Limits...
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.

Show MeSH

Related in: MedlinePlus

The tubular structure identified here was not  a mitochondrion. Neurons  were transfected with adenovirus vectors carrying GAP-43–GFP chimeric DNA after  3 h in culture and were observed using a confocal laser  scan microscope 40 h later.  15 min before observation  Mitotracker was added to the  medium for vital staining of  mitochondria. The medium  was changed just before observation. The axon was observed in both FITC mode  and Texas red mode simultaneously, and the movements of both  GAP-43 transporting vesicles (a and c) and mitochondria (b and  d) were recorded. a–d are the simultaneous double labeling, and  interval between a and b and c and d is 17.25 s. The upper side is  proximal to the cell body. The central area of the axon was photobleached before observation. Note that transporting vesicles  cannot be identified in the peripheral area of the axon because of  the high level of background staining. The GAP-43–containing  vesicles identified in the central area were transported from the  peripheral area during observation. Most of the mitochondria  were stationary and only a few moved into the central region (arrows). Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2140163&req=5

Figure 7: The tubular structure identified here was not a mitochondrion. Neurons were transfected with adenovirus vectors carrying GAP-43–GFP chimeric DNA after 3 h in culture and were observed using a confocal laser scan microscope 40 h later. 15 min before observation Mitotracker was added to the medium for vital staining of mitochondria. The medium was changed just before observation. The axon was observed in both FITC mode and Texas red mode simultaneously, and the movements of both GAP-43 transporting vesicles (a and c) and mitochondria (b and d) were recorded. a–d are the simultaneous double labeling, and interval between a and b and c and d is 17.25 s. The upper side is proximal to the cell body. The central area of the axon was photobleached before observation. Note that transporting vesicles cannot be identified in the peripheral area of the axon because of the high level of background staining. The GAP-43–containing vesicles identified in the central area were transported from the peripheral area during observation. Most of the mitochondria were stationary and only a few moved into the central region (arrows). Bar, 5 μm.

Mentions: We found that the transporting vesicles varied in shape, from tubules to spheres in the axons of GAP-43– or SNAP-25–transfected neurons (see Figs. 5–11). We checked that the observed tubular structures were not mitochondria by double labeling of DRG axons with GFP and Mitotracker. We found that the tubulovesicular organelles were distinct from mitochondria (Fig. 7). These vesicles moved mainly in the anterograde direction at an average speed of 0.76 ± 0.26 μm/s. The speed was independent of vesicle size and was distributed ∼0.9 μm/s with a single peak (see Fig. 9 b). The size distribution of these vesicles varied between neurons. The shape of the vesicles was flexible: for example, tubular vesicles were more elongated in the longitudinal direction when they started to move, and bent when they changed their direction of movement. We often found that tubular vesicles broke into smaller vesicles while they were moving down in axon (Fig. 8 d). Judging from the movement and the elongated shape of the tubular vesicles before they broke down, it was unlikely that the tubular vesicles had consisted of several small independent vesicles. Furthermore, in some neurons, the longitudinal size of vesicles decreased as the vesicles proceeded in the same axons (Fig. 8, a and b). For example, it was 1.71 ± 0.94 μm in length and 0.55 ± 0.22 μm in width in the proximal axon (∼40 μm from the cell body), and 1.16 ± 0.68 μm in length in the distal part (∼110 μm from the cell body) of the same axon (Fig. 9 a). As shown in Fig. 6 a and b, the fluorescent intensity of each vesicle in the distal axon is not as high as that in the proximal axons. This eliminates the possibility that long tubules are simply compressed into small vesicles, because total fluorescence intensity of the vesicles will be unchanged in such case. These results suggest that tubular vesicles divided into smaller vesicles while 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)

The tubular structure identified here was not  a mitochondrion. Neurons  were transfected with adenovirus vectors carrying GAP-43–GFP chimeric DNA after  3 h in culture and were observed using a confocal laser  scan microscope 40 h later.  15 min before observation  Mitotracker was added to the  medium for vital staining of  mitochondria. The medium  was changed just before observation. The axon was observed in both FITC mode  and Texas red mode simultaneously, and the movements of both  GAP-43 transporting vesicles (a and c) and mitochondria (b and  d) were recorded. a–d are the simultaneous double labeling, and  interval between a and b and c and d is 17.25 s. The upper side is  proximal to the cell body. The central area of the axon was photobleached before observation. Note that transporting vesicles  cannot be identified in the peripheral area of the axon because of  the high level of background staining. The GAP-43–containing  vesicles identified in the central area were transported from the  peripheral area during observation. Most of the mitochondria  were stationary and only a few moved into the central region (arrows). Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: The tubular structure identified here was not a mitochondrion. Neurons were transfected with adenovirus vectors carrying GAP-43–GFP chimeric DNA after 3 h in culture and were observed using a confocal laser scan microscope 40 h later. 15 min before observation Mitotracker was added to the medium for vital staining of mitochondria. The medium was changed just before observation. The axon was observed in both FITC mode and Texas red mode simultaneously, and the movements of both GAP-43 transporting vesicles (a and c) and mitochondria (b and d) were recorded. a–d are the simultaneous double labeling, and interval between a and b and c and d is 17.25 s. The upper side is proximal to the cell body. The central area of the axon was photobleached before observation. Note that transporting vesicles cannot be identified in the peripheral area of the axon because of the high level of background staining. The GAP-43–containing vesicles identified in the central area were transported from the peripheral area during observation. Most of the mitochondria were stationary and only a few moved into the central region (arrows). Bar, 5 μm.
Mentions: We found that the transporting vesicles varied in shape, from tubules to spheres in the axons of GAP-43– or SNAP-25–transfected neurons (see Figs. 5–11). We checked that the observed tubular structures were not mitochondria by double labeling of DRG axons with GFP and Mitotracker. We found that the tubulovesicular organelles were distinct from mitochondria (Fig. 7). These vesicles moved mainly in the anterograde direction at an average speed of 0.76 ± 0.26 μm/s. The speed was independent of vesicle size and was distributed ∼0.9 μm/s with a single peak (see Fig. 9 b). The size distribution of these vesicles varied between neurons. The shape of the vesicles was flexible: for example, tubular vesicles were more elongated in the longitudinal direction when they started to move, and bent when they changed their direction of movement. We often found that tubular vesicles broke into smaller vesicles while they were moving down in axon (Fig. 8 d). Judging from the movement and the elongated shape of the tubular vesicles before they broke down, it was unlikely that the tubular vesicles had consisted of several small independent vesicles. Furthermore, in some neurons, the longitudinal size of vesicles decreased as the vesicles proceeded in the same axons (Fig. 8, a and b). For example, it was 1.71 ± 0.94 μm in length and 0.55 ± 0.22 μm in width in the proximal axon (∼40 μm from the cell body), and 1.16 ± 0.68 μm in length in the distal part (∼110 μm from the cell body) of the same axon (Fig. 9 a). As shown in Fig. 6 a and b, the fluorescent intensity of each vesicle in the distal axon is not as high as that in the proximal axons. This eliminates the possibility that long tubules are simply compressed into small vesicles, because total fluorescence intensity of the vesicles will be unchanged in such case. These results suggest that tubular vesicles divided into smaller vesicles while 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