Cell dynamics and the actin cytoskeleton

187

Capping protein: ‘decommissioning’ the old

Capping protein is an abundant heterodimeric protein that binds with high affinity to the barbed ends of actin filaments to block both assembly and disassembly at these ends (Cooper and Pollard, 1985; Isenberg et al., 1980). Vertebrates express multiple isoforms of both the α and β subunits (Hart et al., 1997; Schafer et al., 1994). With the conditions of cells favoring polymerization at free barbed ends, capping protein is essential to control the degree of polymerization. The association rates of capping protein with barbed ends in combination with high cellular concentrations of capping protein ( 2 µM) should only allow a newly crated, unprotected barbed end to grow for1 s (Schafer et al., 1996). On the other hand, because the residency time of capping proteins on barbed ends is 30 minutes (Schafer et al., 1996), short capped filaments will depolymerize from their pointed ends in cells. Capping proteins are thought to be integral to the recycling of monomers in dendritically arranged filaments at the leading edge of cells (Pollard et al., 2000). Consistent with this idea are findings that perturbations of capping activity dramatically alter the geometry of Arp2/3-complex- induced networks in reconstitution studies (Pantaloni et al., 2000; Vignjevic et al., 2003).

In addition to blocking barbed-end dynamics, capping protein diminishes the lag phase of actin polymerization (Pollard and Cooper, 1984). In this ‘nucleating’ activity, capping protein is probably stabilizing small oligomers rather than generating filaments de novo (Schafer and Cooper, 1995). Because the growing filaments are capped at their barbed end, this function is probably not active in cells with abundant sequestering proteins that can prevent assembly at pointed ends. The only known regulation of capping protein activity is by phospholipids. Phospholipids can both inactivate free capping protein (Heiss and Cooper, 1991) and remove bound capping protein from barbed ends (Schafer et al., 1996).

Gelsolin: rapid remodeling in one or two steps

If the job of actin-binding proteins is to remodel the actin cytoskeleton, then gelsolin has exceptional qualifications. Activated by micromolar Ca2+ (Yin and Stossel, 1979), gelsolin binds to the sides of actin filaments and severs them (Yin et al., 1980). However, unlike cofilin, gelsolin remains attached to the new barbed end created by severing to block further polymerization (Yin et al., 1981; Yin et al., 1980). Because gelsolin has nM affinity for barbed ends, it functions as a permanent cap that can only be removed through subsequent binding by phospholipids (Janmey and Stossel, 1987). In platelets and neutrophils, activated gelsolin remodels actin in two steps (Barkalow et al., 1996; Glogauer et al., 2000). Because the majority of filaments in resting cells are capped, cellular activation first leads to gelsolin severing to create a large number of dynamically stable filaments. Shortly thereafter, these filaments become nuclei for new growth as phospholipid levels increase to result in massive uncapping.

While gelsolin seems built for acute remodeling, expression studies clearly indicate a role for gelsolin at steady-state. Gelsolin null fibroblasts have impaired motility, reduced membrane ruffling, slow filament turnover, and abundant stress fibers (Azuma et al., 1998; McGrath et al., 2000a; Witke et al., 1995), and gelsolin overexpression produces the opposite trends (Cunningham et al., 1991). With its high