Slow Axonal Transport of Neurofilament Protein in Cultured Neurons

Koehnle, Thomas J.; Brown, Anthony

doi:10.1083/jcb.144.3.447

Cited by 42 publications

(27 citation statements)

References 68 publications

(81 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We have shown previously that sodium iodoacetate impairs neurofilament transport in cultured DRG neurons (Koehnle and Brown, 1999). In pulseescape experiments, we observed a partial reduction in the loss of fluorescence (data not shown).…”

Section: Metabolic Requirement For Neurofilament Transportsupporting

confidence: 51%

Neurofilaments Switch between Distinct Mobile and Stationary States during Their Transport along Axons

Trivedi

Jung²,

Brown³

2007

J. Neurosci.

139

View full text Add to dashboard Cite

We have developed a novel pulse-escape fluorescence photoactivation technique to investigate the long-term pausing behavior of axonal neurofilaments. Cultured sympathetic neurons expressing a photoactivatable green fluorescent neurofilament fusion protein were illuminated with violet light in a short segment of axon to create a pulse of fluorescent neurofilaments. Neurofilaments departed from the photoactivated regions at rapid velocities, but the overall loss of fluorescence was slow because many of the neurofilaments paused for long periods of time before moving. The frequency of neurofilament departure was more rapid initially and slower at later times, resulting in biphasic decay kinetics. By computational simulation of the kinetics, we show that the neurofilaments switched between two distinct states: a mobile state characterized by intermittent movements and short pauses (average ϭ 30 s) and a stationary state characterized by remarkably long pauses (average ϭ 60 min). On average, the neurofilaments spent 92% of their time in the stationary state. Combining short and long pauses, they paused for 97% of the time, resulting in an average transport rate of 0.5 mm/d. We speculate that the relative proportion of the time that neurofilaments spend in the stationary state may be a principal determinant of their transport rate and distribution along axons, and a potential target of mechanisms that lead to abnormal neurofilament accumulations in disease.

show abstract

Section: Metabolic Requirement For Neurofilament Transportsupporting

confidence: 51%

Neurofilaments Switch between Distinct Mobile and Stationary States during Their Transport along Axons

Trivedi

Jung²,

Brown³

2007

J. Neurosci.

139

View full text Add to dashboard Cite

show abstract

“…12, October 2001 3265 axonally transported neurofilament proteins at axonal constrictions indicate that any net retrograde component does not exceed 5% of the total transported protein (Koehnle and Brown, 1999). This suggests that many of the retrogradely moving neurofilaments that we observed eventually reversed direction and moved in a net anterograde direction.…”

Section: Axonal Transport Of Neurofilamentsmentioning

confidence: 58%

“…Louis,MO;Mw,000) and Matrigel (Collaborative Research, Bedford, MA; 10 g/ml). Cultures were maintained at 37°C in Liebovitz's L-15 medium (GIBCO Life Technologies, Grand Island, NY; phenol red-free) supplemented with 0.6% glucose, 2 mM L-glutamine, 100 ng/ml 2.5S nerve growth factor (Collaborative Research), 10% adult rat serum (prepared by the method of Hawrot and Patterson, 1979), and 0.5% hydroxypropylmethylcellulose (Methocel, Dow Corning, Midland, MI) as described by Koehnle and Brown (1999).…”

Section: Cell Culturementioning

confidence: 99%

Rapid Intermittent Movement of Axonal Neurofilaments Observed by Fluorescence Photobleaching

Wang

Brown

2001

MBoC

Self Cite

119

162

View full text Add to dashboard Cite

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 ...

show abstract

“…Although NFs undergo predominantly anterograde axonal transport (for review, see Shea and Yabe, 2000), many studies in culture and in situ also demonstrate that some NFs undergo retrograde subunit transport (Glass and Griffin, 1991;Watson et al, 1993;Koehnle and Brown, 1999;Yabe et al, 1999Yabe et al, , 2001aRoy et al, 2000;Shah et al, 2000;Wang et al, 2000). Retrograde NF transport may be mediated by the retrograde motor dynein (Shah et al, 2000).…”

Section: Discussionmentioning

confidence: 99%

Neurofilament Transport Is Dependent on Actin and Myosin

Jung¹,

Chylinski²,

Pimenta³

et al. 2004

J. Neurosci.

View full text Add to dashboard Cite

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.

show abstract

Slow Axonal Transport of Neurofilament Protein in Cultured Neurons

Cited by 42 publications

References 68 publications

Neurofilaments Switch between Distinct Mobile and Stationary States during Their Transport along Axons

Neurofilaments Switch between Distinct Mobile and Stationary States during Their Transport along Axons

Rapid Intermittent Movement of Axonal Neurofilaments Observed by Fluorescence Photobleaching

Neurofilament Transport Is Dependent on Actin and Myosin

Contact Info

Product

Resources

About