DataSheet1_The resealing factor S100A11 interacts with annexins and extended synaptotagmin-1 in the course of plasma membrane wound repair.pdf

After damage, cells repair their plasma membrane in an active process that is driven by Ca2+ entering through the wound. This triggers a range of Ca2+-regulated events such as the translocation of different Ca2+-binding proteins to the wound site which likely function in the repair process. The translocated proteins include Ca2+/phospholipid binding proteins of the annexin (ANX) family and S100A11, an EF hand-type Ca2+-binding protein which can interact with ANX. The molecular mechanism by which S100A11 mediates PM wound repair remains poorly understood although it likely involves interactions with ANX. Here, using S100A11 knockout endothelial cells and expression of S100A11 mutants, we show that endothelial S100A11 is essential for efficient plasma membrane wound repair and engages in Ca2+-dependent interactions with ANXA1 and ANXA2 through its C-terminal extension (residues 93–105). ANXA2 but not ANXA1 translocation to the wound is substantially inhibited in the absence of S100A11; however, the repair defect in S100A11 knockout cells is rescued by ectopic expression of an ANX interaction-defective S100A11 mutant, suggesting an ANX-independent role of S100A11 in membrane wound repair. In search for other interaction partners that could mediate this action of S100A11 we identify extended synaptotagmin 1 (E-Syt1), a protein tether that regulates endoplasmic reticulum-plasma membrane contact sites. E-Syt1 binds to S100A11 in the presence of Ca2+ and depletion of E-Syt1 interferes with wound site recruitment of S100A11 and proper membrane resealing. Thus, the role of S100A11 in membrane wound repair does not exclusively dependent on ANX interactions and a Ca2+-regulated S100A11-E-Syt1 complex acts as a yet unrecognized component of the membrane resealing machinery.

细胞受损后，会通过一种由伤口处流入的钙离子（Ca2+）驱动的主动过程修复其细胞膜（plasma membrane）。该过程会触发一系列受钙离子调控的事件，例如多种钙离子结合蛋白向伤口位点的转位，这类蛋白可能在修复进程中发挥功能。发生转位的蛋白包括膜联蛋白（annexin, ANX）家族的Ca2+/磷脂结合蛋白，以及可与膜联蛋白相互作用的EF手型钙离子结合蛋白（EF hand-type Ca2+-binding protein）S100A11。尽管S100A11介导细胞膜伤口修复的分子机制可能涉及与膜联蛋白的相互作用，但目前对其仍知之甚少。本研究利用S100A11敲除内皮细胞模型与S100A11突变体的异位表达实验，证实内皮细胞中的S100A11对于高效完成细胞膜伤口修复至关重要，且其可通过C端延伸区（残基93-105）与ANXA1、ANXA2发生钙离子依赖性相互作用。在缺失S100A11的细胞中，ANXA2向伤口位点的转位会受到显著抑制，而ANXA1的转位则不受影响；不过，异位表达膜联蛋白结合缺陷型S100A11突变体，可挽救S100A11敲除细胞中的修复缺陷，这提示S100A11在细胞膜伤口修复中存在不依赖膜联蛋白的作用通路。为探寻介导S100A11这一功能的其他相互作用伴侣，我们鉴定出了延伸型突触结合蛋白1（extended synaptotagmin 1, E-Syt1）——一种调控内质网-细胞膜接触位点的蛋白拴系因子（protein tether）。在钙离子存在的条件下，E-Syt1可与S100A11结合；而E-Syt1敲低会干扰S100A11向伤口位点的招募以及正常的膜封合过程。综上，S100A11在细胞膜伤口修复中的作用并非完全依赖于与膜联蛋白的相互作用，钙离子调控的S100A11-E-Syt1复合物是膜封合机制中一种尚未被认知的组成成分。