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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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Targeting of the plasma membrane protein–GFP fusion  proteins in mouse DRG neurons. 40 h after infection with each  recombinant virus, living cells were examined by confocal laser  scan microscopy. (a) GAP-43–GFP fusion protein. (b) SNAP-25–GFP fusion protein. (c) TrkA–GFP fusion protein. The proteins were concentrated in the growthcones. (d) Higher magnification of a confocal slice showing that GAP-43–GFP is localized  on the plasma membrane. Arrows indicate varicosities. Bars: (a–c)  50 μm; (d) 5 μm.
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Figure 2: Targeting of the plasma membrane protein–GFP fusion proteins in mouse DRG neurons. 40 h after infection with each recombinant virus, living cells were examined by confocal laser scan microscopy. (a) GAP-43–GFP fusion protein. (b) SNAP-25–GFP fusion protein. (c) TrkA–GFP fusion protein. The proteins were concentrated in the growthcones. (d) Higher magnification of a confocal slice showing that GAP-43–GFP is localized on the plasma membrane. Arrows indicate varicosities. Bars: (a–c) 50 μm; (d) 5 μm.

Mentions: First, we tested whether the membrane protein–GFP fusion proteins are correctly targeted in mouse DRG neurons. 40 h after infection with recombinant adenovirus, we examined DRG neurons by confocal laser scan microscopy. Adenovirus vectors transduced DRG neurons in tissue culture without significant effects on neuronal viability. GAP-43 is a growth cone–associated protein that is localized on the plasma membrane of growth cones (Meiri et al., 1986; Skene, 1989). The GAP-43–GFP fusion protein was targeted to the growth cone (Fig. 2 a), and a confocal slice observed under higher magnification revealed that the protein was localized on the plasma membrane at the growing neurites (Fig. 2 d). Synaptosome-associated protein 25 (SNAP-25) is a soluble NSF attachment protein receptor at the target membrane (t-SNARE) that is localized on the plasma membrane and involved in the docking and fusion of synaptic vesicles to the plasma membrane (Sollner et al., 1993). The SNAP-25–GFP fusion protein was localized in the distal axon (Fig. 2 b), quite similar to the reported localization of endogenous SNAP-25 in nerve cells (Garcia et al., 1995). TrkA, a high affinity receptor for NGF, is expressed on the plasma membrane and internalized after binding to NGF (Klein et al., 1991; Meakin et al., 1992; Ehlers et al., 1995). The trkA-GFP fusion protein was localized on the plasma membrane and accumulated in the growing tips of axons (Fig. 2 c).


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

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

Targeting of the plasma membrane protein–GFP fusion  proteins in mouse DRG neurons. 40 h after infection with each  recombinant virus, living cells were examined by confocal laser  scan microscopy. (a) GAP-43–GFP fusion protein. (b) SNAP-25–GFP fusion protein. (c) TrkA–GFP fusion protein. The proteins were concentrated in the growthcones. (d) Higher magnification of a confocal slice showing that GAP-43–GFP is localized  on the plasma membrane. Arrows indicate varicosities. Bars: (a–c)  50 μm; (d) 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Targeting of the plasma membrane protein–GFP fusion proteins in mouse DRG neurons. 40 h after infection with each recombinant virus, living cells were examined by confocal laser scan microscopy. (a) GAP-43–GFP fusion protein. (b) SNAP-25–GFP fusion protein. (c) TrkA–GFP fusion protein. The proteins were concentrated in the growthcones. (d) Higher magnification of a confocal slice showing that GAP-43–GFP is localized on the plasma membrane. Arrows indicate varicosities. Bars: (a–c) 50 μm; (d) 5 μm.
Mentions: First, we tested whether the membrane protein–GFP fusion proteins are correctly targeted in mouse DRG neurons. 40 h after infection with recombinant adenovirus, we examined DRG neurons by confocal laser scan microscopy. Adenovirus vectors transduced DRG neurons in tissue culture without significant effects on neuronal viability. GAP-43 is a growth cone–associated protein that is localized on the plasma membrane of growth cones (Meiri et al., 1986; Skene, 1989). The GAP-43–GFP fusion protein was targeted to the growth cone (Fig. 2 a), and a confocal slice observed under higher magnification revealed that the protein was localized on the plasma membrane at the growing neurites (Fig. 2 d). Synaptosome-associated protein 25 (SNAP-25) is a soluble NSF attachment protein receptor at the target membrane (t-SNARE) that is localized on the plasma membrane and involved in the docking and fusion of synaptic vesicles to the plasma membrane (Sollner et al., 1993). The SNAP-25–GFP fusion protein was localized in the distal axon (Fig. 2 b), quite similar to the reported localization of endogenous SNAP-25 in nerve cells (Garcia et al., 1995). TrkA, a high affinity receptor for NGF, is expressed on the plasma membrane and internalized after binding to NGF (Klein et al., 1991; Meakin et al., 1992; Ehlers et al., 1995). The trkA-GFP fusion protein was localized on the plasma membrane and accumulated in the growing tips of axons (Fig. 2 c).

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