Eccentric exercise leads to focal disruptions in the myofibrils, referred to as “lesions”. These structures are thought to contribute to the post-exercise muscle weakness, and to represent areas of mechanical damage and/or remodelling. Lesions have been investigated in human biopsies and animal samples after exercise. However, this approach does not examine the mechanisms behind lesion formation, or their behaviour during contraction. To circumvent this, we used electrical pulse stimulation (EPS) to simulate exercise in C2C12 myotubes, combined with live microscopy. EPS application led to the formation of sarcomeric lesions in the myotubes, resembling those seen in exercised mice, increasing in number with the time of application or stimulation intensity. Furthermore, transfection with an EGFP-tagged version of the lesion and Z-disc marker filamin-C allowed us to observe the formation of lesions using live cell imaging. Finally, using the same technique we studied the behaviour of these structures during contraction, and observed them to be passively stretching. This passive behaviour supports the hypothesis that lesions contribute to the post-exercise muscle weakness, protecting against further damage. We conclude that EPS can be reliably used as a model for the induction and study of sarcomeric lesions in myotubes in vitro.
SignificanceStriatin-interacting phosphatases and kinases (STRIPAK) complexes can regulate the cytoskeleton and cell migration in cell lines, but their roles in vivo in mammals are not known. Here, we show that mouse embryos that lack striatin-interacting protein 1 (STRIP1), a core component of STRIPAK complexes, arrest at midgestation with striking morphological defects. Strip1 mutants lack a trunk, and both paraxial and axial mesoderm fail to elongate along the anterior–posterior body axis. Mesodermal cells from Strip1 mutants have defects in actin organization, focal adhesions, and cell migration that can account for the failure of normal mesoderm migration. The findings demonstrate that STRIPAK is a critical regulator of mammalian cell migration and is likely to have important roles in tumor progression as well as development.
The development of complex stratified epithelial barriers in mammals is initiated from single-layered epithelia. How stratification is initiated and fueled are still open questions. Previous studies on skin epidermal stratification suggested a central role for perpendicular/asymmetric cell division orientation of the basal keratinocyte progenitors. Here, we use centrosomes, that organize the mitotic spindle, to test whether cell division orientation and stratification are linked. Genetically ablating centrosomes from the developing epidermis leads to the activation of the p53-, 53BP1- and USP28-dependent mitotic surveillance pathway causing a thinner epidermis and hair follicle arrest. The centrosome/p53-double mutant keratinocyte progenitors significantly alter their division orientation in the later stages without majorly affecting epidermal differentiation. Together with time-lapse imaging and tissue growth dynamics measurements, the data suggest that the first and major phase of epidermal development is boosted by high proliferation rates in both basal and suprabasally-committed keratinocytes as well as cell delamination, whereas the second phase maybe uncoupled from the division orientation of the basal progenitors. The data provide insights for tissue homeostasis and hyperproliferative diseases that may recapitulate developmental programs.
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