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Deep Learning (DL) methods are powerful analytical tools for microscopy and can outperform conventional image processing pipelines. Despite the enthusiasm and innovations fuelled by DL technology, the need to access powerful and compatible resources to train DL networks leads to an accessibility barrier that novice users often find difficult to overcome. Here, we present ZeroCostDL4Mic, an entry-level platform simplifying DL access by leveraging the free, cloud-based computational resources of Google Colab. ZeroCostDL4Mic allows researchers with no coding expertise to train and apply key DL networks to perform tasks including segmentation (using U-Net and StarDist), object detection (using YOLOv2), denoising (using CARE and Noise2Void), super-resolution microscopy (using Deep-STORM), and image-to-image translation (using Label-free prediction - fnet, pix2pix and CycleGAN). Importantly, we provide suitable quantitative tools for each network to evaluate model performance, allowing model optimisation. We demonstrate the application of the platform to study multiple biological processes.
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Aprendizado Profundo , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Animais , Linhagem Celular Tumoral , Computação em Nuvem , Conjuntos de Dados como Assunto , Humanos , Cultura Primária de Células , Ratos , SoftwareRESUMO
[This corrects the article DOI: 10.1016/j.isci.2020.100907.].
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The link between integrin activity regulation and cellular mechanosensing of tissue rigidity, especially on different extracellular matrix ligands, remains poorly understood. Here, we find that primary mouse mammary gland stromal fibroblasts (MSFs) are able to spread efficiently, generate high forces, and display nuclear YAP on soft collagen-coated substrates, resembling the soft mammary gland tissue. We describe that loss of the integrin inhibitor, SHARPIN, impedes MSF spreading specifically on soft type I collagen but not on fibronectin. Through quantitative experiments and computational modeling, we find that SHARPIN-deficient MSFs display faster force-induced unbinding of adhesions from collagen-coated beads. Faster unbinding, in turn, impairs force transmission in these cells, particularly, at the stiffness optimum observed for wild-type cells. Mechanistically, we link the impaired mechanotransduction of SHARPIN-deficient cells on collagen to reduced levels of collagen-binding integrin α11ß1. Thus integrin activity regulation and α11ß1 play a role in collagen-specific mechanosensing in MSFs.
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Somatic stem cells are characterized by their capacity for self-renewal and differentiation, making them integral for normal tissue homeostasis. Different stem cell functions are strongly affected by the specialized microenvironment surrounding the cells. Consisting of soluble signaling factors, extracellular matrix (ECM) ligands and other cells, but also biomechanical cues such as the viscoelasticity and topography of the ECM, these factors are collectively known as the niche. Cell-ECM interactions are mediated largely by integrins, a class of heterodimeric cell adhesion molecules. Integrins bind their ligands in the extracellular space and associate with the cytoskeleton inside the cell, forming a direct mechanical link between the cells and their surroundings. Indeed, recent findings have highlighted the importance of integrins in translating biophysical cues into changes in cell signaling and function, a multistep process known as mechanotransduction. The mechanical properties of the stem cell niche are important, yet the underlying molecular details of integrin-mediated mechanotransduction in stem cells, especially the roles of the different integrin heterodimers, remain elusive. Here, we introduce the reader to the concept of integrin-mediated mechanotransduction, summarize current knowledge on the role of integrin signaling and mechanotransduction in regulation of somatic stem cell functions, and discuss open questions in the field.
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Células-Tronco Adultas/metabolismo , Integrinas/metabolismo , Mecanotransdução Celular , Transdução de Sinais , Animais , HumanosRESUMO
Loss of endothelial integrity promotes capillary leakage in numerous diseases, including sepsis, but there are no effective therapies for preserving endothelial barrier function. Angiopoietin-2 (ANGPT2) is a context-dependent regulator of vascular leakage that signals via both endothelial TEK receptor tyrosine kinase (TIE2) and integrins. Here, we show that antibodies against ß1-integrin decrease LPS-induced vascular leakage in murine endotoxemia, as either a preventative or an intervention therapy. ß1-integrin inhibiting antibodies bound to the vascular endothelium in vivo improved the integrity of endothelial cell-cell junctions and protected mice from endotoxemia-associated cardiac failure, without affecting endothelial inflammation, serum proinflammatory cytokine levels, or TIE receptor signaling. Moreover, conditional deletion of a single allele of endothelial ß1-integrin protected mice from LPS-induced vascular leakage. In endothelial monolayers, the inflammatory agents thrombin, lipopolysaccharide (LPS), and IL-1ß decreased junctional vascular endothelial (VE)-cadherin and induced actin stress fibers via ß1- and α5-integrins and ANGPT2. Additionally, ß1-integrin inhibiting antibodies prevented inflammation-induced endothelial cell contractility and monolayer permeability. Mechanistically, the inflammatory agents stimulated ANGPT2-dependent translocation of α5ß1-integrin into tensin-1-positive fibrillar adhesions, which destabilized the endothelial monolayer. Thus, ß1-integrin promotes endothelial barrier disruption during inflammation, and targeting ß1-integrin signaling could serve as a novel means of blocking pathological vascular leak.
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Células Endoteliais/metabolismo , Endotoxemia/metabolismo , Integrina beta1/metabolismo , Junções Intercelulares/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/patologia , Endotoxemia/induzido quimicamente , Endotoxemia/genética , Endotoxemia/patologia , Integrina alfa5beta1/genética , Integrina alfa5beta1/metabolismo , Integrina beta1/genética , Junções Intercelulares/genética , Junções Intercelulares/patologia , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Transgênicos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Receptor TIE-2/genética , Receptor TIE-2/metabolismoRESUMO
Cell-type-specific functions and identity are tightly regulated by interactions between the cell cytoskeleton and the extracellular matrix (ECM). Human pluripotent stem cells (hPSCs) have ultimate differentiation capacity and exceptionally low-strength ECM contact, yet the organization and function of adhesion sites and associated actin cytoskeleton remain poorly defined. We imaged hPSCs at the cell-ECM interface with total internal reflection fluorescence microscopy and discovered that adhesions at the colony edge were exceptionally large and connected by thick ventral stress fibers. The actin fence encircling the colony was found to exert extensive Rho-ROCK-myosin-dependent mechanical stress to enforce colony morphology, compaction, and pluripotency and to define mitotic spindle orientation. Remarkably, differentiation altered adhesion organization and signaling characterized by a switch from ventral to dorsal stress fibers, reduced mechanical stress, and increased integrin activity and cell-ECM adhesion strength. Thus, pluripotency appears to be linked to unique colony organization and adhesion structure.
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Actinas/metabolismo , Adesões Focais/metabolismo , Células-Tronco Pluripotentes/citologia , Actinas/ultraestrutura , Fenômenos Biomecânicos , Adesão Celular , Diferenciação Celular , Divisão Celular , Linhagem Celular , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Adesões Focais/ultraestrutura , Humanos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/ultraestrutura , Transdução de Sinais , Fibras de Estresse/metabolismo , Fibras de Estresse/ultraestruturaRESUMO
Tight regulation of integrin activity is paramount for dynamic cellular functions such as cell matrix adhesion and mechanotransduction. Integrin activation is achieved through intracellular interactions at the integrin cytoplasmic tails and through integrin-ligand binding. In this study, we identify the metabolic sensor AMP-activated protein kinase (AMPK) as a ß1-integrin inhibitor in fibroblasts. Loss of AMPK promotes ß1-integrin activity, the formation of centrally located active ß1-integrin- and tensin-rich mature fibrillar adhesions, and cell spreading. Moreover, in the absence of AMPK, cells generate more mechanical stress and increase fibronectin fibrillogenesis. Mechanistically, we show that AMPK negatively regulates the expression of the integrin-binding proteins tensin1 and tensin3. Transient expression of tensins increases ß1-integrin activity, whereas tensin silencing reduces integrin activity in fibroblasts lacking AMPK. Accordingly, tensin silencing in AMPK-depleted fibroblasts impedes enhanced cell spreading, traction stress, and fibronectin fiber formation. Collectively, we show that the loss of AMPK up-regulates tensins, which bind ß1-integrins, supporting their activity and promoting fibrillar adhesion formation and integrin-dependent processes.
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Proteínas Quinases Ativadas por AMP/metabolismo , Integrina beta1/metabolismo , Tensinas/metabolismo , Adesão Celular/fisiologia , Linhagem Celular , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Células HEK293 , Humanos , Mecanotransdução Celular/fisiologia , Proteínas dos Microfilamentos/metabolismo , Ligação Proteica/fisiologiaRESUMO
SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial-stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpincpdm), and mice with a stromal (S100a4-Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpincpdm mammary epithelial cells transplanted in vivo into wild-type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpincpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpincpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.
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Proteínas de Transporte/metabolismo , Diferenciação Celular , Colágeno/metabolismo , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Animais , Matriz Extracelular/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos KnockoutRESUMO
Instructive materials are expected to revolutionize stem cell based tissue engineering. As many stem cell cues have adverse effects on normal tissue homeostasis, there is a need to develop bioactive scaffolds which offer locally retained and cell-targeted drug delivery for intracellular release in targeted cell populations. Further, the scaffolds need to support vascularization to promote tissue growth and function. We have developed an electrospun PLA-PANI fiber scaffold, and incorporated mesoporous silica nanoparticles within the scaffold matrix to obtain cell-targeted and localized drug delivery. The isotropy of the scaffold can be tuned to find the optimal morphology for a given application and the scaffold is electroactive to support differentiation of contractile tissues. We demonstrate that there is no premature drug release from particles under physiological conditions over a period of one week and that the drug is released upon internalization of particles by cells within the scaffold. The scaffold is biocompatible, supports muscle stem cell differentiation and cell-seeded scaffolds are vascularized in vivo upon transplantation on the chorioallantoic membrane of chicken embryos. The scaffold is a step towards instructive biomaterials for local control of stem cell differentiation, and tissue formation supported by vascularization and without adverse effects on the homeostasis of adjacent tissues due to diffusion of biological cues.