Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling

Previous observations indicated that the lamellipodium ("leading edge") of fibroblasts contains a dense meshwork, as well as numerous bundles (microspikes) of actin filaments. Most, if not all, of the filaments have a uniform polarity, with the "barbed" end associated with the membrane. I investigated whether and how actin subunits exchange in this region by microinjecting living gerbil fibroma cells (IMR-33) with actin that had been labeled with iodoacetamidotetramethylrhodamine. After incorporation of the labeled actin into the lamellipodium, I used a laser microbeam to photobleach a 3-4-micron region at and surrounding a microspike, without disrupting the integrity of the structure. I then recorded the pattern of fluorescence recovery and analyzed it using a combination of TV image intensification and digital image processing techniques. Fluorescence recovery was first detected near the edge of the cell and then moved toward the cell's center at a constant rate of 0.79 +/- 0.31 micron/min. When only part of the lamellipodium near the edge of the cell was photobleached, the bleached spot also moved toward the cell's center and through an area unbleached by the laser beam. These results indicated that steady state incorporation of actin subunits occurred predominantly at the membrane-associated end of actin filaments, and that actin subunits in the lamellipodium underwent a constant movement toward the center of the cell. I suggest that treadmilling, possibly in combination with other molecular interactions, may provide an effective mechanism for the movement of actin subunits and the protrusion of cytoplasm in the lamellipodium of fibroblasts.