Article |
Address correspondence to Ralph Nixon, Nathan Kline Institute, New York University School of Medicine, 140 Old Orangeburg Rd., Orangeburg, NY 10962. Tel.: (845) 398-5423. Fax: (845) 398-5422. E-mail: nixon{at}nki.rfmh.org
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: myosin Va; dilute; neurofilaments; NF-associated proteins; axonal transport
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Proteins of the myosin V family serve as molecular motors to transport diverse molecules. Analyses of the MYO2 gene implicate yeast myosin V in the assembly and transport of actin, the translocation of specific proteins to sites of polarized growth, and the transport of vesicles (Santos and Snyder, 1997). The yeast MYO4 gene product transports components necessary for regulating gene expression and targets mRNA molecules to sites of polarized growth (Bobola et al., 1996). In rat and chick neuronal tissue, myosin Va mediates actin-dependent movement of synaptic vesicles and ER (Tabb et al., 1998), and supports filopodial extension in growth cones (Wang and Jay, 1997).
Myosin Va protein in brain is highly expressed (Cheney et al., 1993) and broadly distributed regionally (Mercer et al., 1991; Espreafico et al., 1992), with prominent localizations in growth cones and neurites of cultured neurons, astrocyte processes (Espindola et al., 1992), and axons of several sensory organs (Hasson et al., 1997). Additionally, myosin Va has been reported to immunolocalize to the intermediate filament (IF)* compartment in a variety of cultured cell types (Engle and Kennett, 1994). Although these findings document that myosin Va is a significant component of CNS neurons and sensory organs, its neural functions are still poorly defined.
Mutations in the mouse myosin Va gene, dilute, cause not only defective melanosome movement but also profound neurological symptoms, including a convulsive disorder that results in death by 34 wk of age (Searle, 1952). Mutations in the human myosin Va gene cause Griscelli's disease, a childhood disease characterized by pronounced hypopigmentation and a convulsive disorder that leads to premature death (Pastural et al., 1997). A similar phenotype is associated with mutations of the ashen locus harboring the Rab27a gene (Menasche et al., 2000), which regulates myosin Va recruitment to melanosomes (Wu et al., 2001). Some features of the dilute phenotype also result from the flailer mutation, which originates from germ line exon shuffling between the guanine nucleotide binding protein ß 5 and myosin Va genes, leading to the synthesis of a mutant hybrid protein that competes with wild-type myosin Va (Jones et al., 2000). These phenotypes clearly underscore the importance of myosin Va for normal neurological function.
In view of the importance of myosin Va in brain and its possible association with IFs (Engle and Kennett, 1994), we investigated whether myosin Va interacts with neurofilaments and how it influences the properties of neurofilaments in neurons. Neurofilaments predominate in axons and, to a lesser extent, in dendrites and perikarya of the nervous system where they modulate the polar shape and size of the neuron (Nixon and Shea, 1992). Unlike other classes of IFs, neurofilament proteins are heteropolymers formed from three distinct subunits with apparent molecular masses of 200 kD (NF-H), 150 kD (NF-M), and 70 kD (NF-L) on SDSpolyacrylamide gels. In some axons, neurofilaments form a three-dimensional network with microtubules and actin filaments, mediated by one or more members of a family of cross-linking proteins (Svitkina et al., 1996; Yang et al., 1996). How the organization of this network is achieved in axons is not known.
Neurofilaments, microtubules, and microfilaments, as well as their associated proteins, are transported from the perikaryon at speeds of 0.12.0 mm/d, collectively referred to as the slow phase of axonal transport. This range of rates is distinct from that of fast axonal transport, which carries mainly vesicular constituents by a microtubule-dependent mechanism powered by kinesin in the anterograde direction (Hirokawa et al., 1991) and by dynein in the retrograde direction (Schnapp and Reese, 1989). It remains controversial as to whether different filamentous cytoskeletal structures are transported exclusively as polymers, subunits/oligomers, or in both forms under different conditions (Hirokawa et al., 1997; Wang et al., 2000; Yabe et al., 2001). Short-range transport mechanisms also mediate restricted longitudinal or lateral movement of organelles within a specific compartment of the neuron. Roles for myosin Va in this form of transport have been suggested (Huang et al., 1999); however, the molecular mechanisms responsible for the movements of cytoskeleton components within axons are poorly understood.
In the study presented here, we establish by multiple criteria that the neurofilament is a major ligand for myosin Va in mouse central and peripheral nervous tissue. We demonstrate that myosin Va binds selectively to the NF-L subunit of the filament, and that myosin Va content, transport, and distribution in axons are regulated in vivo, in part, by levels of NF-L. Our further observations show that myosin Va moves in the slow phase of axonal transport, prominently associates with neurofilaments, and alters neurofilament density selectively when deleted in dilute-lethal (dl) mice, suggesting that myosin Va plays a role in the behavior of neuronal cytoskeletal elements in addition to its well-recognized role in translocating membranous organelles.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Myosin Va associates only with the NF-L subunit of neurofilaments
Because neurofilaments are heteropolymers consisting of three subunits, NF-H, NF-M, and NF-L, we used a blot overlay analysis to investigate which of the neurofilament subunits bind to myosin Va. Purified neurofilament subunits were separated on SDSpolyacrylamide gels and blotted onto nitrocellulose membranes, which were then incubated in solutions containing decreasing concentrations of myosin Va fused with ß-galactosidase (Fig. 3 A, lanes 25). As a control, purified ß-galactosidase protein was incubated with immobilized neurofilament proteins in a concentration equal to the highest concentration of myosin Vaß-gal fusion protein used (Fig. 3 A, lane 6). Bound myosin Vaß-gal or ß-galactosidase alone was detected by Western blot analysis using an antiß-galactosidase antibody. Fig. 3 A demonstrates that myosin Va bound to the NF-L subunit in a concentration-dependent manner, but did not bind to NF-M or NF-H. ß-Galactosidase alone did not bind (Fig. 4 A, lane 6), indicating that the association of myosin Vaß-gal fusion protein with NF-L is due to the myosin Va portion of the fusion protein.
|
|
Interaction of endogenous myosin Va with NF-L
Because the NF-L subunit of the neurofilament triplet is the prime ligand for myosin Va, it was of interest to determine whether myosin Va would coimmunoprecipitate with NF-L from neuronal tissue. Using the NF-L mAb NR-4 (Sigma-Aldrich), we immunoprecipitated NF-L from Triton-insoluble cytoskeletal preparations of brain and spinal cord and observed that the immunoprecipitates were enriched not only for NF-L (Fig. 3 C, bottom) but also for myosin Va (top, lanes 1, 2, 4, and 5). In the absence of NF-L antibody, no myosin Va or NF-L sedimented after centrifugation of the Sepharose beads (lane 3), indicating that precipitation of myosin Va and NF-L was antibody dependent.
Myosin Va binds selectively to IF proteins of several classes
The specificity of myosin Va binding to cytoskeletal proteins was further investigated by blot overlay analysis of Triton-insoluble cytoskeletal fractions from mouse spinal cord (Fig. 4 A, lanes 15) and sciatic nerve (Fig. 4 A, lanes 610). In both tissues studied, the major binding partner of myosin Va was NF-L (Fig. 4 B); however, we also observed binding of myosin Va to a 45-kD protein in spinal cord (Fig. 4 A, lanes 25) and to a 55-kD protein in sciatic nerve (Fig. 4 A, lanes 69). Based on these apparent molecular masses on SDS gels, we probed the same blots with antibodies to glial fibrillary acidic protein (GFAP), a 45-kD protein (Fig. 4 D), and peripherin (Fig. 4 C), a 55-kD protein, and observed that the immunoreactivity of these proteins overlapped precisely with the positions of the protein bands detected by myosin Va binding. Thus, from the dozens of different proteins present in the cytoskeleton preparations, myosin Va bound specifically to three structurally related IF proteins. It is not surprising that actin was not detected in this overlay assay because this myosin Va clone (Fb8; Engle and Kennett, 1994) does not have a complete actin binding site.
Myosin Va levels in sciatic nerve are influenced by neurofilaments in vivo
Based on the foregoing evidence of a myosin VaNF-L association, we next examined whether the levels of NF-L in axons influence the axonal content of myosin Va by analyzing mice in which neurofilament levels were altered. Targeted mice lacking the NF-L gene are devoid of NF-L and virtually all neurofilaments (Zhu et al., 1997). NF-L is absent in the sciatic nerves of these mice (Fig. 5, A and C, lane 2), and we observed a 55% reduction in the levels of myosin Va (Fig. 5, A and B, lane 2). By contrast, myosin Va levels were not altered in sciatic nerves of NF-Hdeleted mice (Fig. 5, A and C, lane 3), in which NF-L and neurofilament levels in axons are normal (Rao et al., 1998). The effects on myosin Va of increasing NF-L levels and filament number in axons were also examined in mice overexpressing the NF-L protein (Xu et al., 1993). NF-L levels and neurofilament counts in sciatic nerves from NF-Loverexpressing mice have been previously shown to be 1.5-fold higher than the corresponding control mice (Xu et al., 1993). We confirmed the reported increase in NF-L levels (Fig. 5, D and F, lane 2) and observed by quantitative immunoblot analyses that myosin Va levels were comparably increased (50%) in the sciatic nerves of NF-L transgenic mice (Fig. 5 D, lane 2). These results indicate that axonal myosin Va content is influenced by levels of NF-L, neurofilament number, or both.
|
|
|
Neurofilament depletion alters the transport and distribution of myosin Va in axons
Direct evidence for an in vivo interaction of myosin Va with NF-L was sought by investigating whether the elimination of neurofilaments altered the movement or principal location of myosin Va in axons. To visualize radiolabeled myosin Va on gels in pulse-labeling studies, we analyzed axonal transport patterns in optic nerves of mice deleted of both NF-L and NF-H. In these neurofilament-deficient axons, the average rate of myosin Va translocation was significantly faster than that in normal axons at 3 or 7 d after [35S]methionine injection (Fig. 8, A and B). Transport of tubulin and major slow component b proteins was unaltered in neurofilament-deficient mice (Fig. 8, E and F). Immunogold labeling NF-L-null mice showed that, in the absence of neurofilaments, myosin Va distribution is restricted to the subaxolemmal compartment and to the surfaces of membranous vesicles (Fig. 8, G and H). An increased incidence of subaxolemmal labeling implied that a proportion of the neurofilament-associated myosin Va was redistributed in these mice. These observations demonstrate unequivocally that a significant proportion of myosin Va normally interacts with neurofilaments in vivo.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In addition to establishing a novel myosin Vaneurofilament 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 vivolabeled actin and myosin Va coimmunoprecipitate and cotransport and that myosin Va antibodies decorate the actin-rich subaxolemmal compartment (Fig. 2; Fig. 8, G and H; Kobayashi et al., 1986). It is not surprising that the myosin Vaneurofilament 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 VaIF 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 (2.53.8 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 34 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 Vamediated 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.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein isolation, Western blot analysis, and densitometry
Total protein homogenates from brain, spinal cord, sciatic, and optic nerves were made according to Rao et al. (1998). Protein concentration was determined using bicinchoninic acid (BCA) assay kit (Pierce Chemical Co.). Protein extracts of known amounts from different neuronal tissues were fractionated on SDS-PAGE gels containing 6 or 7.5% polyacrylamide and transferred to nitrocellulose membranes. Triton-insoluble and -soluble fractions were made according to Nixon et al. (1990). After protein estimation, equal amounts of Triton-insoluble and -soluble fractions were boiled, run on a 7% SDSpolyacrylamide gel, and immunoblotted with affinity-purified polyclonal antibody directed against the COOH terminus of the myosin Va fusion protein (Evans et al., 1997), NF-L (NR-4), -tubulin (DMIA), and kinesin heavy chain mAbs as specified by the manufacturer. The blots were processed with the ECL system (Amersham Biosciences) or the alkaline phosphatase system (Promega). Band images were quantified with a BioImage whole band analyzer from Kodak.
Immunocytochemistry of mouse tissues
C57BL/6J mice were anesthetized and fixed by cardiac perfusion using 10% neutral buffered formalin in TBS, pH 7.4. The brain, cervical spinal cord, and sciatic nerves were dissected and 40-µm-thick vibratome sections were processed for immunocytochemistry (Cataldo et al., 1990) using a polyclonal antibody directed against myosin Va. Several sections were processed in tandem in the absence of primary antibody and served as controls.
Immunoelectron microscopy and morphometry of optic and sciatic nerves
34-wk-old dl and their control mice and NF-Lnull mice were anesthetized and perfused fixed with 4% paraformaldehyde0.2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). The tissues were fixed for an additional 2 h at room temperature in 10% formalin. The brain and optic pathway were removed and 40-µm-thick vibratome sections of the cerebral peduncle and the optic pathway at a distance of 700 µm from the retina were dehydrated in series of alcohols and embedded in Epon as previously described (DeMey, 1983). Ultrathin sections were placed on nickel-coated grids and processed for postembedding immunostaining (DeMey, 1983) using a polyclonal antibody directed against myosin Va. Grids incubated without primary antibody served as controls. All grids were poststained in uranyl acetate and lead citrate and inspected using a JEOL 100EX electron microscope.
Sciatic nerves were dissected and analyzed morphometrically to determine axonal cross-sectional areas for the entire fiber population and neurofilament numbers for a representative subpopulation of axons, as previously described (Rao et al., 1998).
Blot overlay binding assay
Neurofilament triplet subunits were purified as previously reported (Balin et al., 1991). The myosin Va fusion protein (120 kD) (Engle and Kennett, 1994) consists of a small 3.5-kD piece of ß-galactosidase fused to that portion of myosin Va representing 57% of the full-length protein containing half of the actin binding site, calmodulin binding domains,
helical coiled-coil domains, and a small portion of the COOH-terminal tail domain. The fusion protein was expressed in XL-1 Blue Escherichia coli (Stratagene) cells and purified using an antiß-galactosidase affinity column (Promega) per the manufacturer's protocol. 10 µg of purified neurofilament triplet proteins were run per lane on a 7% SDSpolyacrylamide gel, blotted onto nitrocellulose, and cut into strips. The strips were blocked and incubated in 10 µg/ml, 1 µg/ml, 0.1 µg/ml, 0.01 µg/ml, or, as a control, 0 µg/ml of myosin Va fusion protein in PBS. Bound myosin Vaß-gal was detected with antiß-galactosidase mAb (Boehringer) according to the manufacturer's instructions and blots were processed with the ECL system (Amersham Biosciences) and by exposure to Kodak XAR film. The purified neurofilament triplet proteins immobilized on the nitrocellulose were visualized by Coomassie blue staining. Cytoskeletal blot overlay assays were performed same as indicated above for purified NF proteins.
Coimmunoprecipitation of myosin Va and NF-L fusion proteins
Myosin Vaß-gal fusion protein was expressed as described above and the crude extract was used for immunoprecipitation. NF-L myc-tagged plasmid (Heins et al., 1993) was transfected into BL21-DE3 host cells, induced, NF-Lcontaining lysates were made, and protein concentrations determined. 2 µg of NF-LFP crude extract and myosin VFP crude extract (containing 3 µg of myosin VaFP) were mixed together in 1x RIPA buffer. Protein A/Gagarose (Boehringer) was also added to preclear. The mixture was incubated overnight at 4°C to allow binding between NF-LFP and myosin VaFP. As controls, 2 µg of NF-LFP or 3 µg of myosin VaFP, in 1x RIPA buffer were also precleared with protein A/Gagarose overnight at 4°C. Protein A/Gagarose was removed with centrifugation, and NF-LFP/myosin VaFP complex, NF-LFP alone, and myosin VaFP alone were immunoprecipitated for 8 h at 4°C with either 5 µl of a polyclonal Myc antisera (to precipitate NF-LFP) or with 2.5 µl of 1:300 dilution of antiß-galactosidase mAb (Promega). Protein Gagarose (50 µl) was added and the mixtures were nutated overnight at 4°C. Protein Gagarose was pelleted, washed, boiled in Laemmli buffer, fractionated on 7.5% SDSpolyacrylamide gels, blotted onto nitrocellulose membranes, and probed with a mixture of antiß-gal and anti-Myc antisera.
Immunoprecipitation of myosin Va with NF-L antibody
Triton X-100insoluble fractions were suspended in TBS, and SDS was added to a final concentration of 1%. The samples were diluted 1:4 in neurofilament extraction buffer (60 mM Tris-HCl, pH 7.4, 190 mM NaCl, 6 mM EDTA, 1.25% Triton X-100, 1 mM PMSF). The samples were sonicated for 20 s and protein was estimated by bicinchoninic acid (BCA) method. A monoclonal antibody to NF-L (NR-4) was added to 0.62.4 mg of cytoskeletal fractions at a dilution of 1:10 and the samples were incubated overnight at 4°C. The antigen antibody complex was precipitated using protein A/GSepharose (Santa Cruz Biotechnology, Inc.), washed, boiled in Laemmli buffer, fractionated on 7% SDS gels, and immunoblotted with myosin Va and NF-L antibodies.
Immunoprecipitation of 35S-labeled myosin Va
NF-Hnull mice were injected intravitreally with 100 µCi of [35S]methionine into each eye and killed after 3 d, as previously described (Nixon and Logvinenko, 1986). Cytosolic and cytoskeletal fractions of optic nerves and tracts obtained were used to immunoprecipitate labeled myosin Va using an affinity-purified polyclonal antibody to myosin Va (Evans et al., 1997). The samples were incubated overnight at 4°C. The antigenantibody complex was precipitated using protein A/GSepharose, electrophoresed, electroblotted, and exposed to X-ray film followed by Western blotting for myosin Va, NF-L, and actin.
Slow axonal transport of pulse-labeled myosin Va and neurofilaments in optic axons
The retinal ganglion cells of 34-mo-old NF-H and NF-H/NF-L double null mice were radiolabeled in vivo with 100 µCi of [35S]methionine by intravitreal injection. 3 and 7 d after injection, optic pathways from groups of three animals were cut into eight consecutive 1.1-mm segments. Triton-soluble and -insoluble NF-rich cytoskeleton preparations from each segment were subjected to SDS-PAGE, electrotransfer of proteins, phosphor-imaging, and autoradiography.
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
This work was supported by United States Public Health Service grant P30-DK19525 and by Merit grant AG05604 and National Institutes of Health National Research Service Award training grant in neurodegeneration, No. 2-T32-AG00222 (L.J. Engle).
Submitted: 14 May 2002
Revised: 16 September 2002
Accepted: 16 September 2002
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Berg, J.S., B.C. Powell, and R.E. Cheney. 2001. A millennial myosin census. Mol. Biol. Cell. 12:780794.
Bridgman, P.C. 1999. Myosin Va movements in normal and dilute-lethal axons provide support for a dual filament motor complex. J. Cell Biol. 146:10451060.
Cheney, R.E., M.K. O'Shea, J.E. Heuser, M.V. Coelho, J.S. Wolenski, E.M. Espreafico, P. Forscher, R.E. Larson, and M.S. Mooseker. 1993. Brain myosin-V is a two-headed unconventional myosin with motor activity. Cell. 75:1323.[Medline]
Corpet, F. 1988. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 16:1088110890.[Abstract]
DeMey, J.R. 1983. The preparation of immunoglobulin gold conjugates (IGS reagents) and their use as markers for light and electron microscopic immunocytochemistry. In Immunocytochemistry. A.C. Cuello, editor. John Wiley & Sons Inc., New York. 358366.
Espindola, F.S., E.M. Espreafico, M.V. Coelho, A.R. Marins, F.R.C. Costa, M.S. Mooseker, and R.E. Larson. 1992. Biochemical and immunological characterization of p190calmodulin complex from vertebrate brain: a novel calmodulin-binding myosin. J. Cell Biol. 118:359368.[Abstract]
Espreafico, E.M., R.E. Cheney, M. Matteoli, A.A.C. Nascimento, P.V.D. Camilli, R.E. Larson, and M.S. Mooseker. 1992. Primary structure and cellular localization of chicken brain myosin V (p190), an unconventional myosin with calmodulin light chains. J. Cell Biol. 119:15411557.[Abstract]
Evans, L.L., J. Hammer, and P.C. Bridgman. 1997. Subcellular localization of myosin V in nerve growth cones and outgrowth from dilute-lethal neurons. J. Cell Sci. 110:439449.
Hasson, T., P.G. Gillespie, J.A. Garcia, R.B. MacDonald, Y. Zhao, A.G. Yee, M.S. Mooseker, and D.P. Corey. 1997. Unconventional myosins in inner-ear sensory epithelia. J. Cell Biol. 137:12871307.
Heins, S., P.C. Wong, S. Muller, K. Golide, D.W. Cleveland, and U. Aebi. 1993. The rod domain of NF-L determines neurofilament architecture, whereas the end domains specify filament assembly and network formation. J. Cell Biol. 123:15171533.[Abstract]
Hirokawa, N., R. Sato-Yoshitake, N. Kobayashi, K.K. Pfister, G.S. Bloom, and S.T. Brady. 1991. Kinesin associates with anterogradely transported membranous organelles in vivo. J. Cell Biol. 114:295302.[Abstract]
Huang, J.-D., S.T. Brady, B.W. Richards, D. Stenoien, J.H. Resau, N.G. Copeland, and N.A. Jenkins. 1999. Direct interaction of microtubule-and-actin based transport motors. Nature. 397:267270.[CrossRef][Medline]
Jones, J.M., J.-D. Huang, V. Mermall, B.A. Hamilton, M.S. Mooseker, A. Escayg, N.G. Copeland, N.G. Jenkins, and M.H. Meisler. 2000. The mouse neurological mutant flailer expresses a novel hybrid gene derived by exon shuffling between Gnb5 and Myo5a. Hum. Mol. Genet. 9:821828.
Kayalar, C., T. Ord, M.P. Testa, L.T. Zhong, and D.E. Bredesen. 1996. Cleavage of actin by interleukin 1 ß-converting enzyme to reverse DNase I inhibition. Proc. Natl. Acad. Sci. USA. 93:22342238.
Kim, O.-J., M.A. Ariano, R.A. Lazzarini, M.S. Levine, and D.R. Sibley. 2002. Neurofilament-M interacts with the D1 dopamine receptor to regulate cell surface expression and densensitization. J. Neurosci. 22:59205930.
Kobayashi, T., S. Tsukita, S. Tsukita, Y. Yamamoto, and G. Matsumoto. 1986. Subaxolemmal cytoskeleton in squid giant axon. I. Biochemical analysis of microtubules, microfilaments, and their associated high molecular weight proteins. J. Cell Biol. 102:16991709.[Abstract]
Lewis, S., and R.A. Nixon. 1988. Multiple phosphorylated variants of the high molecular mass subunit of neurofilaments in axons of retinal cell neurons: characterization and evidence for their differential association with stationary and moving neurofilaments. J. Cell Biol. 107:26892701.[Abstract]
Mercer, J.A., P.K. Seperack, M.C. Strobel, N.G. Copeland, and N.A. Jenkins. 1991. Novel myosin heavy chain encoded by murine dilute coat colour locus. Nature. 349:709713.[CrossRef][Medline]
Mercken, M., I. Fischer, K.S. Kosik, and R.A. Nixon. 1995. Three distinct axonal transport rates for tau, tubulin, and other microtubule-associated proteins: evidence for dynamic interactions of tau with microtubules in vivo. J. Neurosci. 15:82598267.[Abstract]
Nixon, R.A. 1991. Axonal transport of cytoskeletal proteins. In The Neuronal Cytoskeleton. R.D. Burgoyne, editor. Wiley-Liss, Inc. New York. 283307.
Nixon, R.A., and K.B. Logvinenko. 1986. Multiple fates of newly synthesized neurofilament proteins: Evidence for a stationary neurofilament network distributed nonuniformly along axons of retinal ganglion cell neurons. J. Cell Biol. 102:647659.[Abstract]
Nixon, R.A., I. Fischer, and S.E. Lewis. 1990. Synthesis, axonal transport, and turnover of the high molecular weight microtubule-associated protein MAP 1A in mouse retinal ganglion cells: tubulin and MAP 1A display distinct transport kinetics. J. Cell Biol. 110:437448.[Abstract]
Rao, M.V., M.K. Houseweart, T.L. Williamson, T.O. Crawford, J. Folmer, and D.W. Cleveland. 1998. Neurofilament dependent radial growth of motor axons and axonal organization of neurofilaments does not require the neurofilament heavy subunit (NF-H) or its phosphorylation. J. Cell Biol. 143:171181.
Rodriguez, O.C., and R.E. Cheney. 2002. Human myosin-Vc is a novel class V myosin expressed in epithelial cells. J. Cell Sci. 115(Pt. 5):9911004.
Sanchez, I., L. Hassinger, P.A. Paskevich, H.D. Shine, and R.A. Nixon. 1996. Oligodendroglia regulate the regional expansion of axon caliber and local accumulation of neurofilaments during development independently of myelin formation. J. Neurosci. 16:50955105.
Santos, B., and M. Snyder. 1997. Targeting of chitin synthase 3 to polarized growth sites in yeast requires Chs5p and Myo2p. J. Cell Biol. 136:95110.
Schnapp, B.J., and T.S. Reese. 1989. Dynein is the motor for retrograde axonal transport of organelles. Proc. Natl. Acad. Sci. USA. 86:15481552.[Abstract]
Searle, A.G. 1952. A lethal allele of dilute in the house mouse. Heredity. 6:395401.
Svitkina, T.M., A.B. Verkhovsky, and G.G. Borisy. 1996. Plectin side arms mediate interaction of intermediate filaments with microtubules and other components of cytoskeleton. J. Cell Biol. 135:9911007.[Abstract]
Tabb, J.S., B.J. Molyneaux, D.L. Cohen, S.A. Kuznetsov, and G.M. Langford. 1998. Transport of ER vesicles on actin filaments in neurons by myosin V. J. Cell Sci. 111:32213234.
Terry-Lorenzo, R.T., M. Inoue, J.H. Connor, T.A. Haystead, B.N. Armbruster, R.P. Gupta, C.J. Oliver, and S. Shenolikar. 2000. Neurofilament-L is a protein phosphatase-1-binding protein associated with neuronal plasma membrane and post-synaptic density. J. Biol. Chem. 275:24392446.
Wang, F.S., and D.G. Jay. 1997. Chromophore-assisted laser inactivation (CALI): probing protein function in situ with a high degree of spatial and temporal resolution. Trends Cell Biol. 6:442445.
Willard, M. 1977. The identification of two intra-axonally transported polypeptides resembling myosin in some respects in the rabbit visual system. J. Cell Biol. 75:111.
Wu, Z., K. Rao, M.B. Bowers, N.G. Copeland, N.A. Jenkins, and J.A. Hammer. 2001. Rab27a enables myosin Va-dependent melanosome capture by recruiting the myosin to the organelle. J. Cell Sci. 114:10911100.
Yabe, J.T., W.K. Chang, H.T.M. Chylinski, S. Lee, A.F. Pimenta, and T.B. Shea. 2001. The predominant form in which neurofilament subunits undergo axonal transport varies during axonal initiation, elongation, and maturation. Cell Motil. Cytoskeleton. 48:6183.[CrossRef][Medline]
Yuan, A., R.G. Mills, C.P. Chia, and J.J. Bray. 2000. Tubulin and neurofilament proteins are transported differently in axons of chicken motoneurons. Cell. Mol. Neurobiol. 20:623632.[CrossRef][Medline]
Related Article