Kondo T, Takeuchi K, Doi Y, Yonemura S, Nagata S, Tsukita S
Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.
J Cell Biol. 1997 Nov 3;139(3):749-58. doi: 10.1083/jcb.139.3.749.
Breakdown of microvilli is a common early event in various types of apoptosis, but its molecular mechanism and implications remain unclear. ERM (ezrin/radixin/moesin) proteins are ubiquitously expressed microvillar proteins that are activated in the cytoplasm, translocate to the plasma membrane, and function as general actin filament/plasma membrane cross-linkers to form microvilli. Immunofluorescence microscopic and biochemical analyses revealed that, at the early phase of Fas ligand (FasL)-induced apoptosis in L cells expressing Fas (LHF), ERM proteins translocate from the plasma membranes of microvilli to the cytoplasm concomitant with dephosphorylation. When the FasL-induced dephosphorylation of ERM proteins was suppressed by calyculin A, a serine/threonine protein phosphatase inhibitor, the cytoplasmic translocation of ERM proteins was blocked. The interleukin-1beta-converting enzyme (ICE) protease inhibitors suppressed the dephosphorylation as well as the cytoplasmic translocation of ERM proteins. These findings indicate that during FasL-induced apoptosis, the ICE protease cascade was first activated, and then ERM proteins were dephosphorylated followed by their cytoplasmic translocation, i.e., microvillar breakdown. Next, to examine the subsequent events in microvillar breakdown, we prepared DiO-labeled single-layered plasma membranes with the cytoplasmic surface freely exposed from FasL-treated or nontreated LHF cells. On single-layered plasma membranes from nontreated cells, ERM proteins and actin filaments were densely detected, whereas those from FasL-treated cells were free from ERM proteins or actin filaments. We thus concluded that the cytoplasmic translocation of ERM proteins is responsible for the microvillar breakdown at an early phase of apoptosis and that the depletion of ERM proteins from plasma membranes results in the gross dissociation of actin-based cytoskeleton from plasma membranes. The physiological relevance of this ERM protein-based microvillar breakdown in apoptosis will be discussed.
微绒毛的崩解是各类细胞凋亡中常见的早期事件，但其分子机制及影响仍不清楚。ERM（埃兹蛋白/根蛋白/膜突蛋白）家族蛋白是普遍表达的微绒毛蛋白，在细胞质中被激活后转移至质膜，作为肌动蛋白丝/质膜的通用交联剂形成微绒毛。免疫荧光显微镜和生化分析显示，在表达Fas的L细胞（LHF）中，Fas配体（FasL）诱导凋亡的早期阶段，ERM蛋白伴随着去磷酸化从微绒毛的质膜转移至细胞质。当丝氨酸/苏氨酸蛋白磷酸酶抑制剂花萼海绵诱癌素A抑制FasL诱导的ERM蛋白去磷酸化时，ERM蛋白的细胞质转移被阻断。白细胞介素-1β转换酶（ICE）蛋白酶抑制剂也抑制了ERM蛋白的去磷酸化及细胞质转移。这些发现表明，在FasL诱导的细胞凋亡过程中，ICE蛋白酶级联反应首先被激活，随后ERM蛋白去磷酸化，接着发生细胞质转移，即微绒毛崩解。接下来，为了研究微绒毛崩解的后续事件，我们制备了细胞质表面自由暴露的DiO标记单层质膜，这些质膜来自FasL处理或未处理的LHF细胞。在未处理细胞的单层质膜上，ERM蛋白和肌动蛋白丝被密集检测到，而在FasL处理细胞的单层质膜上则未检测到ERM蛋白或肌动蛋白丝。因此我们得出结论，ERM蛋白的细胞质转移在细胞凋亡早期导致微绒毛崩解，并且质膜上ERM蛋白的缺失导致基于肌动蛋白的细胞骨架与质膜的整体解离。本文将讨论这种基于ERM蛋白的微绒毛崩解在细胞凋亡中的生理相关性。
