Figure 5

Combined dye staining of protoplasts and correlation analyses. There was no polarization detectable in freshly isolated, intact protoplasts. FM4-64 (A) & BD-SM (B) appeared to be homogeneous in the plasma membrane as viewed in the merged image (C) of the protoplast (transmission image (D)). (E-F)The occurrence of lipid polarization was observed 15 h post cell wall removal; (E) FM4-64 fluorescence, (F) BD-SM fluorescence; at the dorsal side there was a depletion of the FM4-64 fluorescence signal detected (E, G (merged image of E and F); arrows) in a viable protoplast (H (transmission image of the protoplast)). In 15 h old protoplasts these findings were confirmed by a combined use of LRB-PE (I) and BD-SM (J). A depletion of the LRB-PE fluorescence signal was also detected (I, K (merged image of I and J); arrows) in intact protoplasts (L (transmission image)). (M): Pearson and Spearman correlation coefficients resulting from two independent ROIs in freshly isolated, FM4-64/BD-SM treated protoplasts. Both coefficients indicated a colocalization of fluorescence signals. (n = 10 protoplasts). (N): In 15 h old protoplasts the determined correlation coefficients showed a decrease between unpolarized regions (ROI1) and polarized regions of the plasma membrane (ROI2). For ROI1 unpolarized regions were determined. ROI2 reflects the correlation of green BD-SM and red FM4-64 fluorescence signals next to ROI1 in unpolarized regions in (M) and in polarized regions (FM4-64 depleted) in (N) of the plasma membrane (n = 10 protoplasts). (See methods and Figure S2 for details of the analysis in M &N).