Limits...
Slow axonal transport of neurofilament protein in cultured neurons.

Koehnle TJ, Brown A - J. Cell Biol. (1999)

Bottom Line: The average transport rate was estimated to be at least 130 micrometer/h (3.1 mm/d), and approximately 90% of the accumulated neurofilament protein remained in the axon after detergent extraction, suggesting that it was present in a polymerized form.These data suggest that the neurofilament proteins were transported either as assembled polymers or in a nonpolymeric form that assembled locally at the site of accumulation.This study represents the first demonstration of the axonal transport of neurofilament protein in cultured neurons.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Program, Department of Biological Sciences, Ohio University, Athens, Ohio 45701, USA.

ABSTRACT
We have investigated the axonal transport of neurofilament protein in cultured neurons by constricting single axons with fine glass fibers. We observed a rapid accumulation of anterogradely and retrogradely transported membranous organelles on both sides of the constrictions and a more gradual accumulation of neurofilament protein proximal to the constrictions. Neurofilament protein accumulation was dependent on the presence of metabolic substrates and was blocked by iodoacetate, which is an inhibitor of glycolysis. These data indicate that neurofilament protein moves anterogradely in these axons by a mechanism that is directly or indirectly dependent on nucleoside triphosphates. The average transport rate was estimated to be at least 130 micrometer/h (3.1 mm/d), and approximately 90% of the accumulated neurofilament protein remained in the axon after detergent extraction, suggesting that it was present in a polymerized form. Electron microscopy demonstrated that there were an abnormally large number of neurofilament polymers proximal to the constrictions. These data suggest that the neurofilament proteins were transported either as assembled polymers or in a nonpolymeric form that assembled locally at the site of accumulation. This study represents the first demonstration of the axonal transport of neurofilament protein in cultured neurons.

Show MeSH

Related in: MedlinePlus

Morphological changes at the site of constriction.  Phase contrast images of an axon immediately before constriction (A) and after constriction for 1, 30, and 120 min (B–D). The  cell was fixed after constriction for 2 h and then the glass fiber  was removed (E) and the cell was stained for NF-L by immunofluorescence microscopy (F). Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132919&req=5

Figure 2: Morphological changes at the site of constriction. Phase contrast images of an axon immediately before constriction (A) and after constriction for 1, 30, and 120 min (B–D). The cell was fixed after constriction for 2 h and then the glass fiber was removed (E) and the cell was stained for NF-L by immunofluorescence microscopy (F). Bar, 5 μm.

Mentions: Fig. 1 shows the method that we used for constricting axons, which is described in Materials and Methods. Fig. 2, A–D shows an axon before constriction and then after constriction for 1, 30, and 120 min. In most cases, constricted axons showed no sign of injury; growth cones continued to exhibit filopodial and/or lamellipodial activity, large membranous organelles visible by phase contrast microscopy continued to move on both sides of the constriction, and the axon retained its normal refractivity under phase contrast optics. When damage did occur, it generally resulted from sudden movement of the glass fiber and was apparent due to an immediate change in refractivity followed by beading and subsequent fragmentation of the axon.


Slow axonal transport of neurofilament protein in cultured neurons.

Koehnle TJ, Brown A - J. Cell Biol. (1999)

Morphological changes at the site of constriction.  Phase contrast images of an axon immediately before constriction (A) and after constriction for 1, 30, and 120 min (B–D). The  cell was fixed after constriction for 2 h and then the glass fiber  was removed (E) and the cell was stained for NF-L by immunofluorescence microscopy (F). Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132919&req=5

Figure 2: Morphological changes at the site of constriction. Phase contrast images of an axon immediately before constriction (A) and after constriction for 1, 30, and 120 min (B–D). The cell was fixed after constriction for 2 h and then the glass fiber was removed (E) and the cell was stained for NF-L by immunofluorescence microscopy (F). Bar, 5 μm.
Mentions: Fig. 1 shows the method that we used for constricting axons, which is described in Materials and Methods. Fig. 2, A–D shows an axon before constriction and then after constriction for 1, 30, and 120 min. In most cases, constricted axons showed no sign of injury; growth cones continued to exhibit filopodial and/or lamellipodial activity, large membranous organelles visible by phase contrast microscopy continued to move on both sides of the constriction, and the axon retained its normal refractivity under phase contrast optics. When damage did occur, it generally resulted from sudden movement of the glass fiber and was apparent due to an immediate change in refractivity followed by beading and subsequent fragmentation of the axon.

Bottom Line: The average transport rate was estimated to be at least 130 micrometer/h (3.1 mm/d), and approximately 90% of the accumulated neurofilament protein remained in the axon after detergent extraction, suggesting that it was present in a polymerized form.These data suggest that the neurofilament proteins were transported either as assembled polymers or in a nonpolymeric form that assembled locally at the site of accumulation.This study represents the first demonstration of the axonal transport of neurofilament protein in cultured neurons.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Program, Department of Biological Sciences, Ohio University, Athens, Ohio 45701, USA.

ABSTRACT
We have investigated the axonal transport of neurofilament protein in cultured neurons by constricting single axons with fine glass fibers. We observed a rapid accumulation of anterogradely and retrogradely transported membranous organelles on both sides of the constrictions and a more gradual accumulation of neurofilament protein proximal to the constrictions. Neurofilament protein accumulation was dependent on the presence of metabolic substrates and was blocked by iodoacetate, which is an inhibitor of glycolysis. These data indicate that neurofilament protein moves anterogradely in these axons by a mechanism that is directly or indirectly dependent on nucleoside triphosphates. The average transport rate was estimated to be at least 130 micrometer/h (3.1 mm/d), and approximately 90% of the accumulated neurofilament protein remained in the axon after detergent extraction, suggesting that it was present in a polymerized form. Electron microscopy demonstrated that there were an abnormally large number of neurofilament polymers proximal to the constrictions. These data suggest that the neurofilament proteins were transported either as assembled polymers or in a nonpolymeric form that assembled locally at the site of accumulation. This study represents the first demonstration of the axonal transport of neurofilament protein in cultured neurons.

Show MeSH
Related in: MedlinePlus