Production of a unique antibody specific for membrane ruffles and its use to characterize the behavior of two distinct types of ruffles

Abstract

I have produced a new monoclonal antibody, YF-169, against membrane ruffle specific 55-kD protein. YF-169 stained membrane ruffles of chick embryo fibroblasts so definitely that it enabled clear and reliable analyses of membrane ruffles. Fibroblasts organized two distinct types of membrane ruffles. One type of the ruffles were transiently formed in serumstarved cells (Type I) when stimulated by serum or platelet-derived growth factor. After spontaneous degradation of Type I ruffles, the other type of ruffles containing many microspikes were gradually organized at leading edges (Type II). The formation of Type I ruffles was not affected by either nocodazole, a microtubule-disrupting drug, or taxol, a microtubule-stabilizing reagent. However, Type II ruffles were entirely destroyed not only by nocodazole but also by taxol, suggesting that regulated organization of microtubule network is important to maintain Type II ruffles. H8, a protein kinase inhibitor prevented the spontaneous degradation of Type I ruffles and also reduced the destructive effect of nocodazole on Type II ruffles without affecting microtubule-disrupting activity. Protein kinases may be involved in the degradation processes of both types of ruffles. W7, a calmodulin antagonist, strongly inhibited Type I ruffle formation and completely destroyed Type II ruffles. W7 was also found to induce a remarkable change of 55-kD protein localization. After degradation of Type II ruffles, most of 55-kD protein was incorporated into newly formed unusual thick fibers. These results suggest that regulated organization of microtubule network is not necessary to form Type I ruffles but is important to maintain Type II ruffles, while calmodulin function is essential for both types of membrane ruffles.