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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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Progressive staining of the distal axon after labeling of cell body–derived  vesicles. DIC and fluorescence images of the distal  axon at different times  (marked in minutes) after  local labeling of cell body– derived vesicles with DiIC12  molecules. The length of the  axon was ∼1,900 μm. DiIC12-labeled vesicles gradually accumulated at the central domain of the growth cone. The  delay between the staining of  the cell body and accumulation of the vesicles at the distal axon is likely to reflect the  time required for the fast axonal transport of the vesicles.  Notice a gradual increase in  the diffuse staining of the  vesicle-free peripheral growth  cone.
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Figure 2: Progressive staining of the distal axon after labeling of cell body–derived vesicles. DIC and fluorescence images of the distal axon at different times (marked in minutes) after local labeling of cell body– derived vesicles with DiIC12 molecules. The length of the axon was ∼1,900 μm. DiIC12-labeled vesicles gradually accumulated at the central domain of the growth cone. The delay between the staining of the cell body and accumulation of the vesicles at the distal axon is likely to reflect the time required for the fast axonal transport of the vesicles. Notice a gradual increase in the diffuse staining of the vesicle-free peripheral growth cone.

Mentions: Within 10–20 min after the staining of the cell body, we observed an accumulation of DiIC12-labeled vesicles at the central cytoplasmic domain of the growth cone (Fig. 2). Individual vesicles could be resolved as brightly stained puncta both along the axon (Fig. 1) and at the growth cone region (Figs. 2 and 3). In parallel with accumulation of DiIC12-labeled vesicles at the distal axon, we observed a progressive increase in the diffuse staining of the peripheral growth cone region (Fig. 2). In agreement with previously published data (Forscher and Smith, 1988), the cell body–derived vesicles were largely excluded from the peripheral growth cone lamella and from the filopodia (Figs. 2 and 3). Therefore, we interpret the diffuse staining of the axon as incorporation of DiIC12 molecules into the plasma membrane. This diffuse staining gradually spread from the growth cone towards the cell body (Fig. 2), consistent with lateral diffusion of DiIC12 molecules along the plasmalemma.


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

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

Progressive staining of the distal axon after labeling of cell body–derived  vesicles. DIC and fluorescence images of the distal  axon at different times  (marked in minutes) after  local labeling of cell body– derived vesicles with DiIC12  molecules. The length of the  axon was ∼1,900 μm. DiIC12-labeled vesicles gradually accumulated at the central domain of the growth cone. The  delay between the staining of  the cell body and accumulation of the vesicles at the distal axon is likely to reflect the  time required for the fast axonal transport of the vesicles.  Notice a gradual increase in  the diffuse staining of the  vesicle-free peripheral growth  cone.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132946&req=5

Figure 2: Progressive staining of the distal axon after labeling of cell body–derived vesicles. DIC and fluorescence images of the distal axon at different times (marked in minutes) after local labeling of cell body– derived vesicles with DiIC12 molecules. The length of the axon was ∼1,900 μm. DiIC12-labeled vesicles gradually accumulated at the central domain of the growth cone. The delay between the staining of the cell body and accumulation of the vesicles at the distal axon is likely to reflect the time required for the fast axonal transport of the vesicles. Notice a gradual increase in the diffuse staining of the vesicle-free peripheral growth cone.
Mentions: Within 10–20 min after the staining of the cell body, we observed an accumulation of DiIC12-labeled vesicles at the central cytoplasmic domain of the growth cone (Fig. 2). Individual vesicles could be resolved as brightly stained puncta both along the axon (Fig. 1) and at the growth cone region (Figs. 2 and 3). In parallel with accumulation of DiIC12-labeled vesicles at the distal axon, we observed a progressive increase in the diffuse staining of the peripheral growth cone region (Fig. 2). In agreement with previously published data (Forscher and Smith, 1988), the cell body–derived vesicles were largely excluded from the peripheral growth cone lamella and from the filopodia (Figs. 2 and 3). Therefore, we interpret the diffuse staining of the axon as incorporation of DiIC12 molecules into the plasma membrane. This diffuse staining gradually spread from the growth cone towards the cell body (Fig. 2), consistent with lateral diffusion of DiIC12 molecules along the plasmalemma.

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