Figure 8.

VPS2-DN Causes Marker Proteins for TGN/EE and MVB/LE to Colocalize.

Tobacco mesophyll protoplasts were transfected with plasmids encoding for fluorescent markers/reporters as indicated below. Proteins were expressed for 18 h prior to CLSM analysis. For quantification, the PSC coefficients (r_p and r_s) were calculated after analysis of at least 10 individual protoplasts and a minimum of 200 signals. The level of colocalization ranges from +1 for perfect correlation to −1 for negative correlation. For the corresponding scatterplots of the fluorescence values of pixels across the two channels, see Supplemental Figure 5 online.

(A) Coexpression of TGN/EE and MVB/LE markers YFP-SYP61 and mRFP-VSR2 18 h after transfection.

(B) Effect of VPS2-DN on the distribution of TGN/EE and MVB/LE markers 14 h after transfection.

(C) Analysis 18 h after transfection: VPS2-DN causes a change in the signal pattern of the marker proteins. The signals accumulate in bigger but fewer structures.

(D) Quantification of the marker colocalization. The r_p and r_s values increase when VPS2-DN is expressed.

(E) Coexpression of TGN/EE and MVB/LE markers YFP-SYP61 and mRFP-ARA7 18 h after transfection.

(F) Effect of the VPS2-DN coexpression with YFP-SYP61 and mRFP-ARA7 14 h after transfection.

(G) When expressed for 18 h, VPS2-DN increases colocalization of YFP-SYP61 and mRFP-ARA7. As observed in (C), the signals change structurally.

(H) Quantification reveals higher r_p and r_s values for the marker proteins when VPS2-DN is expressed.

(I) to (L) An experiment as described in (E) to (H) was performed, except ARA6-mRFP was used as MVB/LE marker. Here, the highest increase of r_p and r_s values is found (L).

Bars = 5 μm.