The molecular basis for the complexities of neuronal growth cone behavior is beginning to emerge. Highly complex motile phenomena such as turning clearly require ensemble behaviors in which signaling cascades implement the engagement of actin filaments with microtubules as they invade the distal region of the growth cone (Tanaka and Sabry, 1995
). Filopodia exhibit a considerably more limited behavioral repertoire consisting of elongation and retraction. These behaviors are mediated by a single cytoskeletal system: actin filaments. Actin-associated proteins are leading candidates to regulate length changes of filopodia. Myosin is involved in the centripetal transport of actin filaments from the leading edge of the growth cone (Lin et al., 1996
). In addition, myosin-V, a two-headed unconventional myosin present in growth cones that possesses Ca2+
–calmodulin-sensitive mechanochemical activities (Espreafico et al., 1992
; Cheney et al., 1993
) appears to be involved in filopodial extension (Wang et al., 1996
). The local obliteration of this molecule by chromophore-assisted laser inactivation in neuronal growth cones from chick dorsal root ganglia decreased the rate of filopodial extension but did not alter the rate of filopodial retraction, leading us to conclude that extension and retraction are independently regulated. Here we have demonstrated that the retraction phase is dependent on gelsolin.
The observation that neurites from Gsn−
mice contain many more filopodia than those from wt mice could be explained not only by impaired retraction but also by increased filopodial extrusion. However, an alteration in filopodia extrusion in the Gsn−
is unlikely for several reasons. In general, filopodia tend to arise from the leading edge of the growth cone and retract from the base (Lewis and Bridgman, 1992
). As was apparent from the time-lapse video images, most of the filopodia on the consolidated segment under study here arose within the growth cone, and as the growth cone advanced, the filopodia remained along the shaft of the newly consolidated neurite. Only rarely did filopodia arise from the shaft. Thus, even if increased filopodial extrusion did occur within the growth cone, these Gsn−
filopodia failed to coordinate their retraction with the forward advance of the growth cone. Filopodia from Gsn−
mice clearly remained extended for longer times than wt controls. The tendency of Gsn−
mice to undergo a stuttering rather than a smooth retraction as well as a less steep slope of retraction (but normal rates of extension) also indicated that a retraction deficit existed. Although Gsn−
mice had many more filopodia within the segment under study, the mean lengths of the individual filopodia did not differ from wt mice. Therefore, even though retraction was impaired, after achieving a certain length, filopodia did not continue to elongate. Therefore, filopodial growth is an independent process that is limited by factors other than the onset of retraction.
Gelsolin is the founding member of a family of six mammalian genes/proteins (Schafer and Cooper, 1995
) with similar domain structure that are expressed in diverse cell types. All have the ability to bind to actin, and several have actin filament severing activity. Adseverin has the most structural and functional similarity to gelsolin, with Ca2+
-regulated actin filament severing activity, and its actin filament binding is inhibited by PPI, as well as phosphatidylinositol and phosphatidylserine (Maekawa and Sakai, 1990
). Moreover, adseverin has been reported to be expressed in mammalian brain tissues, though its precise cellular localization is unknown (Tchakarov et al., 1990
). Those facets of growth cone motility that are normal in the Gsn−
mice may be due to the presence of adseverin in these regions or the actin regulatory protein ADF (Bamberg and Bray, 1987). ADF has complex effects on actin filaments that are dependent upon conditions of pH and ionic strength (Moon and Drubin, 1995
), so that it may function to sever actin filaments and regulate actin dynamics in neuronal growth cones.
Given the filopodial retraction rates observed here and the known rates of actin depolymerization, it is unlikely that the retraction defect observed in the Gsn−
mice is due to a dysfunction involving an effect of gelsolin on the depolymerization of actin filaments. Filopodial retraction occurs at rates from 0.09 to 0.16 μm/s, which corresponds to 32–59 monomers/s, based on the fact that 1 μm of polymerized actin contains 370 actin monomers (Hanson and Lowy, 1963
). Since the most rapid off rate of actin monomers from an actin filament end is 0.18 monomers/s (Pollard, 1986
), simple monomer dissociation cannot explain filopodia retraction. More likely, the nature of the dysfunction resulting in impaired filopodial retraction is due to a loss of the actin filament-severing activity of gelsolin. One potentially critical functional site may be the base of the filopodia where gelsolin may sever active filaments and thereby allow a coordinately engaged myosin motor to slide the filaments through the proximal actin mesh. In this model, filopodia remain extended as long as actin filaments at their base remain intact and prevent sliding of filaments into the shaft or growth cone. Few studies directly visualize well-preserved actin filaments in neuronal filopodia. In one study, negatively stained and freeze-etched EM of permeabilized growth cone from rat superior cervical ganglia explants contained tightly packed actin bundles in the core of their filopodia (Lewis and Bridgman, 1992
). In quiescent filopodia, the filament bundles ended at the base of the filopodia; in contrast, in putatively active regions the bundles extended into the lamellar region. Although our experiments do not directly address filopodia protrusions from the growth cone itself, our model would predict that gelsolin severs actin filaments at the point where they extend into the lamella. Gelsolin may also release actin from sites of tension along the filopodia, where actin is linked to molecules such as vinculin and talin, which indirectly interact with the substrate. These explanations are consistent with the stuttering retraction pattern seen in the Gsn−
mice and with the variation in retraction times, which may arise due to the quite different detailed organizational patterns of actin filaments around individual filopodia.
The tendency of the filopodia to remain along the consolidated neurite once the growth cone has advanced suggests a forward flow within the central cytoplasm while the filopodia hold on to their attachment sites to create the torque for the advancing growth cone. Because substrate interactions dominate neuronal cell growth under tissue culture conditions, neurons from Gsn− mice, placed in tissue culture, may enhance a phenotype that is less apparent in vivo. The Gsn− mice do not display any obvious neurologic deficits, but they have not been subjected to detailed anatomic and behavioral examination. The approach taken here of knocking out specific genes encoding growth cone proteins, may allow the systematic assignment of function to the many components of the growth cone.