MARCKS REGULATES MEMBRANE RUFFLING AND CELL SPREADING

The dynamic rearrangement of the actin cytoskeleton is fundamental to most biological processes including embryogenesis, morphogenesis, cell movement, wound healing and metastasis [1]. Membrane ruffling and rev ersible cell-substratum interactions underlie actin-driven cell moveme nt. Protein kinase C (PKC) stimulates membrane ruffling and adhesion [ 2], but the mechanism by which this occurs is unknown. Myristoylated a lanine-rich C kinase substrate (MARCKS) is a PKC substrate that cycles on and off membranes by a mechanism termed the myristoyl-electrostati c switch [3-6]. While at the membrane, MARCKS binds to and sequesters acidic phospholipids including phosphatidyl-inositol-4,5-bisphosphate (PIPS) [7]. MARCKS also binds and crosslinks filamentous actin, an act ivity which is regulated by PKC-dependent phosphorylation and calcium- calmodulin [8]. In this report, we demonstrate that expression, in fib roblasts, of MARCKS containing a mutation which abrogates the myristoy l-electrostatic switch prevents cell spreading. The MARCKS mutant arre sts the cell during an early stage of spreading, characterized by prof use membrane blebbing, and prevents the formation of membrane ruffles and lamellae usually found at the leading edge of spreading cells. Thi s defect in the regulation of the actin cytoskeleton is accompanied by a decrease in cell-substratum adhesion. Our results provide direct ev idence that MARCKS and PKC regulate actin-dependent membrane ruffling and cell adhesion, perhaps via a PIPS-dependent mechanism.