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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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Staining of cell  body–derived vesicles with  the fluorescent lipid analogue DiIC12. (A) Schematic  representation of experimental approach. The cell body  of a neuron was locally labeled with DiIC12 molecules  using a pair of perfusion pipettes. One pipette was used  for the delivery of DiIC12-containing solution to the  soma region, and another for  its removal. The pipettes  were withdrawn from the  soma 30–60 s after the onset  of perfusion. (B) Fluorescence images of the soma  and the proximal axon at two  different times (marked in  minutes) after DiIC12 incorporation into the plasmalemma, and corresponding  profiles of fluorescence intensities (arbitrary units)  along the axon. (C) Differential interference contrast  (top) and fluorescence images of the axonal segment  located 0.7 mm from the cell  body 15 min after local labeling of the soma. No plasma membrane staining could be detected. The arrows track two rapidly moving fluorescent organelles (time in  seconds). (D) The distribution of velocities of the DiIC12-labeled organelles. 137 organelles were chosen randomly. Measurements of  the rates of organelle movement were made by determining the displacement of the vesicles for a period of 60 s. All vesicles moved anterogradely. Bars: (B) 100 μm; (C) 25 μm.
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Figure 1: Staining of cell body–derived vesicles with the fluorescent lipid analogue DiIC12. (A) Schematic representation of experimental approach. The cell body of a neuron was locally labeled with DiIC12 molecules using a pair of perfusion pipettes. One pipette was used for the delivery of DiIC12-containing solution to the soma region, and another for its removal. The pipettes were withdrawn from the soma 30–60 s after the onset of perfusion. (B) Fluorescence images of the soma and the proximal axon at two different times (marked in minutes) after DiIC12 incorporation into the plasmalemma, and corresponding profiles of fluorescence intensities (arbitrary units) along the axon. (C) Differential interference contrast (top) and fluorescence images of the axonal segment located 0.7 mm from the cell body 15 min after local labeling of the soma. No plasma membrane staining could be detected. The arrows track two rapidly moving fluorescent organelles (time in seconds). (D) The distribution of velocities of the DiIC12-labeled organelles. 137 organelles were chosen randomly. Measurements of the rates of organelle movement were made by determining the displacement of the vesicles for a period of 60 s. All vesicles moved anterogradely. Bars: (B) 100 μm; (C) 25 μm.

Mentions: We used a local superfusion technique (Popov et al., 1993; Engert and Bonhoeffer, 1997) to stain the plasma membrane at the cell body with fluorescent lipid analogue DiIC12 (Fig. 1 A). Within 2–3 min after the onset of perfusion, the soma was brightly labeled with DiIC12 molecules. Initially, the staining was largely localized to the cell body. With time, plasma membrane staining could be detected at progressively greater distances from the soma (Fig. 1 B), consistent with lateral diffusion of DiIC12 molecules along the axonal plasmalemma (Popov et al., 1993). In the first 20 min after cell body labeling, no plasma membrane staining was observed at the distal axonal segment, in agreement with the rapid drop in the rate of diffusional transport with increasing distance. However, fluorescent microscopy of the axon ∼1 mm from the soma, well beyond the reach of diffusional transport, revealed brightly stained tubovesicular organelles (Fig. 1 C). As judged by the fluorescent microscopy, these organelles ranged from ∼0.2 μm (the diffraction limit of the light microscopy) to ∼4 μm in length. The DiIC12-stained organelles could be detected with a delay of ∼10–15 min after the onset of soma labeling. The organelles were transported in an anterograde direction at the rate of 2.93 ± 0.78 μm/s (mean ± SEM of 137 vesicles in 15 neurons), in agreement with previously reported rates of the fast axonal transport (Allen et al., 1982; Nakata et al., 1998). Occasionally, we observed some “hesitation” in this anterograde transport; however, none of the organelles changed direction of movement (total of 1,578 vesicles in 26 neurons; average observation distance ∼150 μm). This result suggests that the polarity of axonal MTs in growing Xenopus neurites is uniform. Staining of neuronal cultures with rhodamine-123, a mitochondria-specific fluorescent dye, indicated that the movement of mitochondria from the soma to the axon was a very rare event. The majority of mitochondria frequently changed their direction of movement and did not translocate over long distances along the axon (data not shown). Moreover, DiIC12-stained tubovesicular organelles were able to fuse with the plasmalemma (see below). Accordingly, we conclude that the overwhelming majority of DiIC12-stained vesicles detected along the axon are distinct from mitochondria.


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

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

Staining of cell  body–derived vesicles with  the fluorescent lipid analogue DiIC12. (A) Schematic  representation of experimental approach. The cell body  of a neuron was locally labeled with DiIC12 molecules  using a pair of perfusion pipettes. One pipette was used  for the delivery of DiIC12-containing solution to the  soma region, and another for  its removal. The pipettes  were withdrawn from the  soma 30–60 s after the onset  of perfusion. (B) Fluorescence images of the soma  and the proximal axon at two  different times (marked in  minutes) after DiIC12 incorporation into the plasmalemma, and corresponding  profiles of fluorescence intensities (arbitrary units)  along the axon. (C) Differential interference contrast  (top) and fluorescence images of the axonal segment  located 0.7 mm from the cell  body 15 min after local labeling of the soma. No plasma membrane staining could be detected. The arrows track two rapidly moving fluorescent organelles (time in  seconds). (D) The distribution of velocities of the DiIC12-labeled organelles. 137 organelles were chosen randomly. Measurements of  the rates of organelle movement were made by determining the displacement of the vesicles for a period of 60 s. All vesicles moved anterogradely. Bars: (B) 100 μm; (C) 25 μm.
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Figure 1: Staining of cell body–derived vesicles with the fluorescent lipid analogue DiIC12. (A) Schematic representation of experimental approach. The cell body of a neuron was locally labeled with DiIC12 molecules using a pair of perfusion pipettes. One pipette was used for the delivery of DiIC12-containing solution to the soma region, and another for its removal. The pipettes were withdrawn from the soma 30–60 s after the onset of perfusion. (B) Fluorescence images of the soma and the proximal axon at two different times (marked in minutes) after DiIC12 incorporation into the plasmalemma, and corresponding profiles of fluorescence intensities (arbitrary units) along the axon. (C) Differential interference contrast (top) and fluorescence images of the axonal segment located 0.7 mm from the cell body 15 min after local labeling of the soma. No plasma membrane staining could be detected. The arrows track two rapidly moving fluorescent organelles (time in seconds). (D) The distribution of velocities of the DiIC12-labeled organelles. 137 organelles were chosen randomly. Measurements of the rates of organelle movement were made by determining the displacement of the vesicles for a period of 60 s. All vesicles moved anterogradely. Bars: (B) 100 μm; (C) 25 μm.
Mentions: We used a local superfusion technique (Popov et al., 1993; Engert and Bonhoeffer, 1997) to stain the plasma membrane at the cell body with fluorescent lipid analogue DiIC12 (Fig. 1 A). Within 2–3 min after the onset of perfusion, the soma was brightly labeled with DiIC12 molecules. Initially, the staining was largely localized to the cell body. With time, plasma membrane staining could be detected at progressively greater distances from the soma (Fig. 1 B), consistent with lateral diffusion of DiIC12 molecules along the axonal plasmalemma (Popov et al., 1993). In the first 20 min after cell body labeling, no plasma membrane staining was observed at the distal axonal segment, in agreement with the rapid drop in the rate of diffusional transport with increasing distance. However, fluorescent microscopy of the axon ∼1 mm from the soma, well beyond the reach of diffusional transport, revealed brightly stained tubovesicular organelles (Fig. 1 C). As judged by the fluorescent microscopy, these organelles ranged from ∼0.2 μm (the diffraction limit of the light microscopy) to ∼4 μm in length. The DiIC12-stained organelles could be detected with a delay of ∼10–15 min after the onset of soma labeling. The organelles were transported in an anterograde direction at the rate of 2.93 ± 0.78 μm/s (mean ± SEM of 137 vesicles in 15 neurons), in agreement with previously reported rates of the fast axonal transport (Allen et al., 1982; Nakata et al., 1998). Occasionally, we observed some “hesitation” in this anterograde transport; however, none of the organelles changed direction of movement (total of 1,578 vesicles in 26 neurons; average observation distance ∼150 μm). This result suggests that the polarity of axonal MTs in growing Xenopus neurites is uniform. Staining of neuronal cultures with rhodamine-123, a mitochondria-specific fluorescent dye, indicated that the movement of mitochondria from the soma to the axon was a very rare event. The majority of mitochondria frequently changed their direction of movement and did not translocate over long distances along the axon (data not shown). Moreover, DiIC12-stained tubovesicular organelles were able to fuse with the plasmalemma (see below). Accordingly, we conclude that the overwhelming majority of DiIC12-stained vesicles detected along the axon are distinct from mitochondria.

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