RESUMEN
We examined keratin aggregate formation and the possible mechanisms involved. With this aim, we observed the effect that different ratios between mutant and wild-type keratins expressed in cultured keratinocytes may have on aggregate formation in vitro, as well as how keratin aggregate formation affects the mechanical properties of cells at the cell cortex. To this end we prepared clones with expression rates as close as possible to 25%, 50% and 100% of the EGFP-K14 proteins (either WT or R125P and V270M mutants). Our results showed that only in the case of the 25% EGFP-K14 R125P mutant significant differences could be seen. Namely, we observed in this case the largest accumulation of keratin aggregates and a significant reduction in cell stiffness. To gain insight into the possible mechanisms behind this observation, we extended our previous mathematical model of keratin dynamics by implementing a more complex reaction network that considers the coexistence of wild-type and mutant keratins in the cell. The new model, consisting of a set of coupled, non-linear, ordinary differential equations, allowed us to draw conclusions regarding the relative amounts of intermediate filaments and aggregates in cells, and suggested that aggregate formation by asymmetric binding between wild-type and mutant keratins could explain the data obtained on cells grown in culture.
Asunto(s)
Queratinocitos/metabolismo , Queratinas/química , Proteínas Mutantes/química , Agregado de Proteínas , Línea Celular , Simulación por Computador , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Queratinocitos/efectos de los fármacos , Modelos Biológicos , Inhibidores de Proteasoma/farmacología , Agregado de Proteínas/efectos de los fármacosRESUMEN
We have generated MLi004-A, a new induced pluripotent stem cell (iPSC) line derived from skin fibroblasts of a female patient with recessive dystrophic epidermolysis bullosa (RDEB). This iPSC line may be used as a model system for studies on skin integrity, the extracellular matrix and skin barrier function. The characterization of the MLi004-A cell line consisted of molecular karyotyping, next-generation sequencing of the COL7A1 alleles, pluripotency and differentiation potentials testing by immunofluorescence of associated markers in vitro. The MLi-004A line has been also tested for ability to differentiate into fibroblasts.
Asunto(s)
Epidermólisis Ampollosa Distrófica , Células Madre Pluripotentes Inducidas , Colágeno Tipo VII/genética , Epidermólisis Ampollosa Distrófica/genética , Femenino , Fibroblastos , Humanos , Mutación , PielRESUMEN
Keratin intermediate filaments are the principal structural element of epithelial cells. Their importance in providing bulk cellular stiffness is well recognized, but their role in the mechanics of cell cortex is less understood. In this study, we therefore compared the cortical stiffness of three keratinocyte lines: primary wild type cells (NHEK2), immortalized wild type cells (NEB1) and immortalized mutant cells (KEB7). The cortical stiffness was measured by lateral indentation of cells with AOD-steered optical tweezers without employing any moving mechanical elements. The method was validated on fixed cells and Cytochalasin-D treated cells to ensure that the observed variations in stiffness within a single cell line were not a consequence of low measurement precision. The measurements of the cortical stiffness showed that primary wild type cells were significantly stiffer than immortalized wild type cells, which was also detected in previous studies of bulk elasticity. In addition, a small difference between the mutant and the wild type cells was detected, showing that mutation of keratin impacts also the cell cortex. Thus, our results indicate that the role of keratins in cortical stiffness is not negligible and call for further investigation of the mechanical interactions between keratins and elements of the cell cortex.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Filamentos Intermedios/metabolismo , Queratinocitos/metabolismo , Queratinas/metabolismo , Microtúbulos/metabolismo , Citoesqueleto de Actina/ultraestructura , Línea Celular , Citocalasina D/farmacología , Elasticidad/efectos de los fármacos , Expresión Génica , Dureza/efectos de los fármacos , Humanos , Filamentos Intermedios/ultraestructura , Queratinocitos/efectos de los fármacos , Queratinocitos/ultraestructura , Queratinas/genética , Microtúbulos/ultraestructura , Pinzas Ópticas , Especificidad de ÓrganosRESUMEN
Lipoproteins have a vital role in the development of metabolic and cardiovascular diseases ranging from protective to deleterious effects on target tissues. VLDL has been shown to induce lipotoxic lipid accumulation and exert a variety of negative effects on cardiomyocytes. Lipotoxicity and endoplasmic reticulum (ER) stress are proposed to be the mediators of damaging effects of metabolic diseases on cardiovascular system. We treated cardiomyocytes with lipoproteins to evaluate the adaptability of these cells to metabolic stress induced by starvation and excess of lipoproteins, and to evaluate the effect of lipoproteins and lipid accumulation on ER stress. VLDL reversed metabolic stress induced by starvation, while HDL did not. VLDL induced dose-dependent lipid accumulation in cardiomyocytes, which however did not result in reduced cell viability or induction of ER stress. Moreover, VLDL or HDL pre-treatment reduced ER stress in cardiomyocytes induced by tunicamycin and palmitic acid as measured by the expression of ER stress markers, even in conditions of increased lipid accumulation. VLDL and HDL induced activation of pro-survival ERK1/2 in cardiomyocytes; however, this activation was not involved in the protection against ER stress. Additionally, we observed that LDLR and VLDLR are regulated differently by lipoproteins and cellular stress, as lipoproteins induced VLDLR protein independently of the level of lipid accumulation. We conclude that VLDL is not a priori detrimental for cardiomyocytes and can even have beneficial effects, enabling cell survival under starvation and attenuating ER stress.