Extended Data Fig. 8: Sphingolipid function in the MEMB12-compartments dynamic interaction with the medial-Golgi as well as the passage of the luminal cargo secGFP through MEMB12 (additional data and controls for Fig. 7).

(a) Time-lapse airyscan acquisition in root epidermal cells of mCherry-MEMB12 x NAG1-EGFP upon metazachlor (Mz) treatment. The time series provide a representative Golgi-independent movement. (b) As compared to the control wild-type condition, the number of association and dissociation within the population of transient interaction between MEMB12 and the medial-Golgi is strongly decreased upon Mz (n = 40 cells, ^****P < 0.0001). (c) The association time of interaction between MEMB12 and the medial-Golgi is slightly altered upon Mz (n = 40 cells, ^*P = 0.0229). (d) The total number of either MEMB12- or medial-Golgi NAG1-compartments is not altered upon whether FB1, Mz or in the acer-1 mutant (n = 40 cells. For MEMB12, ^nsP = 0.4019 (control/FB1), ^nsP = 0.8821 (control/Mz), ^nsP = 0.5057 (control/acer-1). For NAG1, ^nsP = 0.9276 (control/FB1), ^nsP = 0.92 (control/Mz), ^nsP = 0.2105 (control/acer-1)). (e-g) Overall representation of the repartition of Golgi-associated, Golgi-independent or transient interaction between MEMB12 and the medial-Golgi NAG1 marker in either the acer-1 mutant (e), upon FB1 (f) or upon Mz (g) (data coming from the Fig. 7c–h for the acer-1 mutant and FB1 treatment) (n = 40 cells for each dataset. In e (acer-1), ^*P = 0.013 (associated), ^nsP = 0.3859 (independent), ^nsP = 0.8821 (transient). In f (FB1), ^nsP = 0.061 (associated), ^**P = 0.0013 (independent), ^nsP = 0.4127 (transient). In g (Mz), ^nsP = 0.6153 (associated), ^nsP = 0.9733 (independent), ^nsP = 0.6702 (transient)). (h) The velocity of Golgi-associated or Golgi-independent MEMB12-compartments is not altered upon Mz (n = 40 cells, ^nsP = 0.8146 (associated), ^nsP = 0.92 (independent)). Data are presented as violin plots with median represented as a line. (i–r) Airyscan acquisition of root epidermal cells stably expressing mCherry-MEMB12 together with secGFP-FKBP-RUSH upon Mz treatment. The timing of incubation with the FKBP-ligand is indicated in the upper-left corner of each images. (s) Quantification of the normalized percentage of co-localization between secGFP-FKBP-RUSH and mCherry-MEMB12 upon time after incubation with the FKBP-ligand. As compared to the control (Figs. 4y, 7n), Mz treatment might delay the passage of the secGFP-FKBP-RUSH luminal cargo through MEMB12 compartments, although statistically non-significant (n = 680 cells out of 38 roots, ^nsP = 0.0501 (0-5/5-10 min), ^nsP = 0.3926 (5-10/10-15 min), ^nsP = 0.7116 (10-15/15-20 min), ^nsP = 0.2065 (15-20/20-25 min), ^nsP = 0.5692 (20-25/25-30 min)). All statistical tests were performed by non-parametric Mann-Whitney two-sided test. Scale bars is 1 µm in a and 2 µm in i–r. Source numerical data are available in source data.

Source data