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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: 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.

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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Correlative electron microscopy of  trkA–GFP-transporting vesicles. The axon  was photobleached and moving vesicles were  observed in the living cell (a). Then the axon  was again photobleached and fixed with 2%  paraformaldehyde and 0.1% glutaraldehyde.  Numerous anterogradely moving vesicles as  well as a retrogradely moving large vesicle  (arrow) moved into the area before fixation  (b). (c) The same area of the axon was prepared for electron microscopy. Arrow in c indicates the corresponding endosome in b. (d  and e) Higher magnification of c. Numerous  tubules and vesicles were observed (arrows).  Bars: (c) 10 μm; (d and e) 1 μm.
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Figure 15: Correlative electron microscopy of trkA–GFP-transporting vesicles. The axon was photobleached and moving vesicles were observed in the living cell (a). Then the axon was again photobleached and fixed with 2% paraformaldehyde and 0.1% glutaraldehyde. Numerous anterogradely moving vesicles as well as a retrogradely moving large vesicle (arrow) moved into the area before fixation (b). (c) The same area of the axon was prepared for electron microscopy. Arrow in c indicates the corresponding endosome in b. (d and e) Higher magnification of c. Numerous tubules and vesicles were observed (arrows). Bars: (c) 10 μm; (d and e) 1 μm.

Mentions: To correlate the tubulovesicular organelles observed by laser scan microscopy with those observed by electron microscopy, we examined by electron microscopy the area of an axon in which we had observed extensive vesicle transport. Fig. 15 a shows the numerous transporting vesicles that moved into the photobleached area of a living axon. Fig. 15 shows the same axon after the axon was photobleached again and the medium was changed to the fixative, showing that the transporting vesicles moved into the photobleached area before fixation. Fig. 15 c shows an electron micrograph of the area corresponding precisely to that in Fig. 15 b. This micrograph shows that photobleaching did not damage the fine structure of the axon. Observation at higher magnification revealed numerous tubular or vesicular organelles in the area that correspond to the transporting vesicles observed by laser scan microscopy in number and shapes (Fig. 15, d and e, arrows). However, the widths of the vesicles observed by electron microscopy were smaller than the vesicles observed by laser scan microscopy. Presumably, this is due to the fact that a brightly fluorescent object has a halo that makes the object appear bigger than its actual size. For example, microtubules are 24 nm in diameter, but they appear much wider under fluorescence microscopy. In fact, no other membrane structures that may correspond to the light microscopic image were observed in the electron micrographs. Thus the actual diameter of most of the tubulovesicular organelles will be <0.1 μm. In contrast, the length of tubular vesicles may well reflect on the real length of the vesicles, because the errors arising due to the fluorescent halos are assumed to be the same in both the longitudinal and lateral directions. In fact, long tubules were previously identified in electron microscopic studies of axons (Lindsley and Ellisman, 1985). Whether these tubules were derived from one continuous system from the cell body to the terminal or a system of discontinuous components that move within the axon is a matter of debate (Peters et al., 1991). In this study, we found tubules as long as 25 μm moving down axons.


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

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

Correlative electron microscopy of  trkA–GFP-transporting vesicles. The axon  was photobleached and moving vesicles were  observed in the living cell (a). Then the axon  was again photobleached and fixed with 2%  paraformaldehyde and 0.1% glutaraldehyde.  Numerous anterogradely moving vesicles as  well as a retrogradely moving large vesicle  (arrow) moved into the area before fixation  (b). (c) The same area of the axon was prepared for electron microscopy. Arrow in c indicates the corresponding endosome in b. (d  and e) Higher magnification of c. Numerous  tubules and vesicles were observed (arrows).  Bars: (c) 10 μm; (d and e) 1 μm.
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Related In: Results  -  Collection

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Figure 15: Correlative electron microscopy of trkA–GFP-transporting vesicles. The axon was photobleached and moving vesicles were observed in the living cell (a). Then the axon was again photobleached and fixed with 2% paraformaldehyde and 0.1% glutaraldehyde. Numerous anterogradely moving vesicles as well as a retrogradely moving large vesicle (arrow) moved into the area before fixation (b). (c) The same area of the axon was prepared for electron microscopy. Arrow in c indicates the corresponding endosome in b. (d and e) Higher magnification of c. Numerous tubules and vesicles were observed (arrows). Bars: (c) 10 μm; (d and e) 1 μm.
Mentions: To correlate the tubulovesicular organelles observed by laser scan microscopy with those observed by electron microscopy, we examined by electron microscopy the area of an axon in which we had observed extensive vesicle transport. Fig. 15 a shows the numerous transporting vesicles that moved into the photobleached area of a living axon. Fig. 15 shows the same axon after the axon was photobleached again and the medium was changed to the fixative, showing that the transporting vesicles moved into the photobleached area before fixation. Fig. 15 c shows an electron micrograph of the area corresponding precisely to that in Fig. 15 b. This micrograph shows that photobleaching did not damage the fine structure of the axon. Observation at higher magnification revealed numerous tubular or vesicular organelles in the area that correspond to the transporting vesicles observed by laser scan microscopy in number and shapes (Fig. 15, d and e, arrows). However, the widths of the vesicles observed by electron microscopy were smaller than the vesicles observed by laser scan microscopy. Presumably, this is due to the fact that a brightly fluorescent object has a halo that makes the object appear bigger than its actual size. For example, microtubules are 24 nm in diameter, but they appear much wider under fluorescence microscopy. In fact, no other membrane structures that may correspond to the light microscopic image were observed in the electron micrographs. Thus the actual diameter of most of the tubulovesicular organelles will be <0.1 μm. In contrast, the length of tubular vesicles may well reflect on the real length of the vesicles, because the errors arising due to the fluorescent halos are assumed to be the same in both the longitudinal and lateral directions. In fact, long tubules were previously identified in electron microscopic studies of axons (Lindsley and Ellisman, 1985). Whether these tubules were derived from one continuous system from the cell body to the terminal or a system of discontinuous components that move within the axon is a matter of debate (Peters et al., 1991). In this study, we found tubules as long as 25 μm moving down axons.

Bottom Line: 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.

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