VINCULIN-DEFICIENT PC12 CELL-LINES EXTEND UNSTABLE LAMELLIPODIA AND FILOPODIA AND HAVE A REDUCED RATE OF NEURITE OUTGROWTH

We have studied the role of vinculin in regulating integrin-dependent neurite outgrowth in PC12 cells, a neuronal cell line. Vinculin is a c ytoskeletal protein believed to mediate interactions between integrins and the actin cytoskeleton. In differentiated PC12 cells, the cell bo dy, the neurite, and the growth cone contain vinculin. Within the grow th cone, both the proximal region of ''consolidation'' and the more di stal region consisting of lamellipodia and filopodia contain vinculin. To study the role of vinculin in neurite outgrowth, we generated vinc ulin-deficient isolates of PC12 cell lines by transfection with vector s expressing antisense vinculin RNA. In some of these cell lines, vinc ulin levels were reduced to 18-23% of normal levels. In the vinculin-d eficient cell lines, neurite outgrowth on laminin was significantly re duced. In time-lapse analysis, growth cones advanced much more slowly than normal. Further analysis indicated that this deficit could be exp lained in large part by changes in the behaviors of filopodia and lame llipodia. Filopodia were formed in normal numbers, extended at normal rates, and extended to approximately normal lengths, but were much les s stable in the vinculin deficient compared to control PC12 cells. Sim ilarly, lamellipodia formed and grew nearly normally, but were dramati cally less stable in the vinculin-deficient cells. This can account fo r the reduction in rate of growth cone advance. These results indicate that interactions between integrins and the actin-based cytoskeleton are necessary for stability of both filopodia and lamellipodia.