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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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Electron micrographs of constricted axons.  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 the  axonal swellings on either  side of the constriction. This  section passed through the  approximate center of the  proximal and distal swellings, which was ∼1.2 μm  from the surface of the coverslip. The gap between the  swellings represents the space  occupied by the glass fiber  (black arrowheads), which  was removed after fixation.  (B and C) High magnification  views of proximal swellings.  Note the numerous and  highly disorganized neurofilaments arranged singly or in  clusters and oriented longitudinally, obliquely, and transversely within the axon. (D  and E) High magnification  views of distal swellings. Note  the presence of relatively few  neurofilaments and numerous large multilamellar membranous organelles. The images in B and D correspond  to regions of the swellings  shown in A. ER, endoplasmic  reticulum; MT, microtubule;  mito, mitochondrion; mlo,  multilamellar organelle; NF,  neurofilament; ves, vesicle.  Bars: (A) 2 μm; (B–E)  0.25 μm.
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Figure 8: Electron micrographs of constricted axons. 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 the axonal swellings on either side of the constriction. This section passed through the approximate center of the proximal and distal swellings, which was ∼1.2 μm from the surface of the coverslip. The gap between the swellings represents the space occupied by the glass fiber (black arrowheads), which was removed after fixation. (B and C) High magnification views of proximal swellings. Note the numerous and highly disorganized neurofilaments arranged singly or in clusters and oriented longitudinally, obliquely, and transversely within the axon. (D and E) High magnification views of distal swellings. Note the presence of relatively few neurofilaments and numerous large multilamellar membranous organelles. The images in B and D correspond to regions of the swellings shown in A. ER, endoplasmic reticulum; MT, microtubule; mito, mitochondrion; mlo, multilamellar organelle; NF, neurofilament; ves, vesicle. Bars: (A) 2 μm; (B–E) 0.25 μm.

Mentions: To examine the ultrastructure of constricted axons, we fixed cells after constriction for 2 h and then processed them for electron microscopy. Fig. 8 A shows a longitudinal section of an axon taken parallel to the glass coverslip and passing through the center of the proximal and distal swellings. The swellings contained numerous membrane-bound organelles including mitochondria, small vesicles with light or dark lumens, large heterogeneous multilamellar organelles, and small tubules that resembled smooth endoplasmic reticulum. Distally, large multilamellar organelles predominated, though smaller vesicles and tubules were also observed (Fig. 8, D and E). Proximally, small vesicles and tubules predominated and there were very few multilamellar organelles (Fig. 8, B and C). Mitochondria were observed to accumulate on both sides of the constrictions. The distinct size and appearance of the membranous organelles in the proximal and distal swellings is consistent with previous descriptions of anterogradely and retrogradely moving organelles (e.g., Smith, 1980; Tsukita and Ishikawa, 1980; Fahim et al., 1985; Hirokawa et al., 1990), and this indicates that the constrictions impeded both anterograde and retrograde fast axonal transport.


Slow axonal transport of neurofilament protein in cultured neurons.

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

Electron micrographs of constricted axons.  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 the  axonal swellings on either  side of the constriction. This  section passed through the  approximate center of the  proximal and distal swellings, which was ∼1.2 μm  from the surface of the coverslip. The gap between the  swellings represents the space  occupied by the glass fiber  (black arrowheads), which  was removed after fixation.  (B and C) High magnification  views of proximal swellings.  Note the numerous and  highly disorganized neurofilaments arranged singly or in  clusters and oriented longitudinally, obliquely, and transversely within the axon. (D  and E) High magnification  views of distal swellings. Note  the presence of relatively few  neurofilaments and numerous large multilamellar membranous organelles. The images in B and D correspond  to regions of the swellings  shown in A. ER, endoplasmic  reticulum; MT, microtubule;  mito, mitochondrion; mlo,  multilamellar organelle; NF,  neurofilament; ves, vesicle.  Bars: (A) 2 μm; (B–E)  0.25 μm.
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Figure 8: Electron micrographs of constricted axons. 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 the axonal swellings on either side of the constriction. This section passed through the approximate center of the proximal and distal swellings, which was ∼1.2 μm from the surface of the coverslip. The gap between the swellings represents the space occupied by the glass fiber (black arrowheads), which was removed after fixation. (B and C) High magnification views of proximal swellings. Note the numerous and highly disorganized neurofilaments arranged singly or in clusters and oriented longitudinally, obliquely, and transversely within the axon. (D and E) High magnification views of distal swellings. Note the presence of relatively few neurofilaments and numerous large multilamellar membranous organelles. The images in B and D correspond to regions of the swellings shown in A. ER, endoplasmic reticulum; MT, microtubule; mito, mitochondrion; mlo, multilamellar organelle; NF, neurofilament; ves, vesicle. Bars: (A) 2 μm; (B–E) 0.25 μm.
Mentions: To examine the ultrastructure of constricted axons, we fixed cells after constriction for 2 h and then processed them for electron microscopy. Fig. 8 A shows a longitudinal section of an axon taken parallel to the glass coverslip and passing through the center of the proximal and distal swellings. The swellings contained numerous membrane-bound organelles including mitochondria, small vesicles with light or dark lumens, large heterogeneous multilamellar organelles, and small tubules that resembled smooth endoplasmic reticulum. Distally, large multilamellar organelles predominated, though smaller vesicles and tubules were also observed (Fig. 8, D and E). Proximally, small vesicles and tubules predominated and there were very few multilamellar organelles (Fig. 8, B and C). Mitochondria were observed to accumulate on both sides of the constrictions. The distinct size and appearance of the membranous organelles in the proximal and distal swellings is consistent with previous descriptions of anterogradely and retrogradely moving organelles (e.g., Smith, 1980; Tsukita and Ishikawa, 1980; Fahim et al., 1985; Hirokawa et al., 1990), and this indicates that the constrictions impeded both anterograde and retrograde fast axonal transport.

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