The Arabidopsis SAC9 enzyme is enriched in a cortical population of early endosomes and restricts PI(4,5)P2 at the plasma membrane
- Laboratoire Reproduction et Développement des Plantes, Université de Lyon, France
- University of Tübingen, Center for Plant Molecular Biology (ZMBP), Germany
- Division of Biological Science, University of Montana, United States
- Interfaculty Institute for Cell Biology, Department of Quantitative Proteomics, University of Tübingen, Germany
- Faculty of Biology & Biotechnology, Ruhr-University Bochum, Germany
Figures
Figure 1 with 1 supplement
Structure-function analysis of SUPPRESSOR OF ACTIN9 (SAC9).
(A) Schematic representation of SAC9 protein. The SAC catalytic domain, as well as the WW domain and the coil-coiled domain, are represented. (B) Representative images of the macroscopic phenotype …
Figure 1—figure supplement 1
Complementation analysis of the sac9-3 mutant phenotype.
(A) Representative images of the complementation analysis of the sac9-3 mutant. For each construct, 24 primary T1 transformants were selected. Independent T2 and T3, T4 plants were obtained for …
Figure 2 with 1 supplement
SUPPRESSOR OF ACTIN9 (SAC9) localizes in the cytosol and in intracellular compartment enriched at the cell cortex.
(A) Schematic representation of two root epidermal cells (cell #1 indicated as C1 and cell #2 indicated as C2) imaged at two different focal planes (cortical plane, close to the plasma membrane and …
Figure 2—figure supplement 1
Subcellular localization of SAC9 alleles at different depths in the cell.
(A) Confocal imaging of mCIT-SAC9 (green) together with the nuclear marker CENH3-RFP (magenta). Scale bars: 10 µm. (B) Representative images of the signal corresponding to mCIT-SAC9 and FM4-64 at …
Figure 3 with 2 supplements
SUPPRESSOR OF ACTIN9 (SAC9) localizes to a subpopulation of TGN/EE.
(A, B) Confocal images of SAC9pro:mCIT-SAC9 (A, green) or SAC9pro:mCIT- SAC9C459A (B, green) in vivo in Arabidopsis root epidermis together with the endocytic tracer FM4-64 (magenta). (C, D) …
Figure 3—figure supplement 1
Quantification of the number of endosomes per cell labeled by mCIT-SAC9 variants which colocalized with FM4-64 at Zi.
In the plots, middle horizontal bars represent the median, while the bottom and top of each box represent the 25th and 75th percentiles, respectively. At most, the whiskers extend to 1.5 times the …
Figure 3—figure supplement 2
Confocal images of SAC9C459A with endosomal markers.
(A) Confocal images of Arabidopsis root epidermis co-expressing SAC9pro:mCIT-SAC9C459A (green) and markers such as mCH-RabA1g; mCH-RabD1, mCH-Got1p (magenta). The plane (Zi or Zii) in each image is …
Figure 4 with 2 supplements
SUPPRESSOR OF ACTIN9 (SAC9) restricts PI(4,5)P2 at the plasma membrane.
(A) Confocal images of Arabidopsis root epidermis expressing mCIT-tagged sensors in WT (Col-0) and sac9-3-/- genetic backgrounds. Fluorescence intensity is color-coded (green fire blue scale). (B) …
Figure 4—figure supplement 1
Confocal images of Arabidopsis root epidermis expressing various phosphoinositide biosensors in WT and sac9-3-/-.
(A) Representative confocal images of Arabidopsis root epidermis expressing mCIT-tagged lipid sensors for PI3P in wild-type (Col-0) and sac9-3-/- genetic backgrounds. (B) Confocal images of Arabidops…
Figure 4—figure supplement 2
The intracellular accumulation of mCITRINE-TUBBYc observed in sac9 is sensitive to PI-4 kinase inhibition.
(A) Schematic representation of the mode of action of the PAO on the PI4P and PI(4,5)P2 metabolism based on previously published work (Doumane et al., 2020; Platre et al., 2018). (B) Confocal images …
Figure 5 with 1 supplement
The endocytic flux is perturbed in the sac9 mutant.
(A) Representative images of Col-0 and sac9-3-/- seedlings treated for 30 min with FM4-64, which is endocytosed and labels endocytic intracellular compartments. Scale bars: 10 µm. (B) Quantification …
Figure 5—figure supplement 1
Example of FM4-64 labeling in Col-0 and sac9-3 mutant.
(A) Example of confocal images used for the number of FM4-64 labeled intracellular compartments per cell in Col-0 and sac9-3-/-. Scale bar: 75 µm. (B) Example of confocal images used for the number …
Figure 6 with 1 supplement
SUPPRESSOR OF ACTIN9 (SAC9) interact and colocalize with SH3P2 and regulates it subcellular distribution.
(A) Schematic representation of the yeast two-hybrid screen using SAC9 as a bait, where SH3P2 was found as a protein partner. The selected interaction domain (interacting with bait 1) corresponds to …
Figure 6—figure supplement 1
Western blot analysis of TPLATE-GFP and SH3P2-GFP in WT and sac9-3-/-.
The expression of the transgene was detected using an anti-GFP antibody. Ponceau S staining as well as western blot using the anti-tubulinα antibody served as a loading control. The experiment was …
Figure 7
TPLATE dynamics and density at the plasma membrane is disturbed in the sac9 mutant.
(A) Representatives images of the TPLATE-GFP localization in WT and sac9-3 mutant at the plasma membrane observed by TIRF microscopy in etiolated hypocotyl. Scale = 5 µm. (B) Quantification of the …
Figure 8
Model for the mode of action of SUPPRESSOR OF ACTIN9 (SAC9) in regulating PI(4,5)P2 subcellular patterning.
In wild-type plants, SAC9 restricts the localization of PI(4,5)P2 at the plasma membrane allowing endocytic processes to occur. In the absence of SAC9, endocytosis PI(4,5)P2 accumulates in atypical …
Videos
Video 1
Time-lapse imaging of mCIT-SAC9 using spinning disk confocal microscope (1 s per frame).
Video 2
Time-lapse imaging of mCIT-2xPHPLC in sac9 mutant (1 s per frame) using spinning disk confocal microscope.
Video 3
Time-lapse imaging of TPLATE-GFP at the plasma membrane of WT and in sac9-3 mutant using TIRF microscopy.
Time lapses were acquired during 300 time-points for 300 s (acquisition time 500 ms).
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/73837/elife-73837-mdarchecklist1-v2.docx
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Supplementary file 1
Ressources and statistics.
(A) Reagent and resources. (B) Raw data from ULTImate Y2H SCREEN Arabidopsis thaliana - SAC9 (aa 499–966) vs Universal Arabidopsis Normalized_dT. (C) Details of the statistics corresponding to Figure 1C, D. (D) Details of the statistics corresponding to Figure 2D, G. (E) Details of the statistics corresponding to Figure 3G-F. (F) Details of the statistics corresponding to Figure 4B. (G) Details of the statistics corresponding to Figure 5. (H) Details of the statistics corresponding to Figure 6E, G. (I) Details of the statistics corresponding to Figure 7B. (J) Details of the statistics corresponding to Figure 1—figure supplement 1. (K) Details of the statistics corresponding to Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/73837/elife-73837-supp1-v2.xlsx
