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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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Extraction of neurons with 0.02% saponin. (A)  Cell body of a cultured neuron that was microinjected  with tetramethyl rhodamine  isothiocyanate dextran (average mol wt = 76,000). (B)  The same cell body after extraction with 0.02% saponin  in PHEM + 0.19 M NaCl for  10 min as described in Materials and Methods. Bar, 15  μm. (C) Quantitative data  for five cells imaged before  extraction and after extraction for 2, 5, and 10 min. For  each cell, the total fluorescence intensity in the cell  body after extraction was expressed as a percentage of  the total fluorescence intensity in the cell body before extraction.  The error bars represent the standard deviation about the mean  for each time point. 97% of the fluorescent dextran was extracted  from the cells within 10 min.
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Figure 7: Extraction of neurons with 0.02% saponin. (A) Cell body of a cultured neuron that was microinjected with tetramethyl rhodamine isothiocyanate dextran (average mol wt = 76,000). (B) The same cell body after extraction with 0.02% saponin in PHEM + 0.19 M NaCl for 10 min as described in Materials and Methods. Bar, 15 μm. (C) Quantitative data for five cells imaged before extraction and after extraction for 2, 5, and 10 min. For each cell, the total fluorescence intensity in the cell body after extraction was expressed as a percentage of the total fluorescence intensity in the cell body before extraction. The error bars represent the standard deviation about the mean for each time point. 97% of the fluorescent dextran was extracted from the cells within 10 min.

Mentions: To address the form in which the neurofilament protein accumulated, we constricted axons for 2 h and then permeabilized them with detergent under conditions that stabilize neurofilament polymers in order to extract neurofilament protein subunits and diffusible oligomers. The cells were then fixed, immunostained and analyzed as described above. Previous studies in our laboratory have shown that neurofilament polymers in cultured neurons splay apart from each other when treated with the concentrations of detergent that are normally used to extract cultured cells (Brown, 1997, 1998). This splaying phenomenon presented a problem for our studies because we found that it interfered with creation of the segmented mask that we used for our quantitative analyses. However, we found that cells could be extracted without inducing splaying by using lower concentrations of detergent such as 0.02% saponin, which has been used to extract soluble proteins from cells by Nakata et al. (1987) and Okabe et al. (1993). To confirm that 0.02% saponin was sufficient to permeabilize our cells, we microinjected neurons with 70,000 mol wt fluorescent dextran and then quantified the fluorescence intensity within the cell bodies before and after detergent treatment (see Materials and Methods). Fig. 7 shows that 96% of the fluorescent dextran diffused out of the cells within 2 min after addition of 0.02% saponin. After 10 min, the proportion extracted had increased to 97%. Subsequent treatment with 1% Triton X-100 for 10 min (data not shown) increased the proportion extracted to 98%. Thus treatment with 0.02% saponin for 10 min extracted 99% of the Triton X-100 soluble fluorescence in these neurons.


Slow axonal transport of neurofilament protein in cultured neurons.

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

Extraction of neurons with 0.02% saponin. (A)  Cell body of a cultured neuron that was microinjected  with tetramethyl rhodamine  isothiocyanate dextran (average mol wt = 76,000). (B)  The same cell body after extraction with 0.02% saponin  in PHEM + 0.19 M NaCl for  10 min as described in Materials and Methods. Bar, 15  μm. (C) Quantitative data  for five cells imaged before  extraction and after extraction for 2, 5, and 10 min. For  each cell, the total fluorescence intensity in the cell  body after extraction was expressed as a percentage of  the total fluorescence intensity in the cell body before extraction.  The error bars represent the standard deviation about the mean  for each time point. 97% of the fluorescent dextran was extracted  from the cells within 10 min.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132919&req=5

Figure 7: Extraction of neurons with 0.02% saponin. (A) Cell body of a cultured neuron that was microinjected with tetramethyl rhodamine isothiocyanate dextran (average mol wt = 76,000). (B) The same cell body after extraction with 0.02% saponin in PHEM + 0.19 M NaCl for 10 min as described in Materials and Methods. Bar, 15 μm. (C) Quantitative data for five cells imaged before extraction and after extraction for 2, 5, and 10 min. For each cell, the total fluorescence intensity in the cell body after extraction was expressed as a percentage of the total fluorescence intensity in the cell body before extraction. The error bars represent the standard deviation about the mean for each time point. 97% of the fluorescent dextran was extracted from the cells within 10 min.
Mentions: To address the form in which the neurofilament protein accumulated, we constricted axons for 2 h and then permeabilized them with detergent under conditions that stabilize neurofilament polymers in order to extract neurofilament protein subunits and diffusible oligomers. The cells were then fixed, immunostained and analyzed as described above. Previous studies in our laboratory have shown that neurofilament polymers in cultured neurons splay apart from each other when treated with the concentrations of detergent that are normally used to extract cultured cells (Brown, 1997, 1998). This splaying phenomenon presented a problem for our studies because we found that it interfered with creation of the segmented mask that we used for our quantitative analyses. However, we found that cells could be extracted without inducing splaying by using lower concentrations of detergent such as 0.02% saponin, which has been used to extract soluble proteins from cells by Nakata et al. (1987) and Okabe et al. (1993). To confirm that 0.02% saponin was sufficient to permeabilize our cells, we microinjected neurons with 70,000 mol wt fluorescent dextran and then quantified the fluorescence intensity within the cell bodies before and after detergent treatment (see Materials and Methods). Fig. 7 shows that 96% of the fluorescent dextran diffused out of the cells within 2 min after addition of 0.02% saponin. After 10 min, the proportion extracted had increased to 97%. Subsequent treatment with 1% Triton X-100 for 10 min (data not shown) increased the proportion extracted to 98%. Thus treatment with 0.02% saponin for 10 min extracted 99% of the Triton X-100 soluble fluorescence in these neurons.

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