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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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Tubulovesicular organelles were distinct from endosomes. TrkA was transported by both tubulovesicular organelles  and endosomes, but GAP-43 was transported only by tubulovesicular organelles. (a and b) Double labeling with trkA–GFP (a)  and Texas red–dextran (b). The upper side is proximal to the cell  body. Tubulovesicular organelles from the proximal axon were  labeled only by GFP. Retrogradely moving large globular vesicles were labeled by both GFP and Texas red–dextran (arrows).  Note that some vesicles were labeled by Texas red–dextran but  not GFP. Bright stainings outside of the cell (*) in b were aggregates of Texas red–dextran attached to the coverslip. (c and d)  Double labeling with GAP-43–GFP (c) and Texas red–dextran  (d). Upper-right side is proximal to the cell body. The axon bifurcates, and the upper left side and lower left side are distal to the  cell body. Note that endosomes were labeled by Texas red– dextran (arrows) but not by GFP, whereas tubulovesicular organelles were labeled only by GFP. Bars, 5 μm.
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Figure 10: Tubulovesicular organelles were distinct from endosomes. TrkA was transported by both tubulovesicular organelles and endosomes, but GAP-43 was transported only by tubulovesicular organelles. (a and b) Double labeling with trkA–GFP (a) and Texas red–dextran (b). The upper side is proximal to the cell body. Tubulovesicular organelles from the proximal axon were labeled only by GFP. Retrogradely moving large globular vesicles were labeled by both GFP and Texas red–dextran (arrows). Note that some vesicles were labeled by Texas red–dextran but not GFP. Bright stainings outside of the cell (*) in b were aggregates of Texas red–dextran attached to the coverslip. (c and d) Double labeling with GAP-43–GFP (c) and Texas red–dextran (d). Upper-right side is proximal to the cell body. The axon bifurcates, and the upper left side and lower left side are distal to the cell body. Note that endosomes were labeled by Texas red– dextran (arrows) but not by GFP, whereas tubulovesicular organelles were labeled only by GFP. Bars, 5 μm.

Mentions: To further confirm that the retrograde tubulovesiclar organelles are not endosomes, we performed double labeling experiments with a membrane protein–GFP fusion protein and Texas red–dextran. If our interpretation is correct, the tubulovesicular organelles that transport GAP-43 should not be labeled with Texas red–dextran. In contrast, the cell surface receptor proteins that are endocytosed upon binding to their ligands should be transported by two types of vesicle: tubulovesicular organelles and endosomes. TrkA is a high affinity receptor for NGF (Klein et al., 1991) and is endocytosed on binding to NGF (Ehlers et al., 1995). We found that trkA was transported by both tubulovesicular organelles and endosomes by double labeling with trkA–GFP and Texas red–dextran (Fig. 10, a and b). Tubulovesicular organelles were not labeled with Texas red–dextran while large globular vesicles were double labeled by GFP and Texas red–dextran. This was in clear contrast with the GAP-43–transporting vesicles that consisted of tubulovesicular organelles only and were not stained by Texas red–dextran (Fig. 10, c and d).


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

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

Tubulovesicular organelles were distinct from endosomes. TrkA was transported by both tubulovesicular organelles  and endosomes, but GAP-43 was transported only by tubulovesicular organelles. (a and b) Double labeling with trkA–GFP (a)  and Texas red–dextran (b). The upper side is proximal to the cell  body. Tubulovesicular organelles from the proximal axon were  labeled only by GFP. Retrogradely moving large globular vesicles were labeled by both GFP and Texas red–dextran (arrows).  Note that some vesicles were labeled by Texas red–dextran but  not GFP. Bright stainings outside of the cell (*) in b were aggregates of Texas red–dextran attached to the coverslip. (c and d)  Double labeling with GAP-43–GFP (c) and Texas red–dextran  (d). Upper-right side is proximal to the cell body. The axon bifurcates, and the upper left side and lower left side are distal to the  cell body. Note that endosomes were labeled by Texas red– dextran (arrows) but not by GFP, whereas tubulovesicular organelles were labeled only by GFP. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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Figure 10: Tubulovesicular organelles were distinct from endosomes. TrkA was transported by both tubulovesicular organelles and endosomes, but GAP-43 was transported only by tubulovesicular organelles. (a and b) Double labeling with trkA–GFP (a) and Texas red–dextran (b). The upper side is proximal to the cell body. Tubulovesicular organelles from the proximal axon were labeled only by GFP. Retrogradely moving large globular vesicles were labeled by both GFP and Texas red–dextran (arrows). Note that some vesicles were labeled by Texas red–dextran but not GFP. Bright stainings outside of the cell (*) in b were aggregates of Texas red–dextran attached to the coverslip. (c and d) Double labeling with GAP-43–GFP (c) and Texas red–dextran (d). Upper-right side is proximal to the cell body. The axon bifurcates, and the upper left side and lower left side are distal to the cell body. Note that endosomes were labeled by Texas red– dextran (arrows) but not by GFP, whereas tubulovesicular organelles were labeled only by GFP. Bars, 5 μm.
Mentions: To further confirm that the retrograde tubulovesiclar organelles are not endosomes, we performed double labeling experiments with a membrane protein–GFP fusion protein and Texas red–dextran. If our interpretation is correct, the tubulovesicular organelles that transport GAP-43 should not be labeled with Texas red–dextran. In contrast, the cell surface receptor proteins that are endocytosed upon binding to their ligands should be transported by two types of vesicle: tubulovesicular organelles and endosomes. TrkA is a high affinity receptor for NGF (Klein et al., 1991) and is endocytosed on binding to NGF (Ehlers et al., 1995). We found that trkA was transported by both tubulovesicular organelles and endosomes by double labeling with trkA–GFP and Texas red–dextran (Fig. 10, a and b). Tubulovesicular organelles were not labeled with Texas red–dextran while large globular vesicles were double labeled by GFP and Texas red–dextran. This was in clear contrast with the GAP-43–transporting vesicles that consisted of tubulovesicular organelles only and were not stained by Texas red–dextran (Fig. 10, c and d).

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