Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA.
Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI, USA.
FEBS J. 2021 Jan;288(1):160-174. doi: 10.1111/febs.15556. Epub 2020 Sep 25.
Plasma membrane repair is an evolutionarily conserved mechanism by which cells can seal breaches in the plasma membrane. Mutations in several proteins with putative roles in sarcolemma integrity, membrane repair, and membrane transport result in several forms of muscle disease; however, the mechanisms that are activated and responsible for sarcolemma resealing are not well understood. Using the standard assays for membrane repair, which track the uptake of FM 1-43 dye into adult skeletal muscle fibers following laser-induced sarcolemma disruption, we show that labeling of resting fibers by FM1-43 prior to membrane wounding and the induced FM1-43 dye uptake after sarcolemma wounding occurs via dynamin-dependent endocytosis. Dysferlin-deficient muscle fibers show elevated dye uptake following wounding, which is the basis for the assertion that membrane repair is defective in this model. Our data show that dynamin inhibition mitigates the differences in FM1-43 dye uptake between dysferlin-null and wild-type muscle fibers, suggesting that elevated wound-induced FM1-43 uptake in dysferlin-deficient muscle may actually be due to enhanced dynamin-dependent endocytosis following wounding, though dynamin inhibition had no effect on dysferlin trafficking after wounding. By monitoring calcium flux after membrane wounding, we show that reversal of calcium precedes the sustained, slower increase of dynamin-dependent FM1-43 uptake in WT fibers, and that dysferlin-deficient muscle fibers have persistently increased calcium after wounding, consistent with its proposed role in resealing. These data highlight a previously unappreciated role for dynamin-dependent endocytosis in wounded skeletal muscle fibers and identify overactive dynamin-dependent endocytosis following sarcolemma wounding as a potential mechanism or consequence of dysferlin deficiency.
质膜修复是一种进化上保守的机制，通过该机制，细胞可以封闭质膜的破裂。几种具有推测的质膜完整性、膜修复和膜运输作用的蛋白的突变导致几种形式的肌肉疾病；然而，被激活并负责质膜再封闭的机制尚不清楚。使用标准的膜修复测定法，该方法跟踪激光诱导的质膜破坏后 FM 1-43 染料进入成年骨骼肌纤维的摄取，我们表明，在用 FM1-43 对静息纤维进行标记之前进行膜损伤，以及在用 FM1-43 进行诱导后的染料摄取发生在肌球蛋白依赖性内吞作用之后。肌营养不良蛋白缺乏的肌肉纤维在受伤后显示出升高的染料摄取，这是该模型中膜修复有缺陷的基础。我们的数据表明，肌球蛋白抑制剂可减轻肌营养不良蛋白缺失和野生型肌肉纤维之间在 FM1-43 染料摄取方面的差异，这表明在肌营养不良蛋白缺失的肌肉中，受伤诱导的 FM1-43 摄取增加实际上可能是由于受伤后肌球蛋白依赖性内吞作用增强所致，尽管肌球蛋白抑制剂对受伤后肌营养不良蛋白的运输没有影响。通过监测膜损伤后的钙流，我们表明钙的反转先于 WT 纤维中肌球蛋白依赖性 FM1-43 摄取的持续较慢增加，并且肌营养不良蛋白缺失的肌肉纤维在受伤后持续增加钙，这与其在再封闭中的拟议作用一致。这些数据突出了肌球蛋白依赖性内吞作用在受伤的骨骼肌纤维中的先前未被认识的作用，并确定了肌球蛋白依赖性内吞作用在质膜损伤后过度活跃是肌营养不良蛋白缺乏的潜在机制或后果。
