ERM (EZRIN RADIXIN/MOESIN)-BASED MOLECULAR MECHANISM OF MICROVILLAR BREAKDOWN AT AN EARLY-STAGE OF APOPTOSIS/

Breakdown of microvilli is a common early event in various types of ap optosis, but its molecular mechanism and implications remain unclear. ERM (ezrin/radixin/moesin) proteins are ubiquitously expressed microvi llar proteins that are activated in the cytoplasm, translocate to the plasma membrane, and function as general actin filament/plasma membran e 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 dephosphoryl ation of ERM proteins was suppressed by calyculin A, a serine/threonin e protein phosphatase inhibitor, the cytoplasmic translocation of ERM proteins was blocked. The interleukin-1 beta-converting enzyme (ICE) p rotease inhibitors suppressed the dephosphorylation as well as the cyt oplasmic translocation of ERM proteins. These findings indicate that d uring FasL-induced apoptosis, the ICE protease cascade was first activ ated, and then ERM proteins were dephosphorylated followed by their cy toplasmic translocation, i.e., microvillar breakdown. Next, to examine the subsequent events in microvillar breakdown, we prepared DiO-label ed 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 filamen ts were densely detected, whereas those from FasL-treated cells were f ree from ERM proteins or actin filaments. We thus concluded that the c ytoplasmic translocation of ERM proteins is responsible for the microv illar breakdown at an early phase of apoptosis and that the depletion of ERM proteins from plasma membranes results in the gross dissociatio n of actin-based cytoskeleton from plasma membranes. The physiological relevance of this ERM protein-based microvillar breakdown in apoptosi s will be discussed.