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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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Method of analysis of constricted and control sister axons. This schematic diagram depicts a cultured neuron with a bifurcating axon. One of the sister branches is constricted and the  other serves as a nonconstricted control. After constriction for a  certain period of time, the cell is fixed, neurofilament protein is  visualized by immunofluorescence microscopy, and then the fluorescence intensity is quantified along both axons using the segmented mask method (see Materials and Methods). The rectangles drawn with dashed lines demarcate the measurement  windows located proximal and distal to the constriction (P1 and  D1), and at corresponding distances along the nonconstricted sister axon (P2 and D2). The total fluorescence intensity in each  measurement window is a relative measure of the amount of neurofilament protein in that portion of the axon. To calculate the  proximal and distal accumulation ratios, the total fluorescence intensities in the proximal and distal measurement windows of the  constricted axon (P1 and D1, respectively) are divided by the total  fluorescence intensities in the corresponding measurement windows of the nonconstricted sister axon (P2 and D2, respectively),  i.e., P1/P2 and D1/D2. Thus, the proximal and distal accumulation  ratios are each a measure of the accumulation or depletion of  neurofilament protein in the constricted axon relative to its nonconstricted sister.
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Figure 3: Method of analysis of constricted and control sister axons. This schematic diagram depicts a cultured neuron with a bifurcating axon. One of the sister branches is constricted and the other serves as a nonconstricted control. After constriction for a certain period of time, the cell is fixed, neurofilament protein is visualized by immunofluorescence microscopy, and then the fluorescence intensity is quantified along both axons using the segmented mask method (see Materials and Methods). The rectangles drawn with dashed lines demarcate the measurement windows located proximal and distal to the constriction (P1 and D1), and at corresponding distances along the nonconstricted sister axon (P2 and D2). The total fluorescence intensity in each measurement window is a relative measure of the amount of neurofilament protein in that portion of the axon. To calculate the proximal and distal accumulation ratios, the total fluorescence intensities in the proximal and distal measurement windows of the constricted axon (P1 and D1, respectively) are divided by the total fluorescence intensities in the corresponding measurement windows of the nonconstricted sister axon (P2 and D2, respectively), i.e., P1/P2 and D1/D2. Thus, the proximal and distal accumulation ratios are each a measure of the accumulation or depletion of neurofilament protein in the constricted axon relative to its nonconstricted sister.

Mentions: All the experiments described in this paper were performed on cultures obtained from dorsal root ganglia. Though these ganglia contain exclusively sensory neurons, these cells are not a homogeneous population and they do vary in their size and neurofilament protein content (Lawson, 1992). This makes it difficult to accurately compare the effect of constriction on the neurofilament protein distribution in different cells. To control for this variation, we took advantage of the fact that axons in these cultures typically branch by bifurcation of the growth cone, giving rise to two sister axons that have similar length, diameter, and growth behavior (Bray et al., 1987). By restricting our analyses to axons that branched in this manner we were able to constrict one of the two sister axons and then compare the neurofilament protein distribution with its nonconstricted sister control (Fig. 3).


Slow axonal transport of neurofilament protein in cultured neurons.

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

Method of analysis of constricted and control sister axons. This schematic diagram depicts a cultured neuron with a bifurcating axon. One of the sister branches is constricted and the  other serves as a nonconstricted control. After constriction for a  certain period of time, the cell is fixed, neurofilament protein is  visualized by immunofluorescence microscopy, and then the fluorescence intensity is quantified along both axons using the segmented mask method (see Materials and Methods). The rectangles drawn with dashed lines demarcate the measurement  windows located proximal and distal to the constriction (P1 and  D1), and at corresponding distances along the nonconstricted sister axon (P2 and D2). The total fluorescence intensity in each  measurement window is a relative measure of the amount of neurofilament protein in that portion of the axon. To calculate the  proximal and distal accumulation ratios, the total fluorescence intensities in the proximal and distal measurement windows of the  constricted axon (P1 and D1, respectively) are divided by the total  fluorescence intensities in the corresponding measurement windows of the nonconstricted sister axon (P2 and D2, respectively),  i.e., P1/P2 and D1/D2. Thus, the proximal and distal accumulation  ratios are each a measure of the accumulation or depletion of  neurofilament protein in the constricted axon relative to its nonconstricted sister.
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Related In: Results  -  Collection

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

Figure 3: Method of analysis of constricted and control sister axons. This schematic diagram depicts a cultured neuron with a bifurcating axon. One of the sister branches is constricted and the other serves as a nonconstricted control. After constriction for a certain period of time, the cell is fixed, neurofilament protein is visualized by immunofluorescence microscopy, and then the fluorescence intensity is quantified along both axons using the segmented mask method (see Materials and Methods). The rectangles drawn with dashed lines demarcate the measurement windows located proximal and distal to the constriction (P1 and D1), and at corresponding distances along the nonconstricted sister axon (P2 and D2). The total fluorescence intensity in each measurement window is a relative measure of the amount of neurofilament protein in that portion of the axon. To calculate the proximal and distal accumulation ratios, the total fluorescence intensities in the proximal and distal measurement windows of the constricted axon (P1 and D1, respectively) are divided by the total fluorescence intensities in the corresponding measurement windows of the nonconstricted sister axon (P2 and D2, respectively), i.e., P1/P2 and D1/D2. Thus, the proximal and distal accumulation ratios are each a measure of the accumulation or depletion of neurofilament protein in the constricted axon relative to its nonconstricted sister.
Mentions: All the experiments described in this paper were performed on cultures obtained from dorsal root ganglia. Though these ganglia contain exclusively sensory neurons, these cells are not a homogeneous population and they do vary in their size and neurofilament protein content (Lawson, 1992). This makes it difficult to accurately compare the effect of constriction on the neurofilament protein distribution in different cells. To control for this variation, we took advantage of the fact that axons in these cultures typically branch by bifurcation of the growth cone, giving rise to two sister axons that have similar length, diameter, and growth behavior (Bray et al., 1987). By restricting our analyses to axons that branched in this manner we were able to constrict one of the two sister axons and then compare the neurofilament protein distribution with its nonconstricted sister control (Fig. 3).

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