RESUMEN
The nuclear lamina mechanically integrates the nucleus with the cytoskeleton and extracellular environment and regulates gene expression. These functions are exerted through direct and indirect interactions with the lamina's major constituent proteins, the A-type lamins, which are encoded by the LMNA gene. Using quantitative stable isotope labeling-based shotgun proteomics we have analyzed the proteome of human dermal fibroblasts in which we have depleted A-type lamins by means of a sustained siRNA-mediated LMNA knockdown. Gene ontology analysis revealed that the largest fraction of differentially produced proteins was involved in actin cytoskeleton organization, in particular proteins involved in focal adhesion dynamics, such as actin-related protein 2 and 3 (ACTR2/3), subunits of the ARP2/3 complex, and fascin actin-bundling protein 1 (FSCN1). Functional validation using quantitative immunofluorescence showed a significant reduction in the size of focal adhesion points in A-type lamin depleted cells, which correlated with a reduction in early cell adhesion capacity and an increased cell motility. At the same time, loss of A-type lamins led to more pronounced stress fibers and higher traction forces. This phenotype could not be mimicked or reversed by experimental modulation of the STAT3-IL6 pathway, but it was partly recapitulated by chemical inhibition of the ARP2/3 complex. Thus, our data suggest that the loss of A-type lamins perturbs the balance between focal adhesions and cytoskeletal tension. This imbalance may contribute to mechanosensing defects observed in certain laminopathies.
Asunto(s)
Citoesqueleto/metabolismo , Adhesiones Focales/metabolismo , Lamina Tipo A/metabolismo , Proteína 2 Relacionada con la Actina/genética , Proteína 2 Relacionada con la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/genética , Proteína 3 Relacionada con la Actina/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Adhesión Celular/genética , Adhesión Celular/fisiología , Células Cultivadas , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Fibroblastos , Humanos , Interleucina-6/metabolismo , Lamina Tipo A/genética , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteoma/metabolismo , ARN Interferente Pequeño/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and their removal by cellular antioxidant systems. Especially under pathological conditions, mitochondria constitute a relevant source of cellular ROS. These organelles harbor the electron transport chain, bringing electrons in close vicinity to molecular oxygen. Although a full understanding is still lacking, intracellular ROS generation and mitochondrial function are also linked to changes in mitochondrial morphology. To study the intricate relationships between the different factors that govern cellular redox balance in living cells, we have developed a high-content microscopy-based strategy for simultaneous quantification of intracellular ROS levels and mitochondrial morphofunction. Here, we summarize the principles of intracellular ROS generation and removal, and we explain the major considerations for performing quantitative microscopy analyses of ROS and mitochondrial morphofunction in living cells. Next, we describe our workflow, and finally, we illustrate that a multiparametric readout enables the unambiguous classification of chemically perturbed cells as well as laminopathy patient cells.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Microscopía Fluorescente/métodos , Mitocondrias/ultraestructura , Especies Reactivas de Oxígeno/metabolismo , Animales , Antioxidantes/farmacología , Células Eucariotas/efectos de los fármacos , Células Eucariotas/metabolismo , Células Eucariotas/ultraestructura , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Fluorescente/instrumentación , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Flujo de TrabajoRESUMEN
The cell nucleus is structurally and functionally organized by lamins, intermediate filament proteins that form the nuclear lamina. Point mutations in genes that encode a specific subset of lamins, the A-type lamins, cause a spectrum of diseases termed laminopathies. Recent evidence points to a role for A-type lamins in intracellular redox homeostasis. To determine whether lamin A/C depletion and prelamin A accumulation differentially induce oxidative stress, we have performed a quantitative microscopy-based analysis of reactive oxygen species (ROS) levels and mitochondrial membrane potential (Δψm) in human fibroblasts subjected to sustained siRNA-mediated knockdown of LMNA and ZMPSTE24, respectively. We measured a highly significant increase in basal ROS levels and an even more prominent rise of induced ROS levels in lamin A/C depleted cells, eventually resulting in Δψm hyperpolarization and apoptosis. Depletion of ZMPSTE24 on the other hand, triggered a senescence pathway that was associated with moderately increased ROS levels and a transient Δψm depolarization. Both knockdowns were accompanied by an upregulation of several ROS detoxifying enzymes. Taken together, our data suggest that both persistent prelamin A accumulation and lamin A/C depletion elevate ROS levels, but to a different extent and with different effects on cell fate. This may contribute to the variety of disease phenotypes witnessed in laminopathies.
