Fig. 4: CSI1/POM2 was required for the formation of Golgi-derived SmaCCs/MASCs.

a Representative images of an epidermal cell expressing both GFP-STL2 and mCherry-CSI1/POM2 in the basal region of 4-day-old etiolated hypocotyls. Arrows indicated colocalization of the two proteins at the SmaCCs/MASCs. For analysis, region of interest (ROI) that excluded the Golgi apparatuses was marked by yellow dotted outlines. Bar = 3 μm. b Colocalization analysis of GFP-STL2 and mCherry-CSI1/POM2 in the Golgi and ROI as indicated in (a) using the Pearson correlation coefficient. Values are mean ± SD. n = 10 cells from 4 seedlings; ***P value < 0.001; two-sided Student’s t test. c Time-lapse images showing the Golgi membrane tail-stretching processes labeled by GFP-STL2 and mCherry-CSI1/POM2. Representative events of successful rupture, Golgi reversal and tail-end retraction were showed in the left, middle and right panels, respectively. The successful rupture events included two subgroups: a group with mCherry-CSI1/POM2 signal existing already at the initial phase of the membrane tail-stretching processes (labeled by yellow arrow and arrowheads), and the other group without CSI1/POM2 at the tail end at the beginning of membrane tail-stretching processes (labeled by white arrow and arrowheads). Arrows indicated the Golgi, while the arrowheads indicated the ends of the Golgi tails. The progress of the membrane tail-stretching events at the indicated time points was schematically presented above the images. The dashed lines in the left panel indicated the time point for which the progress was schematically shown. Bars = 3 μm. d Frequency of different groups of Golgi membrane tail-stretching processes in the control cells with CSI1/POM2 at the tail ends (n = 47 events), without CSI1/POM2 at the tail ends (n = 13 events), and in csi1-3 mutants (n = 35 events). e Time-lapse images showing the Golgi membrane tail-stretching processes labeled by GFP-STL2 in csi1-3 mutants. Note that all the Golgi membrane tail-stretching processes ended up as tail-end retraction in csi1-3 mutants. n = 35 events in 9 cells. Bar = 3 μm. f–h Quantification of the maximal tail length (f), the displacement of the tail ends (g) and the tail stretching time (h) in the control cells with CSI1/POM2 at the tail ends, without CSI1/POM2 at the tail ends, and in csi1-3 mutants. Values are mean ± SD. In total, 47 membrane tail-stretching events with CSI1/POM2 at the tail ends, 13 membrane tail-stretching events without CSI1/POM2 at the tail ends, and 35 membrane tail-stretching events in csi1-3 mutants were quantified. **P value < 0.01; ***P value < 0.001; ns, not significant; One-way ANOVA. i Representative images showing the density of GFP-STL2-localized SmaCCs/MASCs in the control and csi1-3 cells. Bar = 3 μm. j Quantification of the density of GFP-STL2-localized SmaCCs/MASCs in the control and csi1-3 cells. Values are mean ± SD. n = 12 cells from 4 WT seedlings; n = 13 cells from 4 csi1-3 seedlings. ***P value < 0.001; two-sided Student’s t test.