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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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Dynamic instability of MTs is required for the insertion of new membrane into the distal axon. (A–D) DIC (top) and fluorescence images of the distal axon at two different times (marked in minutes) after the staining of the soma. 7 nM taxol (A) or 3 nM vinblastine (B and C) was added to the culture medium 30 min before the staining of the soma. Fluorescent vesicles accumulated at the  growth cone region. Filopodia staining in A and B was drastically reduced in comparison with control (D) neurons (P < 0.001, t test). In  C, 30 min after the staining of the cell body, the concentration of vinblastine was increased to 1 μM. This induced a rapid insertion of the  vesicles accumulated at the distal axon into the plasma membrane and staining of the filopodia. (E) Quantitative analysis of the plasma  membrane staining. For each neuron the intensity of the plasma membrane staining at the growth cone region was determined as an  average for at least 20 filopodia. The data are presented as a mean ± SEM for five to seven different neurons. *P < 0.001, t test. Bar,  30 μm.
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Figure 5: Dynamic instability of MTs is required for the insertion of new membrane into the distal axon. (A–D) DIC (top) and fluorescence images of the distal axon at two different times (marked in minutes) after the staining of the soma. 7 nM taxol (A) or 3 nM vinblastine (B and C) was added to the culture medium 30 min before the staining of the soma. Fluorescent vesicles accumulated at the growth cone region. Filopodia staining in A and B was drastically reduced in comparison with control (D) neurons (P < 0.001, t test). In C, 30 min after the staining of the cell body, the concentration of vinblastine was increased to 1 μM. This induced a rapid insertion of the vesicles accumulated at the distal axon into the plasma membrane and staining of the filopodia. (E) Quantitative analysis of the plasma membrane staining. For each neuron the intensity of the plasma membrane staining at the growth cone region was determined as an average for at least 20 filopodia. The data are presented as a mean ± SEM for five to seven different neurons. *P < 0.001, t test. Bar, 30 μm.

Mentions: MTs at the growth cone region display a complex pattern of behavior and appear to be significantly more dynamic than MTs along the axon (Bamburg et al., 1986; Tanaka and Kirschner, 1991; Tanaka et al., 1995). To test whether the high rate of MT turnover at the distal axon contributes to the preferential insertion of the cell body–derived vesicles at the growth cone region, we determined the effects of low concentrations of taxol and vinblastine on the pattern of membrane insertion into the growing axons. Taxol and vinblastine are antimitotic drugs that, in micromolar concentrations, stabilize and disrupt MTs, respectively. In nanomolar concentrations, both drugs decrease the dynamic instability of MTs (Jordan et al., 1992, 1993). In nanomolar concentrations, vinblastine and taxol did not affect the polymerization of MTs in Xenopus neurons (Table I). Neither taxol (7 nM) nor vinblastine (3 nM) affected the delivery of cell body–derived vesicles to the distal axon, as evidenced by the accumulation of the DiIC12-stained organelles at the growth cone region (Fig. 5, A and B). However, the staining of the plasma membrane, and thus insertion of new membrane into the growth cone region, were dramatically inhibited. The quantitative analysis of the plasma membrane staining (Fig. 5 E) was facilitated by the fact that under the cell culture conditions used in this study, axons possess a rich net of filopodia that spans the entire length of the neurite, including the growth cone. Since DiIC12-labeled vesicles were largely excluded from the filopodia, the sampling areas were chosen along the length of individual filopodia at the growth cone. Similar results were obtained when the sampling areas were chosen at the lamellipodium region of the growth cone. In a series of control experiments, neuronal cultures were pretreated with 3 nM vinblastine for 30 min to allow accumulation of the fluorescent vesicles at the growth cone region, after which the concentration of vinblastine was increased to 1 μM. This treatment resulted in the disassembly of axonal MTs (data not shown), and rapid staining of the plasma membrane at the distal axon (Fig. 5 C). Hence, inhibition of membrane insertion by nanomolar concentrations of vinblastine is not related to a nonspecific effect of the drug on vesicular fusion.


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

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

Dynamic instability of MTs is required for the insertion of new membrane into the distal axon. (A–D) DIC (top) and fluorescence images of the distal axon at two different times (marked in minutes) after the staining of the soma. 7 nM taxol (A) or 3 nM vinblastine (B and C) was added to the culture medium 30 min before the staining of the soma. Fluorescent vesicles accumulated at the  growth cone region. Filopodia staining in A and B was drastically reduced in comparison with control (D) neurons (P < 0.001, t test). In  C, 30 min after the staining of the cell body, the concentration of vinblastine was increased to 1 μM. This induced a rapid insertion of the  vesicles accumulated at the distal axon into the plasma membrane and staining of the filopodia. (E) Quantitative analysis of the plasma  membrane staining. For each neuron the intensity of the plasma membrane staining at the growth cone region was determined as an  average for at least 20 filopodia. The data are presented as a mean ± SEM for five to seven different neurons. *P < 0.001, t test. Bar,  30 μm.
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Figure 5: Dynamic instability of MTs is required for the insertion of new membrane into the distal axon. (A–D) DIC (top) and fluorescence images of the distal axon at two different times (marked in minutes) after the staining of the soma. 7 nM taxol (A) or 3 nM vinblastine (B and C) was added to the culture medium 30 min before the staining of the soma. Fluorescent vesicles accumulated at the growth cone region. Filopodia staining in A and B was drastically reduced in comparison with control (D) neurons (P < 0.001, t test). In C, 30 min after the staining of the cell body, the concentration of vinblastine was increased to 1 μM. This induced a rapid insertion of the vesicles accumulated at the distal axon into the plasma membrane and staining of the filopodia. (E) Quantitative analysis of the plasma membrane staining. For each neuron the intensity of the plasma membrane staining at the growth cone region was determined as an average for at least 20 filopodia. The data are presented as a mean ± SEM for five to seven different neurons. *P < 0.001, t test. Bar, 30 μm.
Mentions: MTs at the growth cone region display a complex pattern of behavior and appear to be significantly more dynamic than MTs along the axon (Bamburg et al., 1986; Tanaka and Kirschner, 1991; Tanaka et al., 1995). To test whether the high rate of MT turnover at the distal axon contributes to the preferential insertion of the cell body–derived vesicles at the growth cone region, we determined the effects of low concentrations of taxol and vinblastine on the pattern of membrane insertion into the growing axons. Taxol and vinblastine are antimitotic drugs that, in micromolar concentrations, stabilize and disrupt MTs, respectively. In nanomolar concentrations, both drugs decrease the dynamic instability of MTs (Jordan et al., 1992, 1993). In nanomolar concentrations, vinblastine and taxol did not affect the polymerization of MTs in Xenopus neurons (Table I). Neither taxol (7 nM) nor vinblastine (3 nM) affected the delivery of cell body–derived vesicles to the distal axon, as evidenced by the accumulation of the DiIC12-stained organelles at the growth cone region (Fig. 5, A and B). However, the staining of the plasma membrane, and thus insertion of new membrane into the growth cone region, were dramatically inhibited. The quantitative analysis of the plasma membrane staining (Fig. 5 E) was facilitated by the fact that under the cell culture conditions used in this study, axons possess a rich net of filopodia that spans the entire length of the neurite, including the growth cone. Since DiIC12-labeled vesicles were largely excluded from the filopodia, the sampling areas were chosen along the length of individual filopodia at the growth cone. Similar results were obtained when the sampling areas were chosen at the lamellipodium region of the growth cone. In a series of control experiments, neuronal cultures were pretreated with 3 nM vinblastine for 30 min to allow accumulation of the fluorescent vesicles at the growth cone region, after which the concentration of vinblastine was increased to 1 μM. This treatment resulted in the disassembly of axonal MTs (data not shown), and rapid staining of the plasma membrane at the distal axon (Fig. 5 C). Hence, inhibition of membrane insertion by nanomolar concentrations of vinblastine is not related to a nonspecific effect of the drug on vesicular fusion.

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