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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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Movements of several representative transporting vesicles of GAP-43 (A),  and synaptophysin (B, tubulovesicular organelles, and C,  large globular vesicles that  colocalize with FM1-43 or  Texas red–dextran). Anterograde movement (from the cell  body to the periphery) was set  as plus direction. The net  translocation (μm) of each  vesicle from the starting point  was plotted against the time  (s). Intervals between the  frames were 3.3 s. Comparison  with the tubulovesicular organelles that transport either GAP-43 (A) or synaptophysin (B) revealed that the large globular vesicles often change the direction of the movement (C). Nevertheless, the large globular vesicles have a tendency to move retrogradely.
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Figure 13: Movements of several representative transporting vesicles of GAP-43 (A), and synaptophysin (B, tubulovesicular organelles, and C, large globular vesicles that colocalize with FM1-43 or Texas red–dextran). Anterograde movement (from the cell body to the periphery) was set as plus direction. The net translocation (μm) of each vesicle from the starting point was plotted against the time (s). Intervals between the frames were 3.3 s. Comparison with the tubulovesicular organelles that transport either GAP-43 (A) or synaptophysin (B) revealed that the large globular vesicles often change the direction of the movement (C). Nevertheless, the large globular vesicles have a tendency to move retrogradely.

Mentions: We found two types of organelles that moved in synaptophysin–GFP-transfected cells (Fig. 12, a and b): first, tubulovesicular organelles similar to those transporting plasma membrane proteins in size, shape (1.48 ± 1.01 μm in longitudinal length, 0.46 ± 0.13 μm in width, see Table I), speed (0.69 ± 0.33 μm/s), and behavior in axons; and second, organelles that were much wider and in most cases globular in shape (1.33 ± 0.48 μm in length, and 0.89 ± 0.26 μm in width), more intensely stained with GFP (fluorescence intensity of tubulovesicular organelles was 105 ± 22 [arbitrary units], whereas that of large globular vesicles was 240 ± 21.1), but fewer in number (in most cases, the number of tubulovesicular organelles was 8–14 times higher than that of the large globular vesicles) than the tubulovesicular organelles. They changed their direction of movement more frequently than did the tubulovesicular organelles (25% of large vesicles stop or change their direction of movement within three successive frames [6.9 s], whereas only 3% of tubulovesicular organelles stop or change their direction), and accordingly, their movement was bidirectional, but the net transport was retrograde (Fig. 13).


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

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

Movements of several representative transporting vesicles of GAP-43 (A),  and synaptophysin (B, tubulovesicular organelles, and C,  large globular vesicles that  colocalize with FM1-43 or  Texas red–dextran). Anterograde movement (from the cell  body to the periphery) was set  as plus direction. The net  translocation (μm) of each  vesicle from the starting point  was plotted against the time  (s). Intervals between the  frames were 3.3 s. Comparison  with the tubulovesicular organelles that transport either GAP-43 (A) or synaptophysin (B) revealed that the large globular vesicles often change the direction of the movement (C). Nevertheless, the large globular vesicles have a tendency to move retrogradely.
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Related In: Results  -  Collection

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Figure 13: Movements of several representative transporting vesicles of GAP-43 (A), and synaptophysin (B, tubulovesicular organelles, and C, large globular vesicles that colocalize with FM1-43 or Texas red–dextran). Anterograde movement (from the cell body to the periphery) was set as plus direction. The net translocation (μm) of each vesicle from the starting point was plotted against the time (s). Intervals between the frames were 3.3 s. Comparison with the tubulovesicular organelles that transport either GAP-43 (A) or synaptophysin (B) revealed that the large globular vesicles often change the direction of the movement (C). Nevertheless, the large globular vesicles have a tendency to move retrogradely.
Mentions: We found two types of organelles that moved in synaptophysin–GFP-transfected cells (Fig. 12, a and b): first, tubulovesicular organelles similar to those transporting plasma membrane proteins in size, shape (1.48 ± 1.01 μm in longitudinal length, 0.46 ± 0.13 μm in width, see Table I), speed (0.69 ± 0.33 μm/s), and behavior in axons; and second, organelles that were much wider and in most cases globular in shape (1.33 ± 0.48 μm in length, and 0.89 ± 0.26 μm in width), more intensely stained with GFP (fluorescence intensity of tubulovesicular organelles was 105 ± 22 [arbitrary units], whereas that of large globular vesicles was 240 ± 21.1), but fewer in number (in most cases, the number of tubulovesicular organelles was 8–14 times higher than that of the large globular vesicles) than the tubulovesicular organelles. They changed their direction of movement more frequently than did the tubulovesicular organelles (25% of large vesicles stop or change their direction of movement within three successive frames [6.9 s], whereas only 3% of tubulovesicular organelles stop or change their direction), and accordingly, their movement was bidirectional, but the net transport was retrograde (Fig. 13).

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