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Dynamics of axonal microtubules regulate the topology of new membrane insertion into the growing neurites.

Zakharenko S, Popov S - J. Cell Biol. (1998)

Bottom Line: Suppression of microtubule (MT) dynamic instability did not interfere with the delivery of new membrane material to the growth cone region; however, the insertion of vesicles into the plasma membrane was dramatically inhibited.Local disassembly of MTs by focal application of nocodazole to the middle axonal segment resulted in the addition of new membrane at the site of drug application.Our results suggest that the local destabilization of axonal MTs is necessary and sufficient for the delivery of membrane material to specific neuronal sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612, USA.

ABSTRACT
Nerve growth depends on the delivery of cell body-synthesized material to the growing neuronal processes. The cellular mechanisms that determine the topology of new membrane addition to the axon are not known. Here we describe a technique to visualize the transport and sites of exocytosis of cell body- derived vesicles in growing axons. We found that in Xenopus embryo neurons in culture, cell body-derived vesicles were rapidly transported all the way down to the growth cone region, where they fused with the plasma membrane. Suppression of microtubule (MT) dynamic instability did not interfere with the delivery of new membrane material to the growth cone region; however, the insertion of vesicles into the plasma membrane was dramatically inhibited. Local disassembly of MTs by focal application of nocodazole to the middle axonal segment resulted in the addition of new membrane at the site of drug application. Our results suggest that the local destabilization of axonal MTs is necessary and sufficient for the delivery of membrane material to specific neuronal sites.

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Local disruption of axonal MTs is sufficient for the insertion of cell body–derived vesicles along the axon. (A) Cell body– derived vesicles were stained with DiIC12 molecules as in Fig. 1. Local perfusion of the middle axonal segment with a culture medium  containing 5 μg/ml nocodazole was started 30 min before the soma staining. (B) Fluorescent images of the superfused site at different  times (marked in minutes) after the staining of the cell body with DiIC12. The staining of filopodia, which reflects the insertion of soma-derived vesicles into the plasmalemma, could be detected as soon as 10 min after the onset of cell body staining. The bright staining of  the axon proximal to the perfusion site reflects an accumulation of fluorescent vesicles in this region. (C) Fluorescence intensity profiles  of the filopodia staining 10 min (filled triangles) and 50 min (open squares) after the soma staining. The intensity of individual filopodia  staining (arbitrary units) is plotted vs. the distance from the center of the profile. The center of the superfused zone was located ∼50 μm  distal from the center of the fluorescence profile. The widening of the profiles reflects the lateral diffusion of DiIC12 molecules incorporated into the plasma membrane along the axon. Data from three representative experiments are combined together. (D) Representative immunofluorescent micrograph of the MT array in the axon near the site locally superfused with nocodazole. The cell was fixed and  stained for MTs 30 min after the onset of perfusion. (E) Quantitative analysis of immunofluorescence data. For each axon, the intensity  of fluorescence along the axon was normalized to that ∼100 μm proximal to the center of the superfused zone. Data are presented as  mean ± SEM for 10 different axons. Bars: (B) 50 μm; (D) 30 μm.
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Figure 6: Local disruption of axonal MTs is sufficient for the insertion of cell body–derived vesicles along the axon. (A) Cell body– derived vesicles were stained with DiIC12 molecules as in Fig. 1. Local perfusion of the middle axonal segment with a culture medium containing 5 μg/ml nocodazole was started 30 min before the soma staining. (B) Fluorescent images of the superfused site at different times (marked in minutes) after the staining of the cell body with DiIC12. The staining of filopodia, which reflects the insertion of soma-derived vesicles into the plasmalemma, could be detected as soon as 10 min after the onset of cell body staining. The bright staining of the axon proximal to the perfusion site reflects an accumulation of fluorescent vesicles in this region. (C) Fluorescence intensity profiles of the filopodia staining 10 min (filled triangles) and 50 min (open squares) after the soma staining. The intensity of individual filopodia staining (arbitrary units) is plotted vs. the distance from the center of the profile. The center of the superfused zone was located ∼50 μm distal from the center of the fluorescence profile. The widening of the profiles reflects the lateral diffusion of DiIC12 molecules incorporated into the plasma membrane along the axon. Data from three representative experiments are combined together. (D) Representative immunofluorescent micrograph of the MT array in the axon near the site locally superfused with nocodazole. The cell was fixed and stained for MTs 30 min after the onset of perfusion. (E) Quantitative analysis of immunofluorescence data. For each axon, the intensity of fluorescence along the axon was normalized to that ∼100 μm proximal to the center of the superfused zone. Data are presented as mean ± SEM for 10 different axons. Bars: (B) 50 μm; (D) 30 μm.

Mentions: Under the cell culture conditions used in this study, the majority of DiIC12-stained vesicles are transported all the way down to the growth cone region, where the vesicles fuse with the plasma membrane. The insertion of cell body–derived vesicles along the axon may be inhibited because of the limitations imposed by the axonal cytoskeleton. Alternatively, the plasma membrane along the axon may lack the appropriate membrane receptors (t-SNAREs), which are necessary for vesicular targeting and/or fusion (Rothman, 1994; Calacos and Scheller, 1996; Hanson et al., 1997). To distinguish between these possibilities, we locally applied nocodazole (5 μg/ml) to the middle axonal segment located at least ∼500 μm away both from the soma and the growth cone. This distance was sufficient to exclude the lateral diffusion of DiIC12 molecules from the soma or the growth cone region to the superfused site. 30 min after the onset of perfusion with nocodazole, we stained cell body–derived vesicles with DiIC12 molecules as described above. Within 10–20 min after the staining of the soma, we observed the accumulation of cell body–derived organelles 50–80 μm proximal to the perfusion site, local staining of the plasma membrane, and depolymerization of MTs at the perfusion site (Fig. 6). Thus, plasma membrane components for vesicular exocytosis are likely to be present throughout the whole axonal surface. Local disruption of MTs along the axon is sufficient to induce the fusion of cell body–derived vesicles with the plasma membrane.


Dynamics of axonal microtubules regulate the topology of new membrane insertion into the growing neurites.

Zakharenko S, Popov S - J. Cell Biol. (1998)

Local disruption of axonal MTs is sufficient for the insertion of cell body–derived vesicles along the axon. (A) Cell body– derived vesicles were stained with DiIC12 molecules as in Fig. 1. Local perfusion of the middle axonal segment with a culture medium  containing 5 μg/ml nocodazole was started 30 min before the soma staining. (B) Fluorescent images of the superfused site at different  times (marked in minutes) after the staining of the cell body with DiIC12. The staining of filopodia, which reflects the insertion of soma-derived vesicles into the plasmalemma, could be detected as soon as 10 min after the onset of cell body staining. The bright staining of  the axon proximal to the perfusion site reflects an accumulation of fluorescent vesicles in this region. (C) Fluorescence intensity profiles  of the filopodia staining 10 min (filled triangles) and 50 min (open squares) after the soma staining. The intensity of individual filopodia  staining (arbitrary units) is plotted vs. the distance from the center of the profile. The center of the superfused zone was located ∼50 μm  distal from the center of the fluorescence profile. The widening of the profiles reflects the lateral diffusion of DiIC12 molecules incorporated into the plasma membrane along the axon. Data from three representative experiments are combined together. (D) Representative immunofluorescent micrograph of the MT array in the axon near the site locally superfused with nocodazole. The cell was fixed and  stained for MTs 30 min after the onset of perfusion. (E) Quantitative analysis of immunofluorescence data. For each axon, the intensity  of fluorescence along the axon was normalized to that ∼100 μm proximal to the center of the superfused zone. Data are presented as  mean ± SEM for 10 different axons. Bars: (B) 50 μm; (D) 30 μm.
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Figure 6: Local disruption of axonal MTs is sufficient for the insertion of cell body–derived vesicles along the axon. (A) Cell body– derived vesicles were stained with DiIC12 molecules as in Fig. 1. Local perfusion of the middle axonal segment with a culture medium containing 5 μg/ml nocodazole was started 30 min before the soma staining. (B) Fluorescent images of the superfused site at different times (marked in minutes) after the staining of the cell body with DiIC12. The staining of filopodia, which reflects the insertion of soma-derived vesicles into the plasmalemma, could be detected as soon as 10 min after the onset of cell body staining. The bright staining of the axon proximal to the perfusion site reflects an accumulation of fluorescent vesicles in this region. (C) Fluorescence intensity profiles of the filopodia staining 10 min (filled triangles) and 50 min (open squares) after the soma staining. The intensity of individual filopodia staining (arbitrary units) is plotted vs. the distance from the center of the profile. The center of the superfused zone was located ∼50 μm distal from the center of the fluorescence profile. The widening of the profiles reflects the lateral diffusion of DiIC12 molecules incorporated into the plasma membrane along the axon. Data from three representative experiments are combined together. (D) Representative immunofluorescent micrograph of the MT array in the axon near the site locally superfused with nocodazole. The cell was fixed and stained for MTs 30 min after the onset of perfusion. (E) Quantitative analysis of immunofluorescence data. For each axon, the intensity of fluorescence along the axon was normalized to that ∼100 μm proximal to the center of the superfused zone. Data are presented as mean ± SEM for 10 different axons. Bars: (B) 50 μm; (D) 30 μm.
Mentions: Under the cell culture conditions used in this study, the majority of DiIC12-stained vesicles are transported all the way down to the growth cone region, where the vesicles fuse with the plasma membrane. The insertion of cell body–derived vesicles along the axon may be inhibited because of the limitations imposed by the axonal cytoskeleton. Alternatively, the plasma membrane along the axon may lack the appropriate membrane receptors (t-SNAREs), which are necessary for vesicular targeting and/or fusion (Rothman, 1994; Calacos and Scheller, 1996; Hanson et al., 1997). To distinguish between these possibilities, we locally applied nocodazole (5 μg/ml) to the middle axonal segment located at least ∼500 μm away both from the soma and the growth cone. This distance was sufficient to exclude the lateral diffusion of DiIC12 molecules from the soma or the growth cone region to the superfused site. 30 min after the onset of perfusion with nocodazole, we stained cell body–derived vesicles with DiIC12 molecules as described above. Within 10–20 min after the staining of the soma, we observed the accumulation of cell body–derived organelles 50–80 μm proximal to the perfusion site, local staining of the plasma membrane, and depolymerization of MTs at the perfusion site (Fig. 6). Thus, plasma membrane components for vesicular exocytosis are likely to be present throughout the whole axonal surface. Local disruption of MTs along the axon is sufficient to induce the fusion of cell body–derived vesicles with the plasma membrane.

Bottom Line: Suppression of microtubule (MT) dynamic instability did not interfere with the delivery of new membrane material to the growth cone region; however, the insertion of vesicles into the plasma membrane was dramatically inhibited.Local disassembly of MTs by focal application of nocodazole to the middle axonal segment resulted in the addition of new membrane at the site of drug application.Our results suggest that the local destabilization of axonal MTs is necessary and sufficient for the delivery of membrane material to specific neuronal sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612, USA.

ABSTRACT
Nerve growth depends on the delivery of cell body-synthesized material to the growing neuronal processes. The cellular mechanisms that determine the topology of new membrane addition to the axon are not known. Here we describe a technique to visualize the transport and sites of exocytosis of cell body- derived vesicles in growing axons. We found that in Xenopus embryo neurons in culture, cell body-derived vesicles were rapidly transported all the way down to the growth cone region, where they fused with the plasma membrane. Suppression of microtubule (MT) dynamic instability did not interfere with the delivery of new membrane material to the growth cone region; however, the insertion of vesicles into the plasma membrane was dramatically inhibited. Local disassembly of MTs by focal application of nocodazole to the middle axonal segment resulted in the addition of new membrane at the site of drug application. Our results suggest that the local destabilization of axonal MTs is necessary and sufficient for the delivery of membrane material to specific neuronal sites.

Show MeSH
Related in: MedlinePlus