Differential dynamics of neurofilament-H protein and neurofilament-L protein in neurons.

Takeda, Sén; Okabe, Susumu; Funakoshi, Takeshi; Hirokawa, Nobutaka

doi:10.1083/jcb.127.1.173

Cited by 60 publications

(65 citation statements)

References 70 publications

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“…In our experiments tubulin has a Péclet number of 0.86 after 3 hr, during which it has traveled 281 m, whereas neurofilament has traveled only 142 m in the same time but, with its lower diffusion coefficient, has a Péclet number of 4.4. These numbers suggest that although there is a contribution from diffusion to our measurements in the squid axon, it is not nearly as dominant as in some of the previously reported photobleaching (12,13,(41)(42)(43) and photoactivation (42,44) experiments. In those experiments the short lengths of the detectable photobleached or photoactivation spots, and thus Péclet numbers, are several orders of magnitude less than those available in the squid axon.…”

Section: Discussionmentioning

confidence: 65%

Slow transport of unpolymerized tubulin and polymerized neurofilament in the squid giant axon

Galbraith

Reese

Schlief

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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A major issue in the slow transport of cytoskeletal proteins is the form in which they are transported. We have investigated the possibility that unpolymerized as well as polymerized cytoskeletal proteins can be actively transported in axons. We report the active transport of highly diffusible tubulin oligomers, as well as transport of the less diffusible neurofilament polymers. After injection into the squid giant axon, tubulin was transported in an anterograde direction at an average rate of 2.3 mm͞day, whereas neurofilament was moved at 1.1 mm͞day. Addition of the metabolic poisons cyanide or dinitrophenol reduced the active transport of both proteins to less than 10% of control values, whereas disruption of microtubules by treatment of the axon with cold in the presence of nocodazole reduced transport of both proteins to Ϸ20% of control levels. Passive diffusion of these proteins occurred in parallel with transport. The diffusion coefficient of the moving tubulin in axoplasm was 8.6 m 2 ͞s compared with only 0.43 m 2 ͞s for neurofilament. These results suggest that the tubulin was transported in the unpolymerized state and that the neurofilament was transported in the polymerized state by an energy-dependent nocodazole͞cold-sensitive transport mechanism.

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Section: Discussionmentioning

confidence: 65%

Slow transport of unpolymerized tubulin and polymerized neurofilament in the squid giant axon

Galbraith

Reese

Schlief

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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“…Variations in caliber size also are closely related to changes in neurofilament protein phosphorylation, which promote the local accumulation of neurofilaments within a stationary, but dynamic, axonal network (Lewis and Nixon, 1988;Nixon et al, 1994a;Takeda et al, 1994) and induce neurofilaments to extend sidearms that increase lateral spacing between neurofilaments (DeWaegh et al, 1992;Hsieh et al, 1994;Nixon et al, 1994b;Nakagawa et al, 1995). Our finding that caliber size does not expand in a proximal portion of optic axons proved to be useful in evaluating the possible contributions of these different neurofilament-related variables to axon radial growth.…”

Section: Local Neurofilament Accumulation and Changes In Neurofilamenmentioning

confidence: 89%

Oligodendroglia Regulate the Regional Expansion of Axon Caliber and Local Accumulation of Neurofilaments during Development Independently of Myelin Formation

Sánchez¹,

Hassinger²,

et al. 1996

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Axon caliber may be influenced by intrinsic neuronal factors and extrinsic factors related to myelination. To understand these extrinsic influences, we studied how axon-caliber expansion is related to changes in neurofilament and microtubule organization as axons of retinal ganglion cells interact with oligodendroglia and become myelinated during normal mouse brain development. Caliber expanded and neurofilaments accumulated only along regions of the axon invested with oligodendroglia. Very proximal portions of axons within a region of the optic nerve from which oligodendrocytes are excluded remained unchanged. More distally, these axons rapidly expanded an average of fourfold as soon as they were recruited to become myelinated between postnatal days 9 and 120. Unmyelinated axons remained unchanged. Axons ensheathed by oligodendroglial processes, but not yet myelinated, were intermediate in caliber and neurofilament number. That oligodendrocytes can trigger regional caliber expansion in the absence of myelin was confirmed using three strains of mice with different mutations that prevent myelin formation but allow wrapping of some axons by oligodendroglial processes. Unmyelinated axons persistently wrapped by oligodendrocytes showed full axon caliber expansion, neurofilament accumulation, and appropriately increased lateral spacing between neurofilaments. Thus, signals from oligodendrocytes, independent of myelin formation, are sufficient to induce full axon radial growth primarily by triggering local accumulation and reorganization of the neurofilament network.

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“…For example, Hirokawa and colleagues have used laser photobleaching to investigate the turnover of neurofilament polymers in axons of cultured neurons. The average half-time of recovery was ϳ 34 min for NF-L and ϳ 19 min for NF-H (Takeda et al, 1994), but the recovery was more rapid in growing axons than in nongrowing axons . These data indicated that neurofilaments are dynamic polymers and that the dynamics are modulated by the rate of axonal growth.…”

Section: Fluorescence Recovery After Photobleachingmentioning

confidence: 99%

“…Subsequent photobleaching and photoactivation studies (Sabry et al, 1995;Takeda et al, 1995) on tubulin in the motor neurons of developing grasshopper and zebrafish embryos, as well as photobleaching studies (Okabe and Hirokawa, 1990;Takeda et al, 1994) on actin and neurofilament protein in cultured mouse sensory neurons, also failed to observe movement. Microtubules were observed to move in a slow and synchronous manner in cultured embryonic frog neurons (Reinsch et al, 1991;, but it now appears that this was caused by stretching of the growing axon and that it did not represent bona fide slow axonal transport (Okabe and Hirokawa, 1992;Chang et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

Rapid Intermittent Movement of Axonal Neurofilaments Observed by Fluorescence Photobleaching

Wang

Brown

2001

MBoC

120

162

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Observations on naturally occurring gaps in the axonal neurofilament array of cultured neurons have demonstrated that neurofilament polymers move along axons in a rapid, intermittent, and highly asynchronous manner. In contrast, studies on axonal neurofilaments using laser photobleaching have not detected movement. Here, we describe a modified photobleaching strategy that does permit the direct observation of neurofilament movement. Axons of cultured neurons expressing GFP-tagged neurofilament protein were bleached by excitation with the mercury arc lamp of a conventional epifluorescence microscope for 12-60 s. The length of the bleached region ranged from 10 to 60 m. By bleaching thin axons, which have relatively few neurofilaments, we were able to reduce the fluorescent intensity enough to allow the detection of neurofilaments that moved in from the surrounding unbleached regions. Time-lapse imaging at short intervals revealed rapid, intermittent, and highly asynchronous movement of fluorescent filaments through the bleached regions at peak rates of up to 2.8 m/s. The kinetics of movement were very similar to our previous observations on neurofilaments moving through naturally occurring gaps, which indicates that the movement was not impaired by the photobleaching process. These results demonstrate that fluorescence photobleaching can be used to study the slow axonal transport of cytoskeletal polymers, but only if the experimental strategy is designed to ensure that rapid asynchronous movements can be detected. This may explain the failure of previous photobleaching studies to reveal the movement of neurofilament proteins and other cytoskeletal proteins in axons. INTRODUCTIONSlow axonal transport is the mechanism by which cytoskeletal and cytosolic proteins are transported along axons from their site of synthesis in the nerve cell body. During the past decade there have been numerous efforts to observe this movement directly in living cells, but these studies (Baas and Brown, 1997;Hirokawa et al., 1997) have yielded conflicting results. The two techniques that have been used the most widely are fluorescence photobleaching and photoactivation. In these related approaches, fluorescent or caged fluorescent cytoskeletal subunit proteins are injected into nerve cells and then a laser is used to bleach or activate the fluorescence in a short segment of axon. The bleached or activated proteins then are observed by time-lapse imaging to detect their movement.The first photobleaching study (Keith, 1987) on slow axonal transport reported slow and synchronous movement of tubulin in cultured PC12 cells, but attempts to reproduce this result in cultured PC12 cells and cultured chick and mouse sensory neurons were unsuccessful (Lim et al., 1989;Lim et al., 1990; Hirokawa, 1990, 1992). Subsequent photobleaching and photoactivation studies (Sabry et al., 1995;Takeda et al., 1995) on tubulin in the motor neurons of developing grasshopper and zebrafish embryos, as well as photobleaching studies (Okabe and Hirokawa, 1990;Takeda ...

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Differential dynamics of neurofilament-H protein and neurofilament-L protein in neurons.

Cited by 60 publications

References 70 publications

Slow transport of unpolymerized tubulin and polymerized neurofilament in the squid giant axon

Slow transport of unpolymerized tubulin and polymerized neurofilament in the squid giant axon

Oligodendroglia Regulate the Regional Expansion of Axon Caliber and Local Accumulation of Neurofilaments during Development Independently of Myelin Formation

Rapid Intermittent Movement of Axonal Neurofilaments Observed by Fluorescence Photobleaching

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