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1.
Figure 6

Figure 6. Trafficking at the plasma membrane is dispensable for synaptophysin I-directed sorting of VAMP2. From: Synaptophysin I selectively specifies the exocytic pathway of synaptobrevin 2/VAMP2.

(A) Cells triple-transfected with expression plasmids for VAMP2–YFP (red), SypI–CFP (green) and HA-tagged either wild-type or dominant-negative (K44A mutant) dynamin at 1:1:2. Staining with an anti-HA antibody reveals exogenous dynamin (blue). (B) Cells co-expressing VAMP2–YFP, SypI–CFP and either wild-type dynamin (Dyn WT) or the K44A dynamin mutant (Dyn K44A) were processed for synaptophysin I immunoprecipitation (α-SypI IP). Western blotting with an anti-VAMP2 (α-VAMP2) antibody reveals the presence of co-precipitated VAMP2. Molecular masses are given in kDa. (C) Cells triple-transfected with expression plasmids for VAMP2–YFP (red), SypI–CFP (green) and either wild-type or dominant-negative (L294A mutant) α-SNAP at 1:1:2. The anti-α-SNAP antibody was used at a concentration exclusively allowing identification of the overexpressed proteins (blue). Note that co-localization between VAMP2–YFP and SypI–CFP that have exited the Golgi complex is visible also in cells in which α-SNAP L294A severely affects ER-to-Golgi trafficking (right-hand panel). Insets in (A) and (C) show magnifications of the square-selected regions. Scale bars, 10 μm (A and C); 4 μm (insets of A and C).

Dario Bonanomi, et al. Biochem J. 2007 June 15;404(Pt 3):525-534.
2.
Figure 3

Figure 3. Synaptophysin I reduces the accumulation of VAMP2 at the plasma membrane. From: Synaptophysin I selectively specifies the exocytic pathway of synaptobrevin 2/VAMP2.

(A) Membrane proteins of cells transfected with constructs expressing VAMP2–YFP and either ECFP, SypI–CFP, SypIΔC–CFP or CFP–Rab11 were labelled with biotin at 4°C and isolated by streptavidin–Sepharose precipitation. Arrowheads indicate the position of VAMP2–YFP. Molecular masses are given in kDa. (B and C) Quantification of three independent experiments of cell biotinylation. Results are means±S.D. For each of the proteins, the amount recovered from the streptavidin precipitates was normalized to the amount recovered from the total lysates. The intensity of bands from cells transfected with VAMP2–YFP and ECFP was set to 1. The levels of plasma-membrane-associated VAMP2–YFP are reduced upon expression of SypI–CFP, but not after the expression of either SypIΔC–CFP or CFP–Rab11 (B). In contrast, levels of surface-localized TfR are unchanged under all conditions (C). Molecular masses are as follow: VAMP2–YFP, 40 kDa; SypI–CFP, 60–65 kDa; SypIΔC–CFP, 50–55 kDa; CFP–Rab11, 51 kDa; TfR, 85 kDa.

Dario Bonanomi, et al. Biochem J. 2007 June 15;404(Pt 3):525-534.
3.
Figure 4

Figure 4. Synaptophysin I exerts a selective and dose-dependent effect on VAMP2 sorting to intracellular compartments. From: Synaptophysin I selectively specifies the exocytic pathway of synaptobrevin 2/VAMP2.

(A) Cells co-expressing VAMP2–YFP (green in the merge images) and either SypI–CFP, SypIΔC–CFP or CFP–Rab11 (blue in the merge images) surface-stained with an anti-FP antibody to detect plasma-membrane-associated VAMP2–YFP (red in the merge images). Despite expression of comparable levels of VAMP2–YFP, the amount of surface-exposed chimaera is higher in cells expressing lower levels of wild-type SypI–CFP. (B) Cells co-expressing syntaxin 13–CFP (green in the merge image) and SypI–YFP (blue in the merge image), surface-stained with an anti-FP antibody to detect plasma-membrane-associated syntaxin 13–CFP (red in the merge image). Scale bar, 10 μm. (C) Correlation plots show the surface/total expression ratios for VAMP2–YFP (upper three panels) or syntaxin 13–CFP (Stx13, lower panel) plotted against the levels of SypI–CFP, SypIΔC–CFP or CFP–Rab11. Each dot corresponds to a single cell (200–268 cells for each condition in three to four independent experiments). r2 for exponential fitting is 0.6 for VAMP2–YFP:SypI–CFP (P<0.01) and <0.1 in the other cases. a.u., arbitrary units.

Dario Bonanomi, et al. Biochem J. 2007 June 15;404(Pt 3):525-534.
4.
Figure 1

Figure 1. Exogenous VAMP2 is predominantly associated with the plasma membrane. From: Synaptophysin I selectively specifies the exocytic pathway of synaptobrevin 2/VAMP2.

(A) CFP-F (green) co-expressed with either VAMP2–YFP (left), SytI–YFP (middle) or SypI–YFP (right) (red). (B) Upper panels: unfixed cells expressing VAMP2–YFP (green in the merge images) surface-stained at 4°C with an anti-FP antibody (αYFP; red in the merge images). Lower panels: the plasma-membrane-associated anti-FP antibody was removed by acid-stripping, which also quenched the intrinsic fluorescence of surface-localized VAMP2–YFP. (C) Surface-staining of VAMP2–YFP plotted against the total fluorescence of the chimaera. Each dot corresponds to a single cell (r2=0.82; P<0.01). a.u., arbitrary units. (D) VAMP2–YFP-positive vesicles (green) show partial co-localization (arrowheads) with either TfR or ECFP-tagged Rab5 (red). (E) VAMP2–YFP endocytosis monitored by internalization of surface-associated anti-FP antibody after a 20 min incubation at 37°C. A high magnification of the outlined area is shown in the lower panels. Scale bars, 7 μm (A); 10 μm (B and upper panels of E); 2.5 μm (lower panels of E); 1.6 μm (D).

Dario Bonanomi, et al. Biochem J. 2007 June 15;404(Pt 3):525-534.
5.
Figure 2

Figure 2. Synaptophysin I interacts with VAMP2 and leads to its redistribution to intracellular compartments. From: Synaptophysin I selectively specifies the exocytic pathway of synaptobrevin 2/VAMP2.

(A) Cells co-expressing SypI–CFP (green in the merge images) and either VAMP2–YFP (upper panel) or SytI–YFP (lower panel) (red in the merge images). Synaptophysin I directs VAMP2 to intracellular compartments, whereas it does not influence the plasma membrane localization of synaptotagmin I. (B) A subset of vesicles bearing both VAMP2–YFP and SypI–CFP also contain TfR (arrowheads). (C) Number of VAMP2–YFP-positive intracellular puncta and their co-localization with TfR measured in cells expressing VAMP2–YFP and either soluble ECFP (VAMP2–YFP, light grey bars) or SypI–CFP (VAMP2–YFP/SypI–CFP, dark grey bars) after quenching of surface EYFP fluorescence. Results are means±S.D. for three independent experiments. Synaptophysin I expression increases the amount of VAMP2-positive organelles (Student's t test, P<0.0001, n=72 cells per condition), while it does not affect the extent of overlapping between VAMP2 and TfR (n=46 cells per condition). (D) Cells co-expressing VAMP2–YFP and either ECFP (VAMP2), HA-tagged SypI–CFP (VAMP2:SypI–HA) or HA-tagged C-terminally truncated SypIΔC-CFP (VAMP2:SypIΔC–HA) and cells co-expressing SypI–HA and soluble ECFP (SypI–HA) were processed for immunoprecipitation (IP) with anti-HA antibody (α-HA). Western blotting with anti-synaptophysin I (α-SypI; upper panel) and anti-VAMP2 (α-VAMP2; lower panel) antibodies reveals the presence of heterocomplexes between VAMP2–YFP (arrowhead) and either SypI–HA or SypIΔC–HA. Molecular masses are as follow: VAMP2–YFP, 40 kDa; SypI–CFP–HA, 60–65 kDa; SypIΔC–CFP–HA, 50–55 kDa. Scale bars, 10 μm (A); 3.7 μm (B).

Dario Bonanomi, et al. Biochem J. 2007 June 15;404(Pt 3):525-534.
6.
Figure 5

Figure 5. The rate of VAMP2 endocytosis is not influenced by synaptophysin I. From: Synaptophysin I selectively specifies the exocytic pathway of synaptobrevin 2/VAMP2.

(A) Endocytosis of biotin-labelled membrane proteins in cells expressing VAMP2–YFP and either soluble ECFP (VAMP2) or SypI–CFP (VAMP2:SypI). After surface labelling with biotin at 4°C, cells were incubated at 37°C for the indicated times to allow endocytosis. Before precipitation, surface-associated biotin was removed by cleavage of the disulfide bond. The anti-FP antibody (α-FP) reveals the exogenous proteins. The anti-TfR antibody (α-TfR) detects biotin-labelled receptors precipitated from the same cell extracts. Note that surface levels of VAMP2 are reduced by synaptophysin I. Molecular masses are given in kDa. (B) Quantification of the data from three independent experiments similar to that shown in (A). The data are plotted as the fraction of the amount of protein recovered with respect to cells not treated to remove surface label (surface). In each lane, the amount of protein recovered from the precipitates was normalized to the amount recovered from the total lysates. (C) Comparison between the rates of VAMP2–YFP and SypI–CFP internalization in cells co-expressing the two chimaeras. Results in (B) and (C) are means±S.D. for three independent experiments. (D) VAMP2–YFP endocytosis during a 7 min chase at 37°C in cells co-expressing VAMP2–YFP and SypI–CFP, monitored by internalization of surface-bound anti-FP antibody (α-YFP). Lower panels show magnifications of the regions outlined in the respective upper panels. Synaptophysin I is associated with vesicles containing endocytosed VAMP2 (arrowheads). Scale bars, 10 μm (D, upper panels), 2.5 μm (D, lower panels).

Dario Bonanomi, et al. Biochem J. 2007 June 15;404(Pt 3):525-534.

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