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

Neurofilament protein distribution  along constricted and control sister axons.  Neurons were fixed and stained for neurofilament protein by immunofluorescence microscopy using a rabbit polyclonal antibody to  NF-L and the fluorescence intensity was  quantified along constricted and control sister axons by the segmented mask method  (see Materials and Methods). The three profiles on the left represent axons that were  constricted for 5 s, 30 min, and 2 h. The three  profiles on the right represent the corresponding control sister axons, which were not  constricted. Each graph represents the fluorescence intensity profile along a 300-μm  length of axon. The total axon lengths were  739, 500, and 611 μm for the constricted axons and 700, 435, and 776 μm for the control  axons, respectively. The arrow on each constricted axon profile indicates the segment  that contained the constriction. Each point in  the graphs represents the fluorescence intensity for a single 4.1-μm segment along the  axon, which is a relative measure of the  amount of neurofilament protein in that segment. Proximal is left and distal is right in all  cases. The horizontal bars along the abscissa  of each plot represent the locations of the 41-μm (10 segment) measurement windows that  were used for calculation of the proximal and  distal accumulation ratios.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132919&req=5

Figure 4: Neurofilament protein distribution along constricted and control sister axons. Neurons were fixed and stained for neurofilament protein by immunofluorescence microscopy using a rabbit polyclonal antibody to NF-L and the fluorescence intensity was quantified along constricted and control sister axons by the segmented mask method (see Materials and Methods). The three profiles on the left represent axons that were constricted for 5 s, 30 min, and 2 h. The three profiles on the right represent the corresponding control sister axons, which were not constricted. Each graph represents the fluorescence intensity profile along a 300-μm length of axon. The total axon lengths were 739, 500, and 611 μm for the constricted axons and 700, 435, and 776 μm for the control axons, respectively. The arrow on each constricted axon profile indicates the segment that contained the constriction. Each point in the graphs represents the fluorescence intensity for a single 4.1-μm segment along the axon, which is a relative measure of the amount of neurofilament protein in that segment. Proximal is left and distal is right in all cases. The horizontal bars along the abscissa of each plot represent the locations of the 41-μm (10 segment) measurement windows that were used for calculation of the proximal and distal accumulation ratios.

Mentions: Fig. 4 shows the neurofilament protein distribution along three axons that were constricted for different lengths of time, and along their corresponding nonconstricted sister axons. Constriction for 5 s had no apparent effect; both the constricted and control sister axons exhibited a fairly uniform distribution of neurofilament protein along their entire length, which is typical for axons in these cultures. In contrast, constriction for 30 min resulted in a pronounced increase in the amount of neurofilament protein proximal to the constriction and a smaller increase distally. Constriction for 2 h resulted in a further increase in the amount of neurofilament protein proximal to the constriction, but no further increase distal to the constriction. Proximal to the constriction, the amount of neurofilament protein typically declined over a distance of 5–10 segments (∼20–40 μm) whereas distal to the constriction the decline was more abrupt.


Slow axonal transport of neurofilament protein in cultured neurons.

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

Neurofilament protein distribution  along constricted and control sister axons.  Neurons were fixed and stained for neurofilament protein by immunofluorescence microscopy using a rabbit polyclonal antibody to  NF-L and the fluorescence intensity was  quantified along constricted and control sister axons by the segmented mask method  (see Materials and Methods). The three profiles on the left represent axons that were  constricted for 5 s, 30 min, and 2 h. The three  profiles on the right represent the corresponding control sister axons, which were not  constricted. Each graph represents the fluorescence intensity profile along a 300-μm  length of axon. The total axon lengths were  739, 500, and 611 μm for the constricted axons and 700, 435, and 776 μm for the control  axons, respectively. The arrow on each constricted axon profile indicates the segment  that contained the constriction. Each point in  the graphs represents the fluorescence intensity for a single 4.1-μm segment along the  axon, which is a relative measure of the  amount of neurofilament protein in that segment. Proximal is left and distal is right in all  cases. The horizontal bars along the abscissa  of each plot represent the locations of the 41-μm (10 segment) measurement windows that  were used for calculation of the proximal and  distal accumulation ratios.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Neurofilament protein distribution along constricted and control sister axons. Neurons were fixed and stained for neurofilament protein by immunofluorescence microscopy using a rabbit polyclonal antibody to NF-L and the fluorescence intensity was quantified along constricted and control sister axons by the segmented mask method (see Materials and Methods). The three profiles on the left represent axons that were constricted for 5 s, 30 min, and 2 h. The three profiles on the right represent the corresponding control sister axons, which were not constricted. Each graph represents the fluorescence intensity profile along a 300-μm length of axon. The total axon lengths were 739, 500, and 611 μm for the constricted axons and 700, 435, and 776 μm for the control axons, respectively. The arrow on each constricted axon profile indicates the segment that contained the constriction. Each point in the graphs represents the fluorescence intensity for a single 4.1-μm segment along the axon, which is a relative measure of the amount of neurofilament protein in that segment. Proximal is left and distal is right in all cases. The horizontal bars along the abscissa of each plot represent the locations of the 41-μm (10 segment) measurement windows that were used for calculation of the proximal and distal accumulation ratios.
Mentions: Fig. 4 shows the neurofilament protein distribution along three axons that were constricted for different lengths of time, and along their corresponding nonconstricted sister axons. Constriction for 5 s had no apparent effect; both the constricted and control sister axons exhibited a fairly uniform distribution of neurofilament protein along their entire length, which is typical for axons in these cultures. In contrast, constriction for 30 min resulted in a pronounced increase in the amount of neurofilament protein proximal to the constriction and a smaller increase distally. Constriction for 2 h resulted in a further increase in the amount of neurofilament protein proximal to the constriction, but no further increase distal to the constriction. Proximal to the constriction, the amount of neurofilament protein typically declined over a distance of 5–10 segments (∼20–40 μm) whereas distal to the constriction the decline was more abrupt.

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