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Myosin drives retrograde F-actin flow in neuronal growth cones.

Neuronal growth cones guide axons in the developing nervous system toward distant target sites. Recent evidence suggests that growth cones decode both diffusible and substrate-bound molecular signals during the guidance process (Kennedy and Tessier-Lavigne, 1995; Tessier-Lavigne, 1992), which involves pathfinding, branching, and ultimately target recognition. All of these behaviors depend on precise regulation of growth cone motility. Bray pioneered the investigation of growth cone motility and was the first to observe that inert particles placed on the growth cone surface would move in a retrograde direction, suggestive of an underlying centripetal membrane or actin filament flux (Bray, 1970). Early studies also demonstrated that growth cone motility depends on actin filament assembly (Yamada and Wessells, 1973), and a critical role for actin assembly in axonal guidance emerged when axons in the developing grasshopper nervous system were shown to lose all path-finding capabilities upon treatment with cytochalasin (Bentley and Toroion-Raymond, 1986). Later, studies characterizing actin dynamics in Aplysia growth cones in vitro suggested that the retrograde flux mentioned above is actually due to actin filament assembly at nucleation sites along the leading edge, followed by retrograde translocation and filament disassembly or recycling by means of severing at a proximal site. This process was referred to as "retrograde flow" (Forscher and Lin, 1991; Forscher and Smith, 1988; Smith, 1988).

The peripheral actin ultrastructure of the growth cone has also been well characterized as being composed of two distinct structural domains: filopodia containing arrays of uniformly polarized (barbed end distal) actin filaments, and intervening lamellipodium domains with less polarized filament structure (cf figure 6a; Lewis and Bridgman, 1992). Both domains appear to exhibit uniform retrograde flow relative to an external substrate reference as judged by actin fluorescence photobleaching studies (Lin and Forscher, 1995). Assembly of actin filaments, followed by centripetal displacement relative to the leading cell margin, has been observed in a wide variety of motile cells and appears to be a fundamental property of directed growth processes and cell migration (Cramer et al., 1994; Bray and White, 1988; Fisher et al., 1988; Abercrombie et al., 1970).

In our initial characterization of actin filament dynamics in Aplysia growth cones, we noted that when cones were treated with 2-5 [[micro]molar] cytochalasin, retrograde flow did not appear to be markedly affected. Continued retrograde flow in the absence of new actin assembly resulted in formation of an F-actin-free gap along the growth cone margin and eventual clearance (typically in about 3 min) of F-actin from lamellipodia and filopodia. This persistent retraction of actin filament networks in the absence of actin assembly demonstrated that polymerization could not be supplying the driving force for retrograde F-actin flow; therefore, we suggested that a myosinolike molecular motor might be involved (Forscher and Smith, 1988). Despite recent molecular cloning of several brain myosins (Bahler et al., 1994; Ruppert et al., 1993; Cheney et al., 1993) and localization of myosins to growth cones (Rochlin et al., 1995; Espreafico et al., 1992; Cheng et al., 1992; Miller et al., 1992), little or no information about the functional role of myosins in growth cone motility has emerged (cf Tanaka and Sabry, 1995).

Given the functional implications of our previous work, we designed experiments aimed at global inhibition of myosin activity to test whether any myosin was in fact involved in driving retrograde F-actin flow in growth cones. All known myosins have an evolutionarily conserved N-terminal head domain containing the site for ATP and F-actin binding as well as force generation (Mooseker and Cheney, 1995). Chymotryptic digestion of muscle myosin results in cleavage of a head domain subfragment (S1) from the rest of the molecule (Margoso sian and Lowey, 1982). S1 exhibits ATP-dependent actin-filament binding, but lacking the C-terminal tail, is incapable of generating force unless artificially tethered (e.g., by an antibody) to a substrate. In addition, further treatment of S1 with the sulfhydryl reagent, N-ethylmaleimide (NEM), results in a myosin head species (NEM-S1) that remains tightly bound to actin filaments, even in the presence of ATP, and thus can serve as a potent specific inhibitor of actomyosin function (Cande, 1986: Meeusen and Cande, 1979). Our first experimental approach then was to competitively inhibit myosin function by injection of purified S1 or NEM-S1 and to look at effects on growth cone motility, actin dynamics, and structure.

We compared the results of S1 or NEM-S1 injection with those obtained after treatment with 10-30 mM 2,3-butanedione-2-monoxime (BDM), a pharmacological inhibitor of endogenous myosin ATPase activity [ILLUSTRATION FOR FIGURE 1B OMITTED]. BDM has previously been shown to affect cross-bridge kinetics and to inhibit both conventional muscle and nonmuscle myosin ATPases including myosin V, platelet myosin II, and a drosophila myosin ATPase fraction without affecting kinesin ATPase activity or actin assembly (Cramer and Mitchison, 1995; Backx et al, 1994; McKillop et al., 1994; Schramm et al., 1994; Zhao and Kawai, 1994). We found that both types of myosin inhibition produced essentially the same effects: dose-dependent attenuation of retrograde F-actin flow accompanied by filopodial and leading edge extension at rates directly proportional to the degree of flow inhibition [ILLUSTRATION FOR FIGURE 1C OMITTED]. Filopodial growth stimulated by the myosin antagonists was cytochalasin sensitive, indicating that it is due to barbed end filament assembly. These experiments demonstrate that retrograde actin flow is indeed driven by a myosin ATPase, and that actin filament assembly and myosin motors function independently. Our results suggest that simple superimposition of actin polymerization and the action of myosin motors underlie the process of retrograde F-actin flow.

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Title Annotation:The Future of Aquatic Research in Space: Neurobiology, Cellular and Molecular Biology
Author:Lin, C.H.; Espreafico, E.M.; Mooseker, M.S.; Forscher, P.
Publication:The Biological Bulletin
Date:Feb 1, 1997
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