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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.

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Related in: MedlinePlus

Method for constricting single axons in cell culture. A  fine flexible glass fiber is oriented perpendicular to the axon and  at a 45° angle to the horizontal with its tip touching the substrate  to one side of the intended constriction site (A). The fiber is then  advanced downward against the coverslip, causing it to bend and  flatten out across the axon (B).
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Figure 1: Method for constricting single axons in cell culture. A fine flexible glass fiber is oriented perpendicular to the axon and at a 45° angle to the horizontal with its tip touching the substrate to one side of the intended constriction site (A). The fiber is then advanced downward against the coverslip, causing it to bend and flatten out across the axon (B).

Mentions: To constrict an axon, a glass fiber was attached to a Narashige 3-axis hydraulic micromanipulator at a 45° angle to the coverslip and then oriented perpendicular to the axon by rotating the culture dish (see Fig. 1). All subsequent manipulations were performed while observing the axon using a Zeiss 63×/1.4 NA Phase Apochromat oil immersion objective. The fine flexible fiber was positioned with its tip touching the glass coverslip ∼40 μm to one side of the axon and then advanced downward, causing it to bend and flatten out across the axon. The length of the glass fiber in contact with the coverslip ranged from ∼80–140 μm. Forces generated during bending of the fiber sometimes caused it to slip along the substrate, shearing the axon. When axons were damaged during constriction, this was almost always the cause. To avoid this problem, downward movements in the Z dimension were countered by fine adjustments in the X dimension to keep the tip of the fiber in place. Because of slight drift of the microscope stage (<0.5 μm/h), it was often necessary to make fine adjustments to the micromanipulator in the X and Y dimensions during the course of our observations to prevent movement of the fiber relative to the axon. To release the constriction, essentially the same movements were performed as during application of the constriction, but in reverse. Once out of contact with the axon, the fiber could be moved away rapidly without further incident.


Slow axonal transport of neurofilament protein in cultured neurons.

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

Method for constricting single axons in cell culture. A  fine flexible glass fiber is oriented perpendicular to the axon and  at a 45° angle to the horizontal with its tip touching the substrate  to one side of the intended constriction site (A). The fiber is then  advanced downward against the coverslip, causing it to bend and  flatten out across the axon (B).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Method for constricting single axons in cell culture. A fine flexible glass fiber is oriented perpendicular to the axon and at a 45° angle to the horizontal with its tip touching the substrate to one side of the intended constriction site (A). The fiber is then advanced downward against the coverslip, causing it to bend and flatten out across the axon (B).
Mentions: To constrict an axon, a glass fiber was attached to a Narashige 3-axis hydraulic micromanipulator at a 45° angle to the coverslip and then oriented perpendicular to the axon by rotating the culture dish (see Fig. 1). All subsequent manipulations were performed while observing the axon using a Zeiss 63×/1.4 NA Phase Apochromat oil immersion objective. The fine flexible fiber was positioned with its tip touching the glass coverslip ∼40 μm to one side of the axon and then advanced downward, causing it to bend and flatten out across the axon. The length of the glass fiber in contact with the coverslip ranged from ∼80–140 μm. Forces generated during bending of the fiber sometimes caused it to slip along the substrate, shearing the axon. When axons were damaged during constriction, this was almost always the cause. To avoid this problem, downward movements in the Z dimension were countered by fine adjustments in the X dimension to keep the tip of the fiber in place. Because of slight drift of the microscope stage (<0.5 μm/h), it was often necessary to make fine adjustments to the micromanipulator in the X and Y dimensions during the course of our observations to prevent movement of the fiber relative to the axon. To release the constriction, essentially the same movements were performed as during application of the constriction, but in reverse. Once out of contact with the axon, the fiber could be moved away rapidly without further incident.

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