Our results identify the molecular motor, myosin Va, as a neurofilament-associated protein and establish NF-L as a major ligand of myosin Va in nervous tissue. Multiple lines of evidence support these conclusions. First, more than half of the total myosin Va in CNS tissues coisolates with the neurofilament-rich Triton-insoluble cytoskeleton. Second, immunoreactive myosin Va is abundant in nerve fiber tracts of brain, spinal cord, and peripheral nerves. Immunoelectron microscopic analyses show that the majority of the myosin Va in axons is associated with neurofilaments. Third, electroblot overlay analyses and in vitro and in vivo coimmunoprecipitation studies with either myosin Va or NF-L antibodies establish that the binding of myosin Va to neurofilaments is mediated through a specific association of myosin Va with the NF-L subunit. Fourth, we demonstrate in vivo that myosin Va levels, distribution, and transport in axons are influenced by the level of NF-L subunits. Finally, deletion of the myosin Va gene in dilute mice selectively alters neurofilament content and organization in axons.
In addition to establishing a novel myosin Va–neurofilament association, our results confirm previously observed associations of myosin Va with ER membranes, synaptic vesicles (Tabb et al., 1998
), and actin. Extending observations that myosin Va is an actin binding protein (Espreafico et al., 1992
; Cheney et al., 1993
), we demonstrated that in vivo–labeled actin and myosin Va coimmunoprecipitate and cotransport and that myosin Va antibodies decorate the actin-rich subaxolemmal compartment (; ; Kobayashi et al., 1986
). It is not surprising that the myosin Va–neurofilament interaction has been less well appreciated than the associations of myosin Va with actin or membranous organelles because previous myosin Va localization studies have focused on organelle-rich and neurofilament-poor cellular compartments in cell bodies and dendrites in brain, rather than in axons.
The NF-L subunit is one of only a limited number of proteins that interacts directly with myosin Va. The further observation that the two other major ligands in the spinal cord and sciatic nerve are also IF proteins, GFAP and peripherin, suggests that myosin Va plays a more general role in IF behavior or that IFs mediate important functions of myosin Va. By performing multiple protein sequence alignment with hierarchical clustering (Corpet, 1988
; Multalin version 5.4.1) on NF-L, peripherin, and GFAP, we found 132 amino acid homologies in a sequence of 350 residues corresponding to the head and rod domains. The observation that as many as 82 of these amino acid positions differ in NF-H and NF-M may partly explain the relative selectivity of the myosin Va binding to the three “core” subunits of IFs. The observation that other high abundance proteins, such as tubulin, bound negligible amounts of myosin Va underscores the specificity of the myosin Va–IF interaction. Moreover, that another molecular motor, kinesin, is found in negligible amounts in cytoskeletal fractions indicates that tight association with cytoskeletal structures is not a general feature of motor molecules.
The association of myosin Va with neurofilaments raises new possibilities regarding its function in the nervous system where levels are exceptionally high (Cheney et al., 1993
). One of these possibilities is modulating the organization of the axoplasm. We observed that myosin Va deletion in dilute
mice creates a denser packing of neurofilaments in axons, suggesting a role in neurofilament spacing. Because NF-L and actin bind to myosin Va at separate sites, myosin Va may be capable of dynamically cross-linking IFs and microfilaments. Recent studies have emphasized the role of molecules other than neurofilaments themselves in regulating lateral spacing of filaments in axons. Molecules, including BPAG and plectin, have recently been shown to cross-link IFs, microfilaments, and microtubules (Svitkina et al., 1996
; Yang et al., 1996
), although, unlike myosin Va, these proteins have no known ATPase or motor activity. If myosin Va does in fact link filament systems, it is likely to be in the service of dynamically rearranging these structures within the cytoskeletal network. Neurofilaments could either act as an anchor from which myosin Va could move other proteins or vesicular organelles (e.g., actin) or as a cargo of myosin Va. In regard to the first possibility, neurofilaments provide a three-dimensional lattice interconnecting the microtubule system with the subaxolemmal compartment (Yang et al., 1996
). This stationary network of filaments conceivably could represent a system of tracks well suited for myosin Va to guide microfilaments or membranous organelles laterally within axons to achieve the proper radial organization of these structures. Neurofilaments have also been shown to be possible ligands of membrane-associated enzymes and receptors (Terry-Lorenzo et al., 2000
; Kim et al., 2002
), raising the possibility that movements of molecules of this type along neurofilaments may be mediated by myosin Va. Myosin Va has been implicated in moving actin short distances within growth cones (Evans et al., 1997
; Bridgman, 1999
; Huang et al., 1999
). Finally, short-range rearrangements of neurofilaments within the axon, such as those that occur during early postnatal development (Sanchez et al., 1996
), might also require motor activity.
Actin and neurofilaments also move long distances by slow axonal transport. The average rate of myosin Va transport that we observed along optic axons is similar to that of actin and close to the rate at which purified myosin Va moves along actin cables from dissected Nitella cells in vitro (2.5–3.8 mm/d) (Cheney et al., 1993
). Interestingly, Willard (1977)
identified two polypeptides (195 kD and 200 kD) that cosediment with actin in an ATP-reversible way and were transported along optic axons at slow transport rates. Transport of at least some neurofilaments in growing axons of cultured neurons involves a series of rapid movements punctuated by long periods of immobility (Wang et al., 2000
). This pattern suggests that motors, such as kinesin, which are capable of mediating fast transport rates, may be attractive candidates for powering neurofilament movement. Because myosin Va can bind directly to kinesin (Huang et al., 1999
), the interaction of myosin Va with neurofilaments could represent one mechanism to facilitate neurofilament movement along microtubules. Although the results in dilute
mice imply that myosin Va is not essential for movement of neurofilaments into axons, the considerably increased neurofilament number in axons could reflect impaired slow transport. However, a particular pattern of neurofilament distribution along axons, by itself, is not predictive of transport kinetics. An increased rate of incorporation of transported neurofilaments into the stationary cytoskeletal network along axons (Nixon, 1998
) would yield a similar picture. Definitive tests of these possibilities require long-term labeling studies that are precluded in dilute
mice by the frailty and early death of these mice after 3–4 wk.
In conclusion, these novel interactions of myosin Va with IFs indicate previously unrecognized roles for myosin Va in regulating cytoskeleton dynamics in the nervous system. Although long or short range transport and/or rearrangements of neurofilaments represent several of the possible roles, interactions between neurofilaments and myosin Va might instead, or in addition, be important in modulating myosin Va–mediated movements of other cytoskeletal proteins, membranous organelles, or membrane-associated proteins. A variety of experimental approaches will be required to investigate the range of intriguing possibilities.