Article |
Address correspondence to Mala V. Rao, Nathan Kline Institute, NYU School of Medicine, 140 Old Orangeburg Rd., Orangeburg, NY 10962. Tel.: (845) 398-5547. Fax: (845) 398-5422. email: rao{at}nki.rfmh.org
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: NF-M phosphorylation; axonal transport; microtubules; ES cells; gene knockin
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
With the advent of transgenics and gene deletion technologies, mice that either overexpress or are deleted for neurofilament subunit genes indicate that NFs play a pivotal role in the regulation of normal axonal diameters, and this property is sensitive to the levels of NFs in the axon (Ohara et al., 1993; Eyer and Peterson, 1994; Tu et al., 1995; Marszalek et al., 1996; Wong et al., 1996; Zhu et al., 1997; Elder et al., 1998a; Jacomy et al., 1999). Moreover, overexpression of NF-L with either NF-M or NF-H results in much bigger increases in diameters (Xu et al., 1996; Meier et al., 1999) compared with inhibition of the same growth in single transgenic mice, indicating that normal subunit ratios are critical (Monteiro et al., 1990; Eyer and Peterson, 1994; Tu et al., 1995; Marszalek et al., 1996; Wong et al., 1996; Xu et al., 1996).
Newly synthesized neurofilaments are transported along axons at an average rate of 0.21.0 mm/d (Hoffman and Lasek, 1975; Black and Lasek, 1980), although the kinetic behavior of the entire neurofilament population within axons is complex (Nixon, 1998; Jung et al., 2000a,b; Wang et al., 2000; Rao et al., 2002b; Ackerley et al., 2003; Brown, 2003; Rao and Nixon, 2003). Together with microtubules and microfilaments, neurofilaments create a nonuniform fibrous network along the length of mature axons, which is extensively cross-linked by lateral cross-bridges formed by various linking proteins, including plectin (Errante et al., 1994; Rao et al., 1998), BPAGn/dystonin (Yang et al., 1996; Dalpe et al., 1998), gigaxonin (Bomont et al., 2000), myosin Va (Rao et al., 2002b), and, possibly, extended domains of the neurofilament triplet proteins (Willard and Simon, 1981; Hirokawa et al., 1984). How transported neurofilaments and related cytoskeletal elements accumulate locally and achieve the proper integration into this regionally specialized cytoskeletal network along axons is poorly understood.
The phosphorylation of NF-M and NF-H carboxyl-terminal domains has also been suspected of regulating axon caliber by controlling neurofilament transport (Nixon et al., 1982; Lewis and Nixon, 1988), local accumulation (Nixon, 1998; Sanchez et al., 2000), and spacing in relation to their neighbors (Gotow et al., 1992; Nixon et al., 1994; Pant and Veeranna, 1995; Sanchez et al., 1996). Phosphorylated subunits of NF-M and NF-H contain 15 and 50 mol phosphate, respectively (Jones and Williams, 1982; Julien and Mushynski, 1982; Geisler et al., 1987; Goldstein et al., 1987; Lee et al., 1988), and they are the most extensively phosphorylated proteins in neurons (Julien and Mushynski, 1982, 1983; Carden et al., 1985). The carboxyl-terminal domains of these two subunits have multiple lysine-serine-proline (KSP) repeat motifs, which are variably phosphorylated in the axon mainly after neurofilaments enter the axon (Sternberger and Sternberger, 1983; Glicksman et al., 1987; Nixon et al., 1987; Oblinger et al., 1987). This process is linked to intraaxonal local accumulation of neurofilaments (Nixon et al., 1994), which drives caliber expansion and, in turn, is regulated by signals emanating from myelinating glial cells (de Waegh et al., 1992; Hsieh et al., 1994; Nixon et al., 1994; Sanchez et al., 1996, 2000; Yin et al., 1998). NF-H overexpression in mice (Collard et al., 1995; Marszalek et al., 1996), or hyperphosphorylation of NF-H on its carboxyl-terminal region, slows axonal transport of neurofilaments (Jung et al., 2000b; Ackerley et al., 2003), whereas hypophosphorylated NF-H molecules are transported in primary neuronal cultures at much faster rates than normal (Ackerley et al., 2000, 2003; Jung et al., 2000a). However, recent studies have shown that genetic deletion of NF-H (Elder et al., 1998b; Rao et al., 1998; Zhu et al., 1998) or its carboxyl-terminal domain (Rao et al., 2002a; Rao and Nixon, 2003) in mice has relatively small effects on the overall radial growth of motor axons, spacing between neurofilaments (Elder et al., 1998b; Rao et al., 1998; Zhu et al., 1998), and average neurofilament transport rates (Zhu et al., 1998; Rao et al., 2002a). These observations imply that either NF-H tail phosphorylation by itself does not regulate all the properties that are previously ascribed to it, or compensatory mechanisms involving NF-M (Sanchez et al., 2000) or other molecules replace these putative functions of NF-H in mice lacking NF-H.
NF-M domain function has been less extensively investigated than NF-H, although existing data suggest that NF-M may be the more influential subunit in regulating known neurofilament behaviors. NF-M, unlike NF-H, is essential for filament assembly (Elder et al., 1998a; Jacomy et al., 1999). Deletion of NF-M protein results in a fourfold acceleration of the rate of slow axonal transport of NFs in sciatic nerves (Jacomy et al., 1999). Overexpression of NF-M has complex effects on neurofilament number, transport, spacing, and axon caliber (Tu et al., 1995; Wong et al., 1996; Xu et al., 1996; Xu and Tung, 2001). To investigate the in vivo role of NF-M tails specifically, we constructed NF-M tail deletion mice by the ES cellmediated "gene knockin" approach, which does not alter subunit stoichiometry, and therefore, represents a relatively selective in vivo perturbation of NF-M tail function. This work has revealed a distinct role of NF-M tails in establishing inter-neurofilamentous cross-bridges that control the normal spacing between neurofilaments and modulate axon radial growth. The findings also demonstrate that NF-M and NF-H tails, despite structural similarities, have distinct functions.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
NF-M tails influence the dynamics of microtubules in the axon
Alteration of any of the three NF subunits in the axon results in an increased density of microtubules in the axon (Zhu et al., 1997, 1998; Rao et al., 1998). It was postulated that dephosphorylated NF-H tails bind microtubules more effectively (Hisanaga and Hirokawa, 1990), and this binding is inhibited by the phosphorylation of NF-H tails by CDC2 kinase (Hisanaga et al., 1991). Recently, we have shown that NF-H tails do not influence the tubulin levels or the microtubule numbers in NF-H taildeleted motor or sensory axons, suggesting that NF-H tails do not regulate the microtubule dynamics (Rao et al., 2002a). To study the effect of NF-M tail deletion on microtubule content in the axons, we quantified the tubulin subunits in wild-type and their littermate heterozygous and homozygous mice. The immunoblot results indicate that both -tubulin (Fig. 1 L) and neuron-specific ßIII-tubulin (Fig. 1 M) levels are increased in NF-M taildeleted axons. To investigate the polymerization state of tubulin, we measured the density of microtubules in NF-Mtail
motor axons. Our quantitative data revealed an approximately twofold increase in total number of microtubules in NF-M taildeleted axons (Fig. 1 O; P < 0.003). These observations suggest that NF-M tails influence the dynamics of microtubules in the axon.
NF-M tails are essential for radial growth of motor axons but not for axon survival
To understand the function of NF-M tails and their phosphorylation in the radial growth of motor axons, L5 ventral roots of NF-Mtail homozygous mice and their littermate controls were examined. Axonal profiles at either 2 or 6 mo of age demonstrate that the loss of NF-M tails in homozygous mice has severe inhibition of radial growth of motor axons (Fig. 2 A, compare NF-M+/+ with NF-Mtail
/tail
at 2 and 6 mo of age, respectively). To study the effects of the loss of NF-M tails on the survival of motor neurons, we counted the total number of axons from wild-type, heterozygous, and homozygous NF- Mtail
ventral roots at 2 and 6 mo of age. The total number of axons between wild-type and homozygous NF- Mtail
axons did not change significantly, but the relative proportions of axons (of sizes > or <4 µm) changed significantly (Fig. 2, B and C). Although the total number of axons did not change at either 2 or 6 mo of age (Fig. 2 C), the growth of NF-M taildeficient axons is severely compromised (Fig. 2, D and E). To measure the changes associated with the loss of NF-M tails more precisely in motor axons, all the axons within each ventral root (Fig. 2 A) were measured, 2- and 6-mo-old wild-type and NF-M taildeficient homozygous mice and their effective diameters were calculated. Axons in wild-type mice grew bimodally from 2 to 6 mo with peak values of 5-µm and 13-µm diam, respectively (Fig. 2, D and E). In NF-M taildeleted motor axons, the small diameter axons grew normally at both 2 and 6 mo of age (Fig. 2, B and C; <4-µm-diam axons), but the growth of large diameter axons was severely inhibited by as much as 50% at 2 mo (Fig. 2 D, the peak value was 5 µm for the control and 2.5 µm for NF-Mtail
mice) and 6 mo of age (Fig. 2 E, the peak value for large diameter axons was
6 µm in NF-Mtail
compared with 13 µm in wild-type mice). These results indicate that NF-M tails are important for the radial growth of motor axons.
|
|
|
NF-M tail deletion does not alter average rates of neurofilament transport
To study the role of NF-M tail and its phosphorylation in the regulation of slow axonal transport, optic nerve proteins of the wild-type and NF-M taildeficient mice were labeled with [35S]methionine, and the rates of movement of components in the slow phase of axoplasmic transport were measured. NF-Mtail in mice results in the appearance of a novel 50-kD NF-M taildeleted polypeptide, and eliminates full-length NF-M protein (Fig. 5 B). In cytoskeletal fractions of optic axons, the movement of full-length NF-M in wild mice was indistinguishable from that of truncated NF-M tailless protein in NF- Mtail
optic axons (Fig. 5, A and B, full-length and truncated NF-M are indicated by arrows; and Fig. 5, E and F). Confirming this observation is the finding that NF-L (Fig. 5, G and H) and NF-H (Fig. 5, I and J) from NF-M taildeficient axons also moved at the same speed as the corresponding subunits in wild-type axons. The movement of cytoskeleton-associated (unpublished data), soluble tubulins (Fig. 5 K), and actin (Fig. 5 L) also was not significantly affected by the loss of NF-M tails. Additional transport studies performed at different time points, 3 d (unpublished data) and 1 wk (Fig. 5, E, G, and I), after pulse labeling also revealed no change in the transport pattern as seen at the 2-wk time point (Fig. 5, HL). These results demonstrate that loss of the NF-M tails does not influence the average rate of slow axonal transport of NFs. Coupled with previous studies in NF-H taildeleted mice (Rao et al., 2002a), these findings indicate that deletion of the tail domain of either NF-H or NF-M alone does not alter the average rate of NF axonal transport even though NF-M tail deletion has much more profound effects on radial growth and cytoarchitecture of the axon than NF-H tail deletion. It is possible that the presence of either one of these tails is enough to slow the NF transport. To address this question, axonal transport studies have to be performed in axons of NF-M/H double tailless mice.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We found that the NF-M tail domain regulates axon caliber, in part, by increasing the spacing between neurofilaments. Our data showing that the deletion of NF-M tail domains eliminates most fine long neurofilament cross-bridges are consistent with the concept that the NF-M tail increases interneurofilament spacing by extending these cross-bridges laterally between filaments (Fig. 6) as predicted from earlier antibody decoration studies (Willard and Simon, 1981; Hirokawa et al., 1984). Transfection of carboxyl-terminal mutants of NF-M along with wild-type NF-L into Sf9 cells indicates that the carboxyl-terminal region of NF-M is capable of forming these fine long cross-bridges to organize neurofilaments (Nakagawa et al., 1995). This function of NF-M tails may be shared to some extent with the NF-H tail because deleting this region causes loss of fine cross-bridges (Chen et al., 2000; Rao et al., 2002a). However, NF-H tail deletion has a much smaller effect on the organization of neurofilaments in the axons or on axon caliber than deleting NF-M tails. This may suggest a role of the NF-H tail in stabilizing neurofilament cross-bridging rather than in regulating axonal diameters and cytoskeletal architecture.
|
Neurofilaments and microtubules interconnect in the axonal cytoskeleton (Hirokawa et al., 1984). Microtubule numbers in the axon are sensitive to changes in the subunit composition of neurofilaments (Rao et al., 1998; Zhu et al., 1998; Jacomy et al., 1999), supporting a likely interaction between these two structural elements. NF-H tails, in particular, have been implicated in modulating this microtubule binding activity (Hisanaga and Hirokawa, 1990; Miyasaka et al., 1993), although the deletion of NF-H tails did not affect either total tubulin or microtubules in either motor or sensory axons (Rao et al., 2002a). However, we found here, that the NF-M tail is essential for maintaining the proper number of microtubules in the axon. In the absence of NF-M tails, neurofilaments and other axonal structures may still remain associated by means of additional putative cytoskeletal linker proteins (Errante et al., 1994; Yang et al., 1996; Dalpe et al., 1998; Rao et al., 1998), gigaxonin (Bomont et al., 2000), and the neurofilament-binding motor protein myosin Va (Rao et al., 2002b; Fig. 6).
In this work, we also observed that despite major influences of NF-M tails on axon caliber and neurofilament organization, eliminating this phosphorylated tail region without altering subunit ratios had no significant effect on the average rate of transport of NF-Mtail, NF-L, NF-H, tubulin, and actin in optic axons. These observations demonstrate that appropriate expression and axonal transport of neurofilament proteins are crucial for radial growth, independent neurofilament interactions mediated by NF-M are also essential. The independence from the axonal transport process of key aspects of neurofilament organization is consistent with evidence that most neurofilaments in mature myelinated axons are extensively cross-linked to each other and to various stationary cytoskeletal structures (Fig. 6). The recent in vitro studies indicate that slow axonal transport of neurofilaments is due to rapid movements and frequently long "pauses" seen in growing axons in neuronal cultures (Roy et al., 2000; Wang et al., 2000). It has been argued over the years that NFs are transported as filaments, oligomers, and subunits (Terada et al., 1996; Bass and Brown, 1997; Hirokawa et al., 1997; Yabe et al., 1999; Prahlad et al., 2000; Roy et al., 2000; Shah et al., 2000; Wang et al., 2000; Shah and Cleveland, 2002). However, the motor proteins that modulate NF axonal transport are not known. The correlative evidence suggests that kinesin, dynein/dynactin, and myosin Va motor proteins may be regulating this process (Yabe et al., 1999; Prahlad et al., 2000; Shah et al., 2000; Rao et al., 2002b; Xia et al., 2003).
NF-H carboxyl-terminal domain phosphorylation has been proposed as a key determinant of slow axonal transport rate of neurofilaments (Nixon et al., 1982; Lewis and Nixon, 1988; Jung et al., 2000a,b; Ackerley et al., 2003). In vitro cell culture studies and in vivo NF-H overexpression results indicate that the NF-H modulates the rate of NF axonal transport (Collard et al., 1995; Marszalek et al., 1996). Moreover, reducing the phosphorylation state of some of the KSP repeats of NF-H, in cells overexpressing this subunit, accelerates the rate of slow axonal transport of NFs (Ackerley et al., 2003). Axonal transport studies in sciatic nerves of NF-H null mice show increased NF transport rates (Zhu et al., 1998). However, transport studies in optic nerves of NF-H null and NF-H tail domain deletion mice do not exhibit altered rates of slow axonal transport of NFs (Rao et al., 2002a), indicating the NF transport rate is not modulated by the phosphorylated tail of NF-H in axons under these conditions (Rao et al., 2002a; Rao and Nixon, 2003). The results also indicate that loss of NF-Mphosphorylated tails do not influence the rate of NF transport. In this work, it is possible that the presence of the phosphorylated tail on either the NF-H or NF-M is sufficient to mask effects on neurofilament transport of deleting the tail on the other subunit. This possibility can be tested in mice in which both NF-H and NF-M tails are eliminated. However, independent of the resolution of this issue, the results of this paper clearly define a role for the carboxyl-terminal tail of NF-M in neurofilament organization, which does not require a change in rate of neurofilament transport.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunoblotting of neurofilament and tubulin subunits
Sciatic and optic nerve extracts were made from 2 to 6-mo-old animals as described previously (Rao et al., 1998). Protein concentration was determined using bicinchoninic acid assay kit (Pierce Chemical Co.). Protein extracts, as well as known amounts of neurofilament standards, were separated on 7% polyacrylamide gels with SDS and transferred to nitrocellulose membranes. Full-length and NF-Mtail proteins were detected with an mAb raised against the rod domain of NF-M (Tu et al., 1995). The NF-H (pAb-NF-HCOOH; Xu et al., 1993), the NF-L (pAb-NF-LCOOH; Xu et al., 1993), the Myc-tagged NF-Mtail
subunit with a polyclonal Myc antibody (Gill et al., 1990), and the myosin Va with a polyclonal antibody (pMyo Va, Evans et al., 1997) were probed and followed by secondary antirabbit IgG. mAbs to NF-M (RMO44; Tu et al., 1995), NF-H (SMI-31, 32; Sternberger and Sternberger, 1983; RT-97; Sanchez et al., 2000),
-tubulin (DM1A; Sigma-Aldrich), and neuron-specific ßIII-tubulin (T-8660; Sigma-Aldrich) were used to identify each subunit, followed by goat antimouse IgG (Sigma-Aldrich). After developing the blots with appropriate reagents, the immunoreactive bands were visualized by autoradiography and quantified by phosphorimaging (Molecular Dynamics) using known amounts of purified mouse spinal cord neurofilament standards.
Axonal transport studies
Retinal ganglion cells from 4-mo-old NF-Mtail or their control littermate mice were radiolabeled in situ with 100 µCi [35S]methionine by intravitreal injection with a calibrated micropipette apparatus into anesthetized mice (Nixon, 1980). After injection, mice were killed by cervical dislocation and optic pathways were dissected after 3 d, 1 wk, and 2 wk. Three to four animals were analyzed for each genotype and time point. The optic pathways were frozen and cut into eight or nine consecutive 1.1-mm segments. Each was homogenized with a buffer containing 1% Triton X-100, 50 mM Tris, pH 6.8, 2 mM EDTA, 1 mM PMSF, and 50 µg/ml of protease inhibitor cocktail (Boehringer). After centrifugation, the Triton-insoluble cytoskeleton and soluble protein fractions were analyzed on 515% polyacrylamide gradient gels, transferred to nitrocellulose membranes, and quantified by phosphorimaging.
Morphometric analysis
26-mo-old NF-Mtail and their littermate control mice were perfused transcardially with 4% PFA, 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, and postfixed overnight in the same buffer. Samples were treated with 2% osmium tetroxide, washed, dehydrated, and embedded in Epon-Araldite resin. 0.75-µm-thick sections for light microscopy were stained with toluidine blue, and 700-nm-thin sections for electron microscopy were stained with uranyl acetate and lead acetate. Axons were counted in L5 root cross sections from three to four mice of each genotype and each age group. Axon diameters from four animals of each genotype and age were measured using the Bioquant Software.
Quick freeze deep etch analysis of neurofilaments in sciatic nerves
Sciatic nerves of 6-mo-old NF-Mtail and their control littermate animals were dissected and incubated in oxygenated artificial cerebrospinal fluid, pH 7.3, containing the following (mM): 126 NaCl, 22 NaHCO3, 1 Na2HPO4, 2.8 KCl, 0.88 MgCl2, 1.45 CaCl2, and 3.5 glucose. After sectioning with a razor blade, the tissue was frozen by slamming against a liquid helium-cooled copper block (model E7200; Polaron), as described previously (Gotow et al., 1999). The frozen tissue was mounted onto the freeze fracture apparatus (model BAF 400D; Balzers), fractured, and deep etched and rotary replicated with platinum/carbon at an angle of 25°. The replicas were examined with an electron microscope (model H-300; Hitachi) at 75 kV.
![]() |
Acknowledgments |
---|
NF-M tailless mice were produced by M.V. Rao at Ludwig Cancer Institute, University of California at San Diego, La Jolla, CA, with the financial help of National Institutes of Health (NIH) grant R01 NS 27036 to Professor D. Cleveland. The characterization and analysis of the mice were performed with the support of the NIH/National Institute on Aging grant AG0564 to R.A. Nixon and M.V. Rao. This work was also supported by the start-up funds to M.V. Rao from the Center for Dementia Research at the Nathan Kline Institute.
Submitted: 14 August 2003
Accepted: 17 October 2003
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ackerley, S., A.J. Grierson, J. Brownlees, P. Thornhill, B.H. Anderton, P.N. Leigh, C.E. Shaw, and C.C. Miller. 2000. Glutamate slows axonal transport of neurofilaments in transfected neurons. J. Cell Biol. 150:165176.
Ackerley, S., P. Thornhill, A.J. Grierson, J. Brownlees, B.H. Anderton, P.N. Leigh, C.E. Shaw, and C.C. Miller. 2003. Neurofilament heavy chain side arm phosphorylation regulates axonal transport of neurofilaments. J. Cell Biol. 161:489495.
Black, M.M., and R.J. Lasek. 1980. Slow components of axonal transport: two cytoskeletal networks. J. Cell Biol. 86:616623.[Abstract]
Bass, P.W., and A. Brown. 1997. Slow axonal transport: the polymer transport model. Trends Cell Biol. 7:380384.[CrossRef]
Bomont, P., L. Cavalier, F. Blondeau, C. Ben Hamida, S. Belal, M. Tazir, E. Demir, H. Topaloglu, R. Korinthenberg, B. Tuysuz, et al. 2000. The gene encoding gigaxonin, a new member of the cytoskeletal BTB/kelch repeat family, is mutated in giant axonal neuropathy. Nat. Genet. 26:370374.[CrossRef][Medline]
Brown, A. 2003. Live-cell imaging of slow axonal transport in cultured neurons. Methods Cell Biol. 71:305323.[Medline]
Carden, M.J., W.W. Schlaepfer, and V.M.-Y. Lee. 1985. The structure, biochemical properties, and immunogenicity of neurofilament peripheral regions are determined by phosphorylation state. J. Biol. Chem. 260:98059817.
Chen, J., T. Nakata, Z. Zhang, and N. Hirokowa. 2000. The C-terminal tail domain of neurofilament protein-H (NF-H) forms the cross-bridges and regulates neurofilament bundle formation. J. Cell Sci. 113:38613869.
Ching, G.Y., and R.K. Liem. 1993. Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments. J. Cell Biol. 122:13231335.[Abstract]
Cleveland, D.W., M.J. Monteiro, P.C. Wong, S.R. Gill, J.D. Gearhart, and P.N. Hoffman. 1991. Involvement of neurofilaments in the radial growth of axons. J. Cell Sci. Suppl. 15:8595.[Medline]
Collard, J.F., F. Cote, and J.-P. Julien. 1995. Defective axonal transport in a transgenic mouse model of amyotrophic lateral sclerosis. Nature. 375:614618.
Dalpe, G., N. Leclerc, A. Vallee, A. Messer, M. Mattrieu, Y.-D. Repentigny, and R. Kothary. 1998. Dystonin is essential for maintaining neuronal cytoskeletal organization. Mol. Cell. Neurosci. 10:243247.[CrossRef]
de Waegh, A.M., V.M.-Y. Lee, and S.T. Brady. 1992. Local modulation of neurofilament phosphorylation, axonal caliber, and slow axonal transport by myelinating Schwann cells. Cell. 68:451453.[Medline]
Elder, G.A., V.L. Fredric, Jr., C. Kang, P. Basco, A. Gourav, P.-H. Tu, V.M.-Y. Lee, and R.A. Lazzarini. 1998a. Absence of mid-sized neurofilament subunit decreases axonal caliber, levels of neurofilament (NF-L), and neurofilament content. J. Cell Biol. 141:727739.
Elder, G.A., V.L. Fiedrich, Jr., C. Kang, P. Basco, A. Gourav, P.-H. Tu, B. Zhang, V.M.-Y. Lee, and R.A. Lazzarini. 1998b. Requirement of heavy neurofilament subunit in the development of axons with large calibers. J. Cell Biol. 143:195205.
Errante, L.D., G. Wiche, and G. Shaw. 1994. Distribution of plectin, an intermediate filament associated protein, in the adult rat central nervous system. J. Neurosci. Res. 37:515528.[Medline]
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. 110:43949.
Eyer, J., and A. Peterson. 1994. Neurofilament deficient axons and perikaryal aggregates in viable transgenic mice expressing a neurofilament-ß-galactosidase fusion protein. Neuron. 12:389405.[Medline]
Friede, R.L., and T. Samorajski. 1970. Axon caliber related to neurofilaments and microtubules in sciatic nerve fibers of rats and mice. Anat. Rec. 176:379387.
Gasser, H.S., and H. Grundfest. 1939. Axon diameters in relations to the spike dimensions and the conduction velocity in mammalian A fibers. Am. J. Physiol. 127:393414.
Geisler, N., J. Vandekerckhove, and K. Weber. 1987. Location and sequence characterization of the major phosphorylation sites of the high molecular mass neurofilament proteins M and H. FEBS Lett. 221:403407.[CrossRef][Medline]
Gill, S.R., P.C. Wong, M.J. Monteiro, and D.W. Cleveland. 1990. Assembly properties of dominant and recessive mutations in the small mouse neurofilament (NF-L) subunit. J. Cell Biol. 111:20052019.[Abstract]
Glicksman, M.A., D. Soppet, and M.B. Willard. 1987. Posttranslational modifications of neurofilament polypeptides in rabbit retina. J. Neurobiol. 18:167196.[Medline]
Goldstein, M.E., L.A. Sternberger, and N.H. Sternberger. 1987. Varying degrees of phosphorylation determine microheterogeneity of the heavy neurofilament polypeptide (NF-H). J. Neuroimmunol. 14:135148.[CrossRef][Medline]
Gotow, T., M. Takeda, T. Tanaka, and P.H. Hashimoto. 1992. Macromolecular structure of reassembled neurofilaments as revealed by the quick-freeze deep etch mica method: difference between NF-M and NF-H subunits in their ability to form cross-bridges. Eur. J. Cell Biol. 58:331345.[Medline]
Gotow, T., J.F. Leterrier, Y. Ohsawa, T. Watanabae, K. Isahara, R. Shibata, K. Ikenaka, and Y. Uchiyama. 1999. Abnormal expression of neurofilament proteins in dysmyelinating axons located in the central nervous system of jimpy mutant mice. Eur. J. Neurosci. 11:38933903.[CrossRef][Medline]
Grant, P., and H.C. Pant. 2000. Neurofilament protein synthesis and phosphorylation. J. Neurocytol. 29:843872.[CrossRef][Medline]
Hirokawa, N., M.A. Glicksman, and M.B. Willard. 1984. Organization of mammalian neurofilament polypeptides within neuronal cytoskeleton. J. Cell Biol. 98:15231536.[Abstract]
Hirokawa, N., S. Terada, T. Funmakoshi, and S. Terada. 1997. The slow axonal transport: the subunit model. Trends Cell Biol. 7:384388.[CrossRef]
Hisanaga, S., and N. Hirokawa. 1990. Dephosphorylation-induced interaction of neurofilaments with microtubules. J. Biol. Chem. 265:2185221858.
Hisanaga, S., M. Kusubata, E. Okumura, and T. Kishimoto. 1991. Phosphorylation of neurofilament H subunit at the tail domain by CDC2 kinase dissociates the association to microtubules. J. Biol. Chem. 266:2179321803.
Hoffman, P.N., and R.J. Lasek. 1975. The slow component of axonal transport: identification of major structural polypeptides of the axon and their generality among mammalian neurons. J. Cell Biol. 66:351366.[Abstract]
Hoffman, P.N., D.W. Cleveland, J.W. Griffin, P.W. Landes, N.J. Cowan, and D.L. Price. 1987. Neurofilament gene expression: a major determinant of axonal caliber. Proc. Natl. Acad. Sci. USA. 84:34723476.[Abstract]
Hsieh, S.-T., G.J. Kidd, T.O. Crawford, Z.-S. Xu, B.D. Trapp, D.W. Cleveland, and J.W. Griffin. 1994. Regional modulation of neurofilament organization by myelination in normal axons. J. Neurosci. 14:63926401.[Abstract]
Jacomy, H., Q. Zhu, S. Couillard-Despres, J.-M. Beaulieu, and J.-P. Julien. 1999. Disruption of type IV intermediate filament network in mice lacking the neurofilament medium and heavy subunits. J. Neurochem. 73:972984.[CrossRef][Medline]
Jones, S.M., and R.C. Williams. 1982. Phosphate content of mammalian neurofilaments. J. Biol. Chem. 256:99029905.
Joyner, A.L., editor. 1994. Gene Targeting: A Practical Approach. IRL Press. New York. 234 pp.
Julien, J.-P., and W.E. Mushynski. 1982. Multiple phosphorylation sites in mammalian neurofilament polypeptides. J. Biol. Chem. 257:1046710470.
Julien, J.-P., and W.E. Mushynski. 1983. The distribution of phosphorylation sites among identified proteolytic fragments of mammalian neurofilaments. J. Biol. Chem. 258:40194025.
Jung, C., J.T. Yabe, S. Lee, and T.B. Shea. 2000a. Hypophosphorylated neurofilament subunits undergo axonal transport more rapidly than more extensively phosphorylated subunits in situ. Cell Motil. Cytoskeleton. 47:120129.[CrossRef][Medline]
Jung, C., J.T. Yabe, and T.B. Shea. 2000b. C-terminal phosphorylation of the high molecular weight neurofilament subunit correlates with decreased neurofilament axonal transport velocity. Brain Res. 856:1219.[CrossRef][Medline]
Lee, V.M.-Y., L. Otvos, Jr., M.J. Carden, M. Hollosi, B. Dietzschold, and R.A. Lazzarini. 1988. Identification of the major multiphosphorylation site in mammalian neurofilaments. Proc. Natl. Acad. Sci. USA. 85:19982002.[Abstract]
Lee, M.K., Z. Xu, P.C. Wong, and D.W. Cleveland. 1993. Neurofilaments are obligate heteropolymers in vivo. J. Cell Biol. 122:13371350.[Abstract]
Leung, C.L., D. Sun, M. Zheng, D.R. Knowles, and R.K. Leim. 1999. Microtubule actin cross-linking factor (MACF): a hybrid of dystonin-dystrophin that can interact with the actin and microtubule cytoskeleton. J. Cell Biol. 147:12751286.
Lewis, S.E., and R.A. Nixon. 1988. Multiple phosphorylated variants of high molecular weight subunit of neurofilaments of retinal cell neurons: characterization and evidence for their differential association with stationary and moving neurofilaments. J. Cell Biol. 107:26892701.[Abstract]
Marszalek, J.R., T.L. Williamson, M.K. Lee, Z.-S. Xu, T.O. Crawford, P.N. Hoffman, and D.W. Cleveland. 1996. Neurofilament subunit H modulates axonal diameter by affecting the rate of neurofilament transport. J. Cell Biol. 135:711724.[Abstract]
Meier, J., S. Couillard-Despres, H. Jacomy, C. Gravel, and J.P. Julien. 1999. Extra neurofilament NF-L subunits rescue motor neuron disease caused by overexpression of the human NF-H gene in mice. J. Neuropathol. Exp. Neurol. 58:10991110.[Medline]
Miyasaka, H., S. Okabe, K. Ishiguro, T. Uchida, and N. Hirokawa. 1993. Interaction of the tail domain of high molecular weight subunits of neurofilaments with the COOH-terminal region of tubulin and its regulation by protein kinase II. J. Biol. Chem. 268:2269522702.
Monteiro, M.J., P.N. Hoffman, J.D. Gearhart, and D.W. Cleveland. 1990. Expression of NF-L in both neuronal and nonneuronal cells of transgenic mice: increased neurofilament density without affecting the caliber. J. Cell Biol. 111:15431557.[Abstract]
Nakagawa, T., J. Chen, Z. Zhang, Y. Kanai, and N. Hirokawa. 1995. Two distinct functions of the carboxyl-terminal tail domain of NF-M upon neurofilament assembly: cross-bridge formation and longitudinal elongation of filaments. J. Cell Biol. 129:411429.[Abstract]
Nixon, R.A. 1980. Protein degradation in mouse visual system. I. Degradation of axonally transported and retinal proteins. Brain Res. 200:6983.[CrossRef][Medline]
Nixon, R.A. 1998. Dynamic behavior and organization of cytoskeletal proteins in neurons: reconciling old and new findings. Bioessays. 20:798807.[CrossRef][Medline]
Nixon, R.A., B.A. Brown, and C.A. Morotta. 1982. Posttranslational modification of a neurofilament protein during axoplasmic transport: implications for regional specialization of CNS axons. J. Cell Biol. 94:150158.
Nixon, R.A., S.E. Lewis, and C.A. Morota. 1987. Posttranslational modification of neurofilament proteins by phosphate during axoplasmic transport in retinal ganglion cell neurons. J. Neurosci. 7:11451158.[Abstract]
Nixon, R.A., P.A. Paskevich, R.K. Sihag, and C.Y. Thayer. 1994. Phosphorylation on carboxyl terminus domains of neurofilament proteins in retinal ganglion cell neurons in vivo: influences on regional neurofilament accumulation, interfilament spacing, and axonal caliber. J. Cell Biol. 126:10311046.[Abstract]
Oblinger, M.M., S.T. Brady, I.G. McQuarrie, and R.J. Lasek. 1987. Cytotypic differences in the protein composition of the axonally transported cytoskeleton in mammalian neurons. J. Neurosci. 7:453462.[Abstract]
Ohara, O., Y. Gahara, T. Miyake, H. Teraoka, and T. Kitamura. 1993. Neurofilament deficiency in quail caused by nonsense mutation in neurofilament-L gene. J. Cell Biol. 121:387395.[Abstract]
Pant, H.C., and Veeranna. 1995. Neurofilament phosphorylation. Biochem. Cell Biol. 73:575592.[Medline]
Prahlad, V., B.T. Helfand, G.M. Longford, R.D. Vale, and R.D. Goldman. 2000. Fast transport of neurofilament protein along microtubules in squid axoplasm. J. Cell Sci. 113:39393946.
Rao, M.V., and R.A. Nixon. 2003. Defective neurofilament axonal transport in mouse models of amyotrophic lateral sclerosis: a review. Neurochem. Res. 28:10411047.[CrossRef][Medline]
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.
Rao, M.V., M.L. Garcia, Y. Miyazaki, T. Gotow, A. Yuan, S. Mattina, C.M. Ward, N.S. Calcutt, Y. Uchiyama, R.A. Nixon, and D.W. Cleveland. 2002a. Gene replacement in mice reveals that the heavily phosphorylated tail of neurofilament heavy subunit (NF-H) does not affect axonal caliber or the transit of cargoes in slow axonal transport. J. Cell Biol. 158:681693.
Rao, M.V., L.J. Engle, P.S. Mohan, A. Yuan, D. Qiu, A. Cataldo, L. Hassinger, S. Jacobsen, V.M.-Y. Lee, A. Andreadis, et al. 2002b. Myosin Va binding to neurofilaments is essential for correct myosin Va distribution and transport and neurofilament density. J. Cell Biol. 159:279289.
Roy, S., P. Coffee, G. Smith, R.K.H. Liem, S.T. Brady, and M.M. Black. 2000. Neurofilaments are transported rapidly but intermittently in axons: implications for slow axonal transport. J. Neurosci. 20:68496861.
Sakaguchi, T., M. Okada, T. Kitamura, and K. Kawasaki. 1993. Reduced diameter and conduction velocity of myelinated fibers in the sciatic nerve of a neurofilament-deficient mutant quail. Neurosci. Lett. 153:6568.[CrossRef][Medline]
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.
Sanchez, I., L. Hassinger, R.K. Sihag, D.W. Cleveland, P. Mohan, and R.A. Nixon. 2000. Local control of neurofilament accumulation during radial growth of myelinating axons in vivo: selective role of site-specific phosphorylation. J. Cell Biol. 151:10131024.
Shah, J.V., and D.W. Cleveland. 2002. Slow axonal transport: fast motors in slow lane. Curr. Opin. Cell Biol. 14:5862.[CrossRef][Medline]
Shah, J.V., L.A. Flanagan, P.A. Janemy, and J.F. Leterrier. 2000. Bidirectional translocation of neurofilaments along microtubules mediated in part by Dynein/dynactin. Mol. Biol. Cell. 11:34953508.
Strack, S., R.S. Wesphal, R.J. Colbran, F.F. Ebner, and B.E. Wadzinski. 1997. Protein seine/threonine phosphatase 1 and 2A associate with and dephosphorylated neurofilaments. Brain Res. Mol. Brain Res. 49:1528.[CrossRef][Medline]
Sternberger, L.A., and N.H. Sternberger. 1983. Monoclonal antibodies distinguish phosphorylated and nonphosphorylated form of neurofilaments in situ. Proc. Natl. Acad. Sci. USA. 80:61266130.[Abstract]
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]
Terada, S., T. Nakata, A.C. Peterson, and N. Hirokawa. 1996. Visualization of slow axonal transport in vivo. Science. 273:784788.[Abstract]
Tu, P.-H., G. Elder, R.A. Lazzarini, D. Nelson, J.Q. Trojanowski, and V.M. Lee. 1995. Overexpression of human NF-M subunit in transgenic mice modifies the level of endogenous NF-L and the phosphorylation state of NF-H subunits. J. Cell Biol. 129:16291640.[Abstract]
Veeranna., K.T. Shetty, M. Takahashi, P. Grant, and H.C. Pant. 2000. Cdk5 and MAPK are associated with complexes of cytoskeletal proteins in rat brain. Brain. Res. Mol. Brain Res. 76:229236.[Medline]
Wang, L., C.-I. Ho, D. Sun, R.K.H. Liem, and A. Brown. 2000. Rapid movement of axonal neurofilaments interrupted by prolonged pauses. Nat. Cell Biol. 2:137141.[CrossRef][Medline]
Waxman, S.G. 1980. Determinants of conduction velocity in myelinated nerve fibers. Muscle Nerve. 3:141150.[Medline]
Willard, M., and C. Simon. 1981. Antibody decoration of neurofilaments. J. Cell Biol. 89:198205.[Abstract]
Wong, P.C., J. Marszalek, T.O. Crawford, Z. Xu, S.T. Hsieh, J.W. Griffin, and D.W. Cleveland. 1996. Increasing neurofilament subunit M expression reduces axonal NF-H, inhibits radial growth, and results in neurofilamentous accumulation in motor neurons. J. Cell Biol. 130:14131422.
Xia, C.-H., E.A. Roberts, L.-S. Her, X. Liu, D.S. Williams, D.W. Cleveland, and L.S.B. Goldstein. 2003. Abnormal neurofilament transport caused by targeted disruption of neuronal kinesin heavy chain KIF5A. J. Cell Biol. 161:5566.
Xu, Z., and V.W. Tung. 2001. Overexpression of neurofilament M subunit accelerates axonal transport of neurofilaments. Brain Res. 866:326332.
Xu, Z., L.C. Cork, J.W. Griffin, and D.W. Cleveland. 1993. Increased expression of neurofilament subunit L produces morphological alterations that resemble the pathology of human motor neuron disease. Cell. 73:2333.[Medline]
Xu, Z.-S., J.R. Marszalek, M.K. Lee, P.C. Wong, J. Folmer, T.O. Crawford, S.-T. Hsieh, J.W. Griffin, and D.W. Cleveland. 1996. Subunit composition of neurofilaments specifies axonal diameter. J. Cell Biol. 133:10611069.[Abstract]
Yabe, J.T., A. Pimenta, and T.B. Shea. 1999. Kinesin-mediated transport of neurofilament protein oligomers in growing axons. J. Cell Sci. 112:37993814.
Yang, Y., J. Dowling, Q.C. Yu, P. Kouklis, D.W. Cleveland, and E. Fuchs. 1996. An essential cytoskeletal linker protein connecting actin microfilaments to intermediate filaments. Cell. 86:655665.[Medline]
Yin, X., T.O. Crawford, J.W. Griffin, P.H. Tu, V.M.-Y. Lee, C. Li, R. Roder, and B.D. Trapp. 1998. Myelin-associated glycoprotein is a myelin signal that modulates the caliber of myelinated axons. J. Neurosci. 18:19531962.
Zhu, Q., S. Couillard-Despres, and J.-P. Julien. 1997. Delayed maturation of regenerating myelinated axons in mice lacking neurofilaments. Exp. Neurol. 148:299316.[CrossRef][Medline]
Zhu, Q., M. Lindenbaum, F. Levavasseur, H. Jacomy, and J.-P. Julien. 1998. Disruption of the NF-H gene increases axonal microtubule content and velocity of neurofilament transport: relief of axonopathy resulting from the toxin ß,ß'-iminodipropionitrile. J. Cell Biol. 143:183193.
Related Article