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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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Related in: MedlinePlus

Schematic diagrams of membrane protein–GFP fusion  proteins used in this study. GAP-43 and SNAP-25 do not contain  transmembrane domain, but are associated with the plasma  membrane by lipid moiety at their NH2-terminal and central regions, respectively. Synaptophysin has four transmembrane domains and its COOH-terminal region is located on the cytoplasmic side of the synaptic vesicles. TrkA and TGN-38 contain one  transmembrane domain and their COOH-terminal regions are  located on the cytoplasmic side of the plasma membrane and the  TGN. GFPs were fused with the COOH-terminal region of these  membrane proteins, and thus located on the cytoplasmic sides.
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Figure 1: Schematic diagrams of membrane protein–GFP fusion proteins used in this study. GAP-43 and SNAP-25 do not contain transmembrane domain, but are associated with the plasma membrane by lipid moiety at their NH2-terminal and central regions, respectively. Synaptophysin has four transmembrane domains and its COOH-terminal region is located on the cytoplasmic side of the synaptic vesicles. TrkA and TGN-38 contain one transmembrane domain and their COOH-terminal regions are located on the cytoplasmic side of the plasma membrane and the TGN. GFPs were fused with the COOH-terminal region of these membrane proteins, and thus located on the cytoplasmic sides.

Mentions: Rat GAP-43 (Karns et al., 1987), mouse SNAP-25 (Oyler et al., 1989), rat synaptophysin (Sudhof et al., 1987), and rat TGN-38 (Luzio et al., 1990) cDNAs were cloned by RT-PCR and the sequences were confirmed by sequencing. TrkA cDNA was kindly provided by Dr. E.M. Shooter (Stanford University, Stanford, CA) and Dr. T. Hunter (Salk Institute, LaJolla, CA; Meakin et al., 1992). Chimeric cDNAs were constructed to encode full-length GAP-43–S65T-mutated GFP-flag tag (cDNA of S65T-mutated GFP was kindly provided by Dr. R. Tsien, University of California San Diego, La Jolla, CA), full-length SNAP-25–S65T-mutated GFP-flag tag, full-length synaptophysin linker–EGFP (CLONTECH Laboratories, Inc., Palo Alto, CA) HA tag, full-length trkA linker–EGFP-flag tag, and full-length TGN-38–EGFP. All the constructs were made to fuse the GFP molecules with the COOH-terminal of the membrane proteins with/without the intervening linker sequence (see Fig. 1). The aminoacid sequence of the linker was KGVEPKTYCYYSS (Grote et al., 1995). In some constructs, flag-tag (DYKDDDDK) or HA-tag (CYPYDVPDYASL) was added to the COOH-terminal of the GFP. The cDNAs were subcloned into pAdexCAw1 at the SwaI site and cotransfected with viral genome fragments into HEK 293 cells (CRL 1573; American Type Culture Collection [ATCC], Rockville, MD; Miyake et al., 1996). The recombinant adenoviruses were amplified and CsCl-purified (Kanegae et al., 1994). They were then transfected into mouse dorsal root ganglion (DRG) cells over a period of 60 min, 3 h after plating of the cells.


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

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

Schematic diagrams of membrane protein–GFP fusion  proteins used in this study. GAP-43 and SNAP-25 do not contain  transmembrane domain, but are associated with the plasma  membrane by lipid moiety at their NH2-terminal and central regions, respectively. Synaptophysin has four transmembrane domains and its COOH-terminal region is located on the cytoplasmic side of the synaptic vesicles. TrkA and TGN-38 contain one  transmembrane domain and their COOH-terminal regions are  located on the cytoplasmic side of the plasma membrane and the  TGN. GFPs were fused with the COOH-terminal region of these  membrane proteins, and thus located on the cytoplasmic sides.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Schematic diagrams of membrane protein–GFP fusion proteins used in this study. GAP-43 and SNAP-25 do not contain transmembrane domain, but are associated with the plasma membrane by lipid moiety at their NH2-terminal and central regions, respectively. Synaptophysin has four transmembrane domains and its COOH-terminal region is located on the cytoplasmic side of the synaptic vesicles. TrkA and TGN-38 contain one transmembrane domain and their COOH-terminal regions are located on the cytoplasmic side of the plasma membrane and the TGN. GFPs were fused with the COOH-terminal region of these membrane proteins, and thus located on the cytoplasmic sides.
Mentions: Rat GAP-43 (Karns et al., 1987), mouse SNAP-25 (Oyler et al., 1989), rat synaptophysin (Sudhof et al., 1987), and rat TGN-38 (Luzio et al., 1990) cDNAs were cloned by RT-PCR and the sequences were confirmed by sequencing. TrkA cDNA was kindly provided by Dr. E.M. Shooter (Stanford University, Stanford, CA) and Dr. T. Hunter (Salk Institute, LaJolla, CA; Meakin et al., 1992). Chimeric cDNAs were constructed to encode full-length GAP-43–S65T-mutated GFP-flag tag (cDNA of S65T-mutated GFP was kindly provided by Dr. R. Tsien, University of California San Diego, La Jolla, CA), full-length SNAP-25–S65T-mutated GFP-flag tag, full-length synaptophysin linker–EGFP (CLONTECH Laboratories, Inc., Palo Alto, CA) HA tag, full-length trkA linker–EGFP-flag tag, and full-length TGN-38–EGFP. All the constructs were made to fuse the GFP molecules with the COOH-terminal of the membrane proteins with/without the intervening linker sequence (see Fig. 1). The aminoacid sequence of the linker was KGVEPKTYCYYSS (Grote et al., 1995). In some constructs, flag-tag (DYKDDDDK) or HA-tag (CYPYDVPDYASL) was added to the COOH-terminal of the GFP. The cDNAs were subcloned into pAdexCAw1 at the SwaI site and cotransfected with viral genome fragments into HEK 293 cells (CRL 1573; American Type Culture Collection [ATCC], Rockville, MD; Miyake et al., 1996). The recombinant adenoviruses were amplified and CsCl-purified (Kanegae et al., 1994). They were then transfected into mouse dorsal root ganglion (DRG) cells over a period of 60 min, 3 h after plating of the cells.

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