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Visualization of the dynamics of synaptic vesicle and plasma membrane proteins in living axons.

Nakata T, Terada S, Hirokawa N - J. Cell Biol. (1998)

Bottom Line: Newly synthesized membrane proteins are transported by fast axonal flow to their targets such as the plasma membrane and synaptic vesicles.We found that all of these proteins are transported by tubulovesicular organelles of various sizes and shapes that circulate within axons from branch to branch and switch the direction of movement.These organelles are distinct from the endosomal compartments and constitute a new entity of membrane organelles that mediate the transport of newly synthesized proteins from the trans-Golgi network to the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Hongo, Tokyo, Japan, 113.

ABSTRACT
Newly synthesized membrane proteins are transported by fast axonal flow to their targets such as the plasma membrane and synaptic vesicles. However, their transporting vesicles have not yet been identified. We have successfully visualized the transporting vesicles of plasma membrane proteins, synaptic vesicle proteins, and the trans-Golgi network residual proteins in living axons at high resolution using laser scan microscopy of green fluorescent protein-tagged proteins after photobleaching. We found that all of these proteins are transported by tubulovesicular organelles of various sizes and shapes that circulate within axons from branch to branch and switch the direction of movement. These organelles are distinct from the endosomal compartments and constitute a new entity of membrane organelles that mediate the transport of newly synthesized proteins from the trans-Golgi network to the plasma membrane.

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Immunogold electron microscopy of GAP-43–GFP-transporting vesicles. The axons were incubated with antiflag antibody followed by 5-nm colloidal gold conjugated second antibody. pl and m in a indicate plasma membrane and mitochondria,  respectively. Arrows indicate some of the tubulovesicular organelles. Plasma membrane and tubular or vesicular organelles  were labeled with gold particles. (c) Noninfected DRG neuron as  a control. Only a few gold particles were seen. Bars, 0.2 μm.
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Figure 16: Immunogold electron microscopy of GAP-43–GFP-transporting vesicles. The axons were incubated with antiflag antibody followed by 5-nm colloidal gold conjugated second antibody. pl and m in a indicate plasma membrane and mitochondria, respectively. Arrows indicate some of the tubulovesicular organelles. Plasma membrane and tubular or vesicular organelles were labeled with gold particles. (c) Noninfected DRG neuron as a control. Only a few gold particles were seen. Bars, 0.2 μm.

Mentions: Because there was a discrepancy between the width of the tubulovesicular organelles observed in laser scan microscopy and electron microscopy, we next examined whether the GFP-fusion proteins are localized on the tubulovesicular organelles in electron microscopy. We designed GAP-43–GFP construct to contain flag epitope tag in its COOH terminus. Using antiflag monoclonal antibody, we performed immunogold electron microscopy of DRG neurons 40 h after infection. 5-nm colloidal gold stained the plasma membrane of the infected neurons (Fig. 16 a), consistent with the light microscopic observation (Fig. 2 d). In addition, tubulovesicular organelles were stained with the gold (Fig. 16, a and b), while areas enriched in cytoskeletons or mitochondria were not (Fig. 16 a). A few gold labels were observed in noninfected neurons (Fig. 16 c). The results indicate that the tubulovesicular organelles that were observed in the laser scan microscopy were identical to the tubulovesicular organelles observed in the electron microscopy (Figs. 15 and 16).


Visualization of the dynamics of synaptic vesicle and plasma membrane proteins in living axons.

Nakata T, Terada S, Hirokawa N - J. Cell Biol. (1998)

Immunogold electron microscopy of GAP-43–GFP-transporting vesicles. The axons were incubated with antiflag antibody followed by 5-nm colloidal gold conjugated second antibody. pl and m in a indicate plasma membrane and mitochondria,  respectively. Arrows indicate some of the tubulovesicular organelles. Plasma membrane and tubular or vesicular organelles  were labeled with gold particles. (c) Noninfected DRG neuron as  a control. Only a few gold particles were seen. Bars, 0.2 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140163&req=5

Figure 16: Immunogold electron microscopy of GAP-43–GFP-transporting vesicles. The axons were incubated with antiflag antibody followed by 5-nm colloidal gold conjugated second antibody. pl and m in a indicate plasma membrane and mitochondria, respectively. Arrows indicate some of the tubulovesicular organelles. Plasma membrane and tubular or vesicular organelles were labeled with gold particles. (c) Noninfected DRG neuron as a control. Only a few gold particles were seen. Bars, 0.2 μm.
Mentions: Because there was a discrepancy between the width of the tubulovesicular organelles observed in laser scan microscopy and electron microscopy, we next examined whether the GFP-fusion proteins are localized on the tubulovesicular organelles in electron microscopy. We designed GAP-43–GFP construct to contain flag epitope tag in its COOH terminus. Using antiflag monoclonal antibody, we performed immunogold electron microscopy of DRG neurons 40 h after infection. 5-nm colloidal gold stained the plasma membrane of the infected neurons (Fig. 16 a), consistent with the light microscopic observation (Fig. 2 d). In addition, tubulovesicular organelles were stained with the gold (Fig. 16, a and b), while areas enriched in cytoskeletons or mitochondria were not (Fig. 16 a). A few gold labels were observed in noninfected neurons (Fig. 16 c). The results indicate that the tubulovesicular organelles that were observed in the laser scan microscopy were identical to the tubulovesicular organelles observed in the electron microscopy (Figs. 15 and 16).

Bottom Line: Newly synthesized membrane proteins are transported by fast axonal flow to their targets such as the plasma membrane and synaptic vesicles.We found that all of these proteins are transported by tubulovesicular organelles of various sizes and shapes that circulate within axons from branch to branch and switch the direction of movement.These organelles are distinct from the endosomal compartments and constitute a new entity of membrane organelles that mediate the transport of newly synthesized proteins from the trans-Golgi network to the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Hongo, Tokyo, Japan, 113.

ABSTRACT
Newly synthesized membrane proteins are transported by fast axonal flow to their targets such as the plasma membrane and synaptic vesicles. However, their transporting vesicles have not yet been identified. We have successfully visualized the transporting vesicles of plasma membrane proteins, synaptic vesicle proteins, and the trans-Golgi network residual proteins in living axons at high resolution using laser scan microscopy of green fluorescent protein-tagged proteins after photobleaching. We found that all of these proteins are transported by tubulovesicular organelles of various sizes and shapes that circulate within axons from branch to branch and switch the direction of movement. These organelles are distinct from the endosomal compartments and constitute a new entity of membrane organelles that mediate the transport of newly synthesized proteins from the trans-Golgi network to the plasma membrane.

Show MeSH
Related in: MedlinePlus