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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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The axon is continuous across the constriction  site. Axons were constricted  for 2 h and then fixed and  processed for electron microscopy as described in Materials and Methods. Sections  were cut parallel to the glass  coverslip and longitudinal  with respect to the axis of the  axon. Proximal is left and distal is right. (A) Low magnification montage showing continuity of the axon beneath  the glass fiber. This section  was located ∼75–165 nm  from the surface of the coverslip. (B) High magnification  view of the region of continuity beneath the glass fiber in  A. Note the presence of numerous neurofilaments and  microtubules passing beneath the glass fiber. The  black arrowheads indicate the  location of the glass fiber,  which was removed after fixation. See legend to Fig. 8 for  key to abbreviations. Bars:  (A) 2 μm; (B) 0.25 μm.
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Figure 9: The axon is continuous across the constriction site. Axons were constricted for 2 h and then fixed and processed for electron microscopy as described in Materials and Methods. Sections were cut parallel to the glass coverslip and longitudinal with respect to the axis of the axon. Proximal is left and distal is right. (A) Low magnification montage showing continuity of the axon beneath the glass fiber. This section was located ∼75–165 nm from the surface of the coverslip. (B) High magnification view of the region of continuity beneath the glass fiber in A. Note the presence of numerous neurofilaments and microtubules passing beneath the glass fiber. The black arrowheads indicate the location of the glass fiber, which was removed after fixation. See legend to Fig. 8 for key to abbreviations. Bars: (A) 2 μm; (B) 0.25 μm.

Mentions: Serial longitudinal sections taken parallel to the glass coverslip revealed that the constricted axons were continuous and undamaged beneath the glass fiber (Fig. 9). By noting the interference colors of the sections as they came off the diamond knife and subsequently examining each section in the electron microscope it was possible to estimate the thickness of the axon at the constriction site. For the two axons that we analyzed in this way, the thicknesses were estimated to be ∼90 and 290 nm, which indicates that the axons were only partially constricted and that there may have been considerable variability in the extent of constriction for different axons (see Discussion). Within the region of continuity beneath the glass fiber, we observed neurofilaments, microtubules, and a variety of membranous organelles (Fig. 9 B). Some of the neurofilaments and microtubules were oriented longitudinally and passed directly under the fiber, whereas others were oriented transversely. These observations indicate that constriction did not completely disrupt the continuity of the axonal cytoskeleton and thus it is possible that the movement of axonally transported materials, including neurofilament proteins, may have been only partially impaired in our experiments (see Discussion).


Slow axonal transport of neurofilament protein in cultured neurons.

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

The axon is continuous across the constriction  site. Axons were constricted  for 2 h and then fixed and  processed for electron microscopy as described in Materials and Methods. Sections  were cut parallel to the glass  coverslip and longitudinal  with respect to the axis of the  axon. Proximal is left and distal is right. (A) Low magnification montage showing continuity of the axon beneath  the glass fiber. This section  was located ∼75–165 nm  from the surface of the coverslip. (B) High magnification  view of the region of continuity beneath the glass fiber in  A. Note the presence of numerous neurofilaments and  microtubules passing beneath the glass fiber. The  black arrowheads indicate the  location of the glass fiber,  which was removed after fixation. See legend to Fig. 8 for  key to abbreviations. Bars:  (A) 2 μm; (B) 0.25 μm.
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Figure 9: The axon is continuous across the constriction site. Axons were constricted for 2 h and then fixed and processed for electron microscopy as described in Materials and Methods. Sections were cut parallel to the glass coverslip and longitudinal with respect to the axis of the axon. Proximal is left and distal is right. (A) Low magnification montage showing continuity of the axon beneath the glass fiber. This section was located ∼75–165 nm from the surface of the coverslip. (B) High magnification view of the region of continuity beneath the glass fiber in A. Note the presence of numerous neurofilaments and microtubules passing beneath the glass fiber. The black arrowheads indicate the location of the glass fiber, which was removed after fixation. See legend to Fig. 8 for key to abbreviations. Bars: (A) 2 μm; (B) 0.25 μm.
Mentions: Serial longitudinal sections taken parallel to the glass coverslip revealed that the constricted axons were continuous and undamaged beneath the glass fiber (Fig. 9). By noting the interference colors of the sections as they came off the diamond knife and subsequently examining each section in the electron microscope it was possible to estimate the thickness of the axon at the constriction site. For the two axons that we analyzed in this way, the thicknesses were estimated to be ∼90 and 290 nm, which indicates that the axons were only partially constricted and that there may have been considerable variability in the extent of constriction for different axons (see Discussion). Within the region of continuity beneath the glass fiber, we observed neurofilaments, microtubules, and a variety of membranous organelles (Fig. 9 B). Some of the neurofilaments and microtubules were oriented longitudinally and passed directly under the fiber, whereas others were oriented transversely. These observations indicate that constriction did not completely disrupt the continuity of the axonal cytoskeleton and thus it is possible that the movement of axonally transported materials, including neurofilament proteins, may have been only partially impaired in our experiments (see Discussion).

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