C-terminal phosphorylation of the high molecular weight neurofilament subunit correlates with decreased neurofilament axonal transport velocity (original) (raw)
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The Journal of Neuroscience, 2001
We examined the steady-state distribution and axonal transport of neurofilament (NF) subunits within growing axonal neurites of NB2a/d1 cells. Ultrastructural analyses demonstrated a longitudinally oriented "bundle" of closely apposed NFs that was surrounded by more widely spaced individual NFs. NF bundles were recovered during fractionation and could be isolated from individual NFs by sedimentation through sucrose. Immunoreactivity toward the restrictive C-terminal phospho-dependent antibody RT97 was significantly more prominent on bundled than on individual NFs. Microinjected biotinylated NF subunits, GFP-tagged NF subunits expressed after transfection, and radiolabeled endogenous subunits all associated with individual NFs before they associated with bundled NFs. Biotinylated and GFP-tagged NF subunits did not accumulate uniformly along bundled NFs; they initially appeared within the proximal portion of the NF bundle and only subsequently were observed along the entire length of bundled NFs. These findings demonstrate that axonal NFs are not homogeneous but, rather, consist of distinct populations. One of these is characterized by less extensive C-terminal phosphorylation and a relative lack of NF-NF interactions. The other is characterized by more extensive C-terminal NF phosphorylation and increased NF-NF interactions and either undergoes markedly slower axonal transport or does not transport and undergoes turnover via subunit and/or filament exchange with individual NFs. Inhibition of phosphatase activities increased NF-NF interactions within living cells. These findings collectively suggest that C-terminal phosphorylation and NF-NF interactions are responsible for slowing NF axonal transport.
Dissociation of Axonal Neurofilament Content from Its Transport Rate
PloS one, 2015
The axonal cytoskeleton of neurofilament (NF) is a long-lived network of fibrous elements believed to be a stationary structure maintained by a small pool of transported cytoskeletal precursors. Accordingly, it may be predicted that NF content in axons can vary independently from the transport rate of NF. In the present report, we confirm this prediction by showing that human NFH transgenic mice and transgenic mice expressing human NFL Ser55 (Asp) develop nearly identical abnormal patterns of NF accumulation and distribution in association with opposite changes in NF slow transport rates. We also show that the rate of NF transport in wild-type mice remains constant along a length of the optic axon where NF content varies 3-fold. Moreover, knockout mice lacking NFH develop even more extreme (6-fold) proximal to distal variation in NF number, which is associated with a normal wild-type rate of NF transport. The independence of regional NF content and NF transport is consistent with pr...
Brain Research, 1993
Neurofilaments (NFs) were radiolabeled in the optic systems of mice. The leading edge of the radiolabeled NF waveform was distinguished near the injection site (the eye) both by liquid scintillation spectroscopy and visually from fluorographs. The fastest NFs were found to be translocated at rates of between 72 and 144 mm/day. It appears that the continuous (maximal) operation of the slow axonal transport machinery can move polymers intra-axonally at rates one hundred times greater than those previously reported.
Does neurofilament phosphorylation regulate axonal transport?
Trends in Neurosciences, 2003
Phosphorylation of neurofilaments has long been considered to regulate their axonal transport rate and, in doing so, to provide stability to mature axons. Interpretation of data recently obtained following C-terminal deletion experiments has prompted a challenge to this hypothesis. We present evidence that these deletion studies remain consistent with, rather than refute, a role for C-terminal phosphorylation in regulation of neurofilament axonal transport.
Slow axonal transport mechanisms move neurofilaments relentlessly in mouse optic axons
The Journal of Cell Biology, 1992
Pulse-labeling studies of slow axonal transport in many kinds of axons (spinal motor, sensory ganglion, oculomotor, hypoglossal, and olfactory) have led to the inference that axonal transport mechanisms move neurofilaments (NFs) unidirectionally as a single continuous kinetic population with a diversity of individual transport rates. One study in mouse optic axons (Nixon, R. A., and K. B. Logvinenko. 1986. J. Cell Biol. 102:647-659) has given rise to the different suggestion that a significant and distinct population of NFs may be entirely stationary within axons. In mouse optic axons, there are relatively few NFs and the NF proteins are more lightly labeled than other slowly transported slow component b (SCb) proteins (which, however, move faster than the NFs); thus, in mouse optic axons, the radiolabel of some of these faster-moving SCb proteins may confuse NF protein analyses that use one dimensional (1-D) SDS-PAGE, which separates proteins by size only. To test this possibility,...
Neurofilaments can undergo axonal transport and cytoskeletal incorporation in a discontinuous manner
Cell Motility and The Cytoskeleton, 2005
Neurofilaments (NFs) are thought to provide structural support for axons. Some NFs exhibit an extended residence time along axons, the nature of which remains unclear. In prior studies in NB2a/d1 cells, hypophosphorylated NFs were demonstrated to be dispersed throughout the axon and to undergo relatively rapid axonal transport, while extensively phosphorylated NFs organized into a “bundle” localized along the center of the axon. It was not conclusively determined whether bundled NFs underwent transport or instead underwent turnover via exchange with transporting individual NFs. Herein, using transfection with multiple constructs and regional photobleaching, we demonstrate that bundled NFs undergo relatively slow transport as well as exchange with surrounding individual NFs. We also demonstrate that newly synthesized NFs disperse nonhomogenously throughout axonal neurites and perikarya. These findings provide a mechanism by which some NFs exhibit extended residence time within axons, which lessens the metabolic burden of cytoskeletal turnover. Cell Motil. Cytoskeleton 62:166–179, 2005. © 2005 Wiley-Liss, Inc.
Slow axonal transport conveys cytoskeletal proteins from cell body to axon tip. This transport provides the axon with the architectural elements that are required to generate and maintain its elongate shape and also generates forces within the axon that are necessary for axon growth and navigation. The mechanisms of cytoskeletal transport in axons are unknown. One hypothesis states that cytoskeletal proteins are transported within the axon as polymers. We tested this hypothesis by visualizing individual cytoskeletal polymers in living axons and determining whether they undergo vectorial movement. We focused on neurofilaments in axons of cultured sympathetic neurons because individual neurofilaments in these axons can be visualized by optical microscopy. Cultured sympathetic neurons were infected with recombinant adenovirus containing a construct encoding a fusion protein combining green fluorescent protein (GFP) with the heavy neurofilament protein subunit (NFH). The chimeric GFP-NFH coassembled with endogenous neurofilaments. Time lapse imaging revealed that individual GFP-NFH-labeled neurofilaments undergo vigorous vectorial transport in the axon in both anterograde and retrograde directions but with a strong anterograde bias. NF transport in both directions exhibited a broad spectrum of rates with averages of Ϸ0.6-0.7 m/sec. However, movement was intermittent, with individual neurofilaments pausing during their transit within the axon. Some NFs either moved or paused for the most of the time they were observed, whereas others were intermediate in behavior. On average, neurofilaments spend at most 20% of the time moving and rest of the time paused. These results establish that the slow axonal transport machinery conveys neurofilaments.
The Journal of Neuroscience, 2007
We generated mice with doxycycline control of a human neurofilament light (NF-L) transgene in the context of the absence (tTA;hNF-L;NF-L−/−) or presence (tTA;hNF-L;NF-L+/−) of endogenous mouse NF-L proteins. Doxycycline treatment caused the rapid disappearance of human NF-L (hNF-L) mRNA in tTA;hNF-L mice, but the hNF-L proteins remained with a half-life of 3 weeks in the brain. In the sciatic nerve, the disappearance of hNF-L proteins after doxycycline treatment occurred in synchrony along the sciatic nerve, suggesting a proteolysis of NF proteins along the entire axon. The presence of permanent NF network in tTA;hNF-L;NF-L+/−mice further stabilized and extended longevity of hNF-L proteins by several months. Surprisingly, after cessation of doxycycline treatment, there was no evidence of leading front of newly synthesized hNF-L proteins migrating into sciatic nerve axons devoid of NF structures. The hNF-L proteins detected at weekly intervals reappeared and accumulated in synchrony ...
Neurofilament Transport Is Dependent on Actin and Myosin
Journal of Neuroscience, 2004
Real-time analyses have revealed that some newly synthesized neurofilament (NF) subunits translocate into and along axonal neurites by moving along the inner plasma membrane surface, suggesting that they may translocate against the submembrane actin cortex. We therefore examined whether or not NF axonal transport was dependent on actin and myosin. Perturbation of filamentous actin in NB2a/d1 cells with cytochalasin B inhibited translocation of subunits into axonal neurites and inhibited bidirectional translocation of NF subunits within neurites. Intravitreal injection of cytochalasin B inhibited NF axonal transport in optic axons in a dose-response manner. NF subunits were coprecipitated from NB2a/d1 cells by an anti-myosin antibody, and myosin colocalized with NFs in immunofluorescent analyses. The myosin light chain kinase inhibitor ML-7 and the myosin ATPase inhibitor 2,3-butanedione-2-monoxime perturbed NF translocation within NB2a/d1 axonal neurites. These findings suggest that some NF subunits may undergo axonal transport via myosinmediated interactions with the actin cortex.
The Journal of Neuroscience, 1986
Investigations of slow axonal transport reveal variation in both protein composition and the rate of movement. However, these studies involve a variety of nerve preparations in different species, and most lack the resolution needed to determine the kinetics of identified proteins. We have compared the axonal transport of slow-transported proteins in retinal ganglion cells and spinal motor neurons of young rats. Nine proteins that contribute to axonal structures were examined: the neurofilament triplet (NFT), alpha and beta tubulin, actin, fodrin, calmodulin, and clathrin. Axonally transported proteins were pulse- labeled by intraocular or intracord injections of 35S-methionine. After allowing sufficient time for labeled slow-component proteins to enter the spinal or optic nerves, consecutive 2–3 mm nerve segments were subjected to SDS-PAGE. Fluorographs were used as templates for locating the gel regions containing the above polypeptides, and the radioactivity in these regions was m...