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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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Axonal transport of vesicles containing GAP-43–GFP  chimeric protein in mouse DRG neurons. Neurons were transfected with adenovirus vectors carrying GAP-43–GFP chimeric  DNA after 3 h in culture and were observed using a laser scan microscope 40 h later. Note that numerous vesicles of various sizes  were transported from the proximal side of the axon. (a and b)  Proximal and distal area of the same axon. a shows an axon ∼40  μm from the cell body, b shows the same axon ∼110 μm from the  cell body. The upper side of both micrographs is proximal to the  cell body. Note the difference in the sizes of GAP-43–transporting vesicles even though the degree of magnification of both figures is the same. (c and d) Time course images of the underlined  area of the axon in a. The intervals between frames are 3.45 s. The  upper side of both micrographs is proximal to the cell body. In c,  tubular and spherical vesicles of various sizes and shapes are  moving. The vesicle indicated by diamonds showed representative continuous anterograde movement. The vesicle indicated by  arrowheads switched back to retrograde movement, and again  switched direction anterogradely. In d, the vesicle indicated by  stars broke down into smaller spheres while moving anterogradely in the axon. Bars: (b and d) 5 μm.
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Figure 8: Axonal transport of vesicles containing GAP-43–GFP chimeric protein in mouse DRG neurons. Neurons were transfected with adenovirus vectors carrying GAP-43–GFP chimeric DNA after 3 h in culture and were observed using a laser scan microscope 40 h later. Note that numerous vesicles of various sizes were transported from the proximal side of the axon. (a and b) Proximal and distal area of the same axon. a shows an axon ∼40 μm from the cell body, b shows the same axon ∼110 μm from the cell body. The upper side of both micrographs is proximal to the cell body. Note the difference in the sizes of GAP-43–transporting vesicles even though the degree of magnification of both figures is the same. (c and d) Time course images of the underlined area of the axon in a. The intervals between frames are 3.45 s. The upper side of both micrographs is proximal to the cell body. In c, tubular and spherical vesicles of various sizes and shapes are moving. The vesicle indicated by diamonds showed representative continuous anterograde movement. The vesicle indicated by arrowheads switched back to retrograde movement, and again switched direction anterogradely. In d, the vesicle indicated by stars broke down into smaller spheres while moving anterogradely in the axon. Bars: (b and d) 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)

Axonal transport of vesicles containing GAP-43–GFP  chimeric protein in mouse DRG neurons. Neurons were transfected with adenovirus vectors carrying GAP-43–GFP chimeric  DNA after 3 h in culture and were observed using a laser scan microscope 40 h later. Note that numerous vesicles of various sizes  were transported from the proximal side of the axon. (a and b)  Proximal and distal area of the same axon. a shows an axon ∼40  μm from the cell body, b shows the same axon ∼110 μm from the  cell body. The upper side of both micrographs is proximal to the  cell body. Note the difference in the sizes of GAP-43–transporting vesicles even though the degree of magnification of both figures is the same. (c and d) Time course images of the underlined  area of the axon in a. The intervals between frames are 3.45 s. The  upper side of both micrographs is proximal to the cell body. In c,  tubular and spherical vesicles of various sizes and shapes are  moving. The vesicle indicated by diamonds showed representative continuous anterograde movement. The vesicle indicated by  arrowheads switched back to retrograde movement, and again  switched direction anterogradely. In d, the vesicle indicated by  stars broke down into smaller spheres while moving anterogradely in the axon. Bars: (b and d) 5 μm.
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Figure 8: Axonal transport of vesicles containing GAP-43–GFP chimeric protein in mouse DRG neurons. Neurons were transfected with adenovirus vectors carrying GAP-43–GFP chimeric DNA after 3 h in culture and were observed using a laser scan microscope 40 h later. Note that numerous vesicles of various sizes were transported from the proximal side of the axon. (a and b) Proximal and distal area of the same axon. a shows an axon ∼40 μm from the cell body, b shows the same axon ∼110 μm from the cell body. The upper side of both micrographs is proximal to the cell body. Note the difference in the sizes of GAP-43–transporting vesicles even though the degree of magnification of both figures is the same. (c and d) Time course images of the underlined area of the axon in a. The intervals between frames are 3.45 s. The upper side of both micrographs is proximal to the cell body. In c, tubular and spherical vesicles of various sizes and shapes are moving. The vesicle indicated by diamonds showed representative continuous anterograde movement. The vesicle indicated by arrowheads switched back to retrograde movement, and again switched direction anterogradely. In d, the vesicle indicated by stars broke down into smaller spheres while moving anterogradely in the axon. Bars: (b and d) 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: 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