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Nuclear homeostasis requires a balance of forces between the cytoskeleton and nucleus. Variants in LMNA disrupt this balance by weakening the nuclear lamina, resulting in nuclear damage in contractile tissues and ultimately muscle disease. Intriguingly, disrupting the LINC complex that connects the cytoskeleton to the nucleus has emerged as a promising strategy to ameliorate LMNA cardiomyopathy. Yet how LINC disruption protects the cardiomyocyte nucleus remains unclear. To address this, we developed an assay to quantify the coupling of cardiomyocyte contraction to nuclear deformation and interrogated its dependence on the lamina and LINC complex. We found that the LINC complex was surprisingly dispensable for transferring the majority of contractile strain into the nucleus, and that increased nuclear strain in Lmna-deficient myocytes was not rescued by LINC disruption. However, LINC disruption eliminated the cage of microtubules encircling the nucleus, and disrupting microtubules was sufficient to prevent nuclear damage induced by LMNA deficiency. Through computational modeling we simulated the mechanical stress fields surrounding cardiomyocyte nuclei and show how microtubule compression exploits local vulnerabilities to damage LMNA-deficient nuclei. Our work pinpoints localized, microtubule-dependent force transmission through the LINC complex as a pathological driver and therapeutic target for LMNA cardiomyopathy.
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LMNA- related dilated cardiomyopathy ( LMNA -DCM) is one of the most severe forms of DCM. The incomplete understanding of the molecular disease mechanisms results in lacking treatment options, leading to high mortality amongst patients. Here, using an inducible, cardiomyocyte-specific lamin A/C depletion mouse model, we conducted a comprehensive transcriptomic study, combining both bulk and single nucleus RNA sequencing, and spanning LMNA -DCM disease progression, to identify potential disease drivers. Our refined analysis pipeline identified 496 genes already misregulated early in disease. The expression of these genes was largely driven by disease specific cardiomyocyte sub-populations and involved biological processes mediating cellular response to DNA damage, cytosolic pattern recognition, and innate immunity. Indeed, DNA damage in LMNA -DCM hearts was significantly increased early in disease and correlated with reduced cardiomyocyte lamin A levels. Activation of cytosolic pattern recognition in cardiomyocytes was independent of cGAS, which is rarely expressed in cardiomyocytes, but likely occurred downstream of other pattern recognition sensors such as IFI16. Altered gene expression in cardiac fibroblasts and immune cell infiltration further contributed to tissue-wide changes in gene expression. Our transcriptomic analysis further predicted significant alterations in cell-cell communication between cardiomyocytes, fibroblasts, and immune cells, mediated through early changes in the extracellular matrix (ECM) in the LMNA -DCM hearts. Taken together, our work suggests a model in which nuclear damage in cardiomyocytes leads to activation of DNA damage responses, cytosolic pattern recognition pathway, and other signaling pathways that activate inflammation, immune cell recruitment, and transcriptional changes in cardiac fibroblasts, which collectively drive LMNA -DCM pathogenesis.
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The vertebrate intestine forms by asymmetric gut rotation and elongation, and errors cause lethal obstructions in human infants. Rotation begins with tissue deformation of the dorsal mesentery, which is dependent on left-sided expression of the Paired-like transcription factor Pitx2. The conserved morphogen Nodal induces asymmetric Pitx2 to govern embryonic laterality, but organ-level regulation of Pitx2 during gut asymmetry remains unknown. We found Nodal to be dispensable for Pitx2 expression during mesentery deformation. Intestinal rotation instead required a mechanosensitive latent transforming growth factor-ß (TGFß), tuning a second wave of Pitx2 that induced reciprocal tissue stiffness in the left mesentery as mechanical feedback with the right side. This signaling regulator, an accelerator (right) and brake (left), combines biochemical and biomechanical inputs to break gut morphological symmetry and direct intestinal rotation.
Assuntos
Gastrulação , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio , Intestinos , Mecanotransdução Celular , Proteína Nodal , Fatores de Transcrição , Fator de Crescimento Transformador beta , Animais , Embrião de Galinha , Gastrulação/genética , Gastrulação/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/farmacologia , Intestinos/embriologia , Mecanotransdução Celular/genética , Mecanotransdução Celular/fisiologia , Camundongos , Proteína Nodal/genética , Fatores de Transcrição/genética , Fatores de Transcrição/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Proteína Homeobox PITX2RESUMO
Aberrations in nuclear size and shape are commonly used to identify cancerous tissue. However, it remains unclear whether the disturbed nuclear structure directly contributes to the cancer pathology or is merely a consequence of other events occurring during tumorigenesis. Here, we show that highly invasive and proliferative breast cancer cells frequently exhibit Akt-driven lower expression of the nuclear envelope proteins lamin A/C, leading to increased nuclear deformability that permits enhanced cell migration through confined environments that mimic interstitial spaces encountered during metastasis. Importantly, increasing lamin A/C expression in highly invasive breast cancer cells reflected gene expression changes characteristic of human breast tumors with higher LMNA expression, and specifically affected pathways related to cell-ECM interactions, cell metabolism, and PI3K/Akt signaling. Further supporting an important role of lamins in breast cancer metastasis, analysis of lamin levels in human breast tumors revealed a significant association between lower lamin A levels, Akt signaling, and decreased disease-free survival. These findings suggest that downregulation of lamin A/C in breast cancer cells may influence both cellular physical properties and biochemical signaling to promote metastatic progression.
Assuntos
Neoplasias da Mama , Lamina Tipo A , Neoplasias da Mama/patologia , Movimento Celular , Feminino , Humanos , Lamina Tipo A/genética , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-aktRESUMO
Mechanobiology is a rapidly growing research area focused on how mechanical forces and properties influence biological systems at the cell, molecular, and tissue level, and how those biological systems, in turn, control mechanical parameters. Recently, it has become apparent that disrupted mechanobiology has a significant role in many diseases, from cardiovascular disease to muscular dystrophy and cancer. An improved understanding of this intricate process could be harnessed toward developing alternative and more targeted treatment strategies, and to advance the fields of regenerative and personalized medicine. Modulating the mechanical properties of the cellular microenvironment has already been used successfully to boost antitumor immune responses and to induce cardiac and spinal regeneration, providing inspiration for further research in this area.
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Neoplasias , Biofísica , Humanos , Mecanotransdução Celular , Microambiente TumoralRESUMO
Nuclei within cells are constantly subjected to compressive, tensile, and shear forces, which regulate nucleoskeletal and cytoskeletal remodeling, activate signaling pathways, and direct cell-fate decisions. Multiple rheological methods have been adapted for characterizing the response to applied forces of isolated nuclei and nuclei within intact cells. However, in vitro measurements fail to capture the viscoelastic modulation of nuclear stress-strain relationships by the physiological tethering to the surrounding cytoskeleton, extracellular matrix and cells, and tissue-level architectures. Using an equiaxial stretching apparatus, we applied a step stress and measured nucleus deformation dynamics within living Caenorhabditis elegans nematodes. Nuclei deformed nonmonotonically under constant load. Nonmonotonic deformation was conserved across tissues and robust to nucleoskeletal and cytoskeletal perturbations, but it required intact linker of nucleoskeleton and cytoskeleton complex attachments. The transition from creep to strain recovery fits a tensile-compressive linear viscoelastic model that is indicative of nucleoskeletal-cytoskeletal decoupling under high load. Ce-lamin (lmn-1) knockdown softened the nucleus, whereas nematode aging stiffened the nucleus and decreased deformation recovery rate. Recovery lasted minutes rather than seconds due to physiological damping of the released mechanical energy, thus protecting nuclear integrity and preventing chromatin damage.
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Núcleo Celular/metabolismo , Imagem Molecular/métodos , Reologia/métodos , Animais , Fenômenos Biomecânicos , Caenorhabditis elegans , Cromatina/metabolismo , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Membrana Nuclear/metabolismo , Matriz Nuclear/metabolismo , Estresse MecânicoRESUMO
Cancer is one of the most devastating diseases of our time, with 17 million new cancer cases and 9.5 million cancer deaths in 2018 worldwide. The mortality associated with cancer results primarily from metastasis, i.e. the spreading of cancer cells from the primary tumour to other organs. The invasion and migration of cells through basement membranes, tight interstitial spaces and endothelial cell layers are key steps in the metastatic cascade. Recent studies demonstrated that cell migration through three-dimensional environments that mimic the in vivo conditions significantly differs from their migration on two-dimensional surfaces. Here, we review recent technological advances made in the field of cancer research that provide more 'true to the source' experimental platforms and measurements for the study of cancer cell invasion and migration in three-dimensional environments. These include microfabrication, three-dimensional bioprinting and intravital imaging tools, along with force and stiffness measurements of cells and their environments. These techniques will enable new studies that better reflect the physiological environment found in vivo, thereby producing more robust results. The knowledge achieved through these studies will aid in the development of new treatment options with the potential to ultimately lighten the devastating cost cancer inflicts on patients and their families. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.