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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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Preferential insertion of cell body–derived vesicles into the growth cone region. (A and B) DIC (top)  and fluorescence (bottom)  images of the middle (A) and  distal (B) segments of the  same axon 30 min after staining of the cell body. The  length of the axon was  ∼1,600 μm, and the middle  segment was ∼900 μm away  from the soma. Staining of  the plasma membrane with  DiIC12 molecules, reflecting  the insertion of DiIC12- labeled vesicles into the plasmalemma, could be observed  at the growth cone (B). No  plasma membrane staining  was detected at the middle  axon, as well as at sufficiently  large distances from the  growth cone in the distal segment (A and B). (C–E) Differential interference contrast and fluorescent micrographs of the distal axons 60 min after the staining of the  cell body. Before the staining of the soma, neuronal cultures were pretreated for 1 h with brefeldin A (10 μg/ml, C), nocodazole (5 μg/ ml, D), or cytochalasin D (5 μM, E). The drugs were present in the culture medium throughout the experiment. Very few (C) or no (D)  fluorescent vesicles and no plasma membrane staining (C and D) could be detected. Cytochalasin treatment (E) had no obvious effect  on the delivery of the vesicles to the growth cone and their incorporation into the plasma membrane. Bars, 30 μm.
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Figure 4: Preferential insertion of cell body–derived vesicles into the growth cone region. (A and B) DIC (top) and fluorescence (bottom) images of the middle (A) and distal (B) segments of the same axon 30 min after staining of the cell body. The length of the axon was ∼1,600 μm, and the middle segment was ∼900 μm away from the soma. Staining of the plasma membrane with DiIC12 molecules, reflecting the insertion of DiIC12- labeled vesicles into the plasmalemma, could be observed at the growth cone (B). No plasma membrane staining was detected at the middle axon, as well as at sufficiently large distances from the growth cone in the distal segment (A and B). (C–E) Differential interference contrast and fluorescent micrographs of the distal axons 60 min after the staining of the cell body. Before the staining of the soma, neuronal cultures were pretreated for 1 h with brefeldin A (10 μg/ml, C), nocodazole (5 μg/ ml, D), or cytochalasin D (5 μM, E). The drugs were present in the culture medium throughout the experiment. Very few (C) or no (D) fluorescent vesicles and no plasma membrane staining (C and D) could be detected. Cytochalasin treatment (E) had no obvious effect on the delivery of the vesicles to the growth cone and their incorporation into the plasma membrane. Bars, 30 μm.

Mentions: Since no plasma membrane staining at the middle axonal segment could be detected (Fig. 4 A), the staining of the peripheral growth cone was not due to the diffusion of DiIC12 molecules from the soma along the plasmalemma. Hence, the diffuse staining of the growth cone reflected the fusion of DiIC12-labeled vesicles with the plasma membrane at the distal axon. To test this model, before the staining of the soma with DiIC12, we pretreated neuronal cultures for 1 h with brefeldin A (10 μg/ml), a drug that inhibits the supply of the new membrane to the axon (Lippincott-Schwartz et al., 1989; Jareb and Banker, 1997). As expected, the number of DiIC12-labeled vesicles at the distal axon was dramatically reduced, and no plasma membrane staining could be detected (Fig. 4 C). No fluorescent vesicles could be detected at the distal axon after the treatment of neuronal cultures with 5 μg/ml nocodazole, a drug that promotes MT disassembly (Fig. 4 D). This is consistent with the idea that the transport of the cell body– derived vesicles from the soma crucially depends on the integrity of axonal MTs. Cytochalasin D (5 μM), a drug that inhibits actin polymerization, had no obvious effect on organelle delivery to, and incorporation into, the distal axon (Fig. 4 E). Similar results were obtained with latrunculin A (5 μM), a drug that induces depolymerization of actin filaments (data not shown). To investigate whether the drugs used in this study induced significant disassembly or assembly of axonal MTs, we loaded fluorescently labeled tubulin into neurons by embryo injection (Reinch et al., 1991; Chang et al., 1998) and examined the neurons with fluorescent microscopy. Detergent extraction of the neurons in an MT-stabilizing buffer revealed that 76.6 ± 4.4% (mean ± SEM, data from 15 axons) of the total tubulin was in the polymer form. 1 h after the treatment of neuronal cultures with brefeldin A (10 μg/ml) or with cytochalasin D (5 μM), this value was not significantly different from control (Table I). On the contrary, 1 h after the treatment with nocodazole (5 μg/ml), the fraction of the tubulin in polymer form decreased to ∼11% (Table I).


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

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

Preferential insertion of cell body–derived vesicles into the growth cone region. (A and B) DIC (top)  and fluorescence (bottom)  images of the middle (A) and  distal (B) segments of the  same axon 30 min after staining of the cell body. The  length of the axon was  ∼1,600 μm, and the middle  segment was ∼900 μm away  from the soma. Staining of  the plasma membrane with  DiIC12 molecules, reflecting  the insertion of DiIC12- labeled vesicles into the plasmalemma, could be observed  at the growth cone (B). No  plasma membrane staining  was detected at the middle  axon, as well as at sufficiently  large distances from the  growth cone in the distal segment (A and B). (C–E) Differential interference contrast and fluorescent micrographs of the distal axons 60 min after the staining of the  cell body. Before the staining of the soma, neuronal cultures were pretreated for 1 h with brefeldin A (10 μg/ml, C), nocodazole (5 μg/ ml, D), or cytochalasin D (5 μM, E). The drugs were present in the culture medium throughout the experiment. Very few (C) or no (D)  fluorescent vesicles and no plasma membrane staining (C and D) could be detected. Cytochalasin treatment (E) had no obvious effect  on the delivery of the vesicles to the growth cone and their incorporation into the plasma membrane. Bars, 30 μm.
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Figure 4: Preferential insertion of cell body–derived vesicles into the growth cone region. (A and B) DIC (top) and fluorescence (bottom) images of the middle (A) and distal (B) segments of the same axon 30 min after staining of the cell body. The length of the axon was ∼1,600 μm, and the middle segment was ∼900 μm away from the soma. Staining of the plasma membrane with DiIC12 molecules, reflecting the insertion of DiIC12- labeled vesicles into the plasmalemma, could be observed at the growth cone (B). No plasma membrane staining was detected at the middle axon, as well as at sufficiently large distances from the growth cone in the distal segment (A and B). (C–E) Differential interference contrast and fluorescent micrographs of the distal axons 60 min after the staining of the cell body. Before the staining of the soma, neuronal cultures were pretreated for 1 h with brefeldin A (10 μg/ml, C), nocodazole (5 μg/ ml, D), or cytochalasin D (5 μM, E). The drugs were present in the culture medium throughout the experiment. Very few (C) or no (D) fluorescent vesicles and no plasma membrane staining (C and D) could be detected. Cytochalasin treatment (E) had no obvious effect on the delivery of the vesicles to the growth cone and their incorporation into the plasma membrane. Bars, 30 μm.
Mentions: Since no plasma membrane staining at the middle axonal segment could be detected (Fig. 4 A), the staining of the peripheral growth cone was not due to the diffusion of DiIC12 molecules from the soma along the plasmalemma. Hence, the diffuse staining of the growth cone reflected the fusion of DiIC12-labeled vesicles with the plasma membrane at the distal axon. To test this model, before the staining of the soma with DiIC12, we pretreated neuronal cultures for 1 h with brefeldin A (10 μg/ml), a drug that inhibits the supply of the new membrane to the axon (Lippincott-Schwartz et al., 1989; Jareb and Banker, 1997). As expected, the number of DiIC12-labeled vesicles at the distal axon was dramatically reduced, and no plasma membrane staining could be detected (Fig. 4 C). No fluorescent vesicles could be detected at the distal axon after the treatment of neuronal cultures with 5 μg/ml nocodazole, a drug that promotes MT disassembly (Fig. 4 D). This is consistent with the idea that the transport of the cell body– derived vesicles from the soma crucially depends on the integrity of axonal MTs. Cytochalasin D (5 μM), a drug that inhibits actin polymerization, had no obvious effect on organelle delivery to, and incorporation into, the distal axon (Fig. 4 E). Similar results were obtained with latrunculin A (5 μM), a drug that induces depolymerization of actin filaments (data not shown). To investigate whether the drugs used in this study induced significant disassembly or assembly of axonal MTs, we loaded fluorescently labeled tubulin into neurons by embryo injection (Reinch et al., 1991; Chang et al., 1998) and examined the neurons with fluorescent microscopy. Detergent extraction of the neurons in an MT-stabilizing buffer revealed that 76.6 ± 4.4% (mean ± SEM, data from 15 axons) of the total tubulin was in the polymer form. 1 h after the treatment of neuronal cultures with brefeldin A (10 μg/ml) or with cytochalasin D (5 μM), this value was not significantly different from control (Table I). On the contrary, 1 h after the treatment with nocodazole (5 μg/ml), the fraction of the tubulin in polymer form decreased to ∼11% (Table I).

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