we investigated the onset of collective migration in groups of cells as they expand from a few cells into large colonies as a function of extracellular matrix (ECM) protein coating. By varying the amount of ECM presented to the cells, we observe that the mode of colony expansion, as well as their overall geometry, is strongly dependent on substrate adhesiveness. On high ECM protein coated surfaces, cells at the edges of the colonies are well spread exhibiting large outward-pointing protrusive activity, whereas cellular colonies display more circular and convex shapes on less adhesive surfaces. Actin structures at the edge of the colonies also show different organizations with the formation of lamellipodial structures on highly adhesive surfaces and a pluricellular actin cable on less adhesive ones. The analysis of traction forces and cell velocities within the cellular assemblies confirm these results. By increasing ECM protein density, cells exert higher traction forces together with a higher outward motility at the edges. Furthermore, tuning cell-cell adhesion of epithelial cells modified the mode of expansion of the colonies. Finally, we used a recently developed computational model to recapitulate the emergent experimental behaviors of expanding cell colonies and extract that the main effect of the different cell-substrate interactions is on the ability of edge cells to form outward lamellipodia-driven motility. Overall, our data suggest that switching behaviors of epithelial cell assemblies result in a tug-of-war between friction forces at the cell-substrate interface and cell-cell interactions.
Asunto(s)
Adhesión Celular/fisiología , Movimiento Celular/fisiología , Células Epiteliales/citología , Células Epiteliales/fisiología , Actomiosina/fisiología , Animales , Fenómenos Biomecánicos , Comunicación Celular/fisiología , Materiales Biocompatibles Revestidos , Simulación por Computador , Perros , Proteínas de la Matriz Extracelular/fisiología , Fibronectinas/fisiología , Células de Riñón Canino Madin Darby , Microscopía de Fuerza Atómica , Modelos Biológicos , Seudópodos/fisiología , Propiedades de SuperficieRESUMEN
Closure of wounds and gaps in tissues is fundamental for the correct development and physiology of multicellular organisms and, when misregulated, may lead to inflammation and tumorigenesis. To re-establish tissue integrity, epithelial cells exhibit coordinated motion into the void by active crawling on the substrate and by constricting a supracellular actomyosin cable. Coexistence of these two mechanisms strongly depends on the environment. However, the nature of their coupling remains elusive because of the complexity of the overall process. Here we demonstrate that epithelial gap geometry in both in vitro and in vivo regulates these collective mechanisms. In addition, the mechanical coupling between actomyosin cable contraction and cell crawling acts as a large-scale regulator to control the dynamics of gap closure. Finally, our computational modelling clarifies the respective roles of the two mechanisms during this process, providing a robust and universal mechanism to explain how epithelial tissues restore their integrity.