Myosin Va binding to neurofilaments is essential for correct myosin Va distribution and transport and neurofilament density

Abstract

The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive. Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves. Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with neurofilaments, and that the NF-L subunit is its major ligand. A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L–null mice lacking neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H–null mice. In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va. Eliminating neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles. Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased neurofilament number and levels of neurofilament proteins without altering axon caliber. These results identify myosin Va as a neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of neurofilaments in axons.

Neurofilament Transport Is Dependent on Actin and Myosin

“…This possibility was supported by the demonstration that NF-L is a ligand for myosin in axons (Rao et al, 2002). Consistent with the observed colocalization of myosin and NFs in vivo (Rao et al, 2002), myosin also was associated with NFs in differentiated NB2a/d1 cells. NF subunits were coprecipitated from NB2a/d1 cells with an anti-myosin antibody (Fig.…”

“…Notably, coprecipitation of NF subunits by antimyosin and colocalization of NFs with myosin with axons [Rao et al (2002); this work] suggest that some NFs may interact directly with, and undergo translocation along the actin cortex via, myosin without the need for dynein-mediated microtubule transport. Myosin Va also binds directly to kinesin (Huang et al, 1999), leaving open the possibility that myosin may link NFs to kinesin as well as to actin (for review, see Bridgman, 2004;Brown and Bridgman, 2004).…”

“…Note the reactivity of immunoreactive species corresponding to the migratory positions of the intermediate chain of dynein and those of NF-M and NF-H (arrows) in blots reacted with SMI-31. In addition, note the presence of a 190 kDa immunoreactive species (the expected migratory position of neuronal myosin) (Rao et al, 2002) in the blot reacted with anti-myosin; this blot also contains a prominent immunoreactive species at ϳ125 kDa, which may represent a breakdown product of the 190 kDa species or a novel myosin-immunoreactive species unique to these cells. As anticipated, the IgG derived from immunoprecipitation with anti-myosin also is stained prominently (1°ab) by the goat secondary antibody used in antimyosin staining.…”

“…Recent observations identify NFs as a major ligand of myosin in situ (Rao et al, 2002). NFs also have been shown to bind the actin-associated protein spectrin (Frappier et al, 1987(Frappier et al, , 1991.…”

Deficiency in ubiquitin ligase TRIM2 causes accumulation of neurofilament light chain and neurodegeneration

“…The binding partner of TRIM2, myosin V, has been shown to bind neurofilament light chain (NF-L) and through this interaction to modulate neurofilament organization in axons (13). Because changes in NF-L metabolism have been shown to trigger neurodegeneration in mice (14), we tested whether TRIM2 could interact with NF-L and affect its metabolism.…”