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Figures
- Fig. 1.
Genetic interactions identified by synthetic genetic array screens. (A) Summary of genetic interaction results for pil1Δ and sle1Δ; the table is limited to shared hits that were verified by tetrad dissection. (B) Description of key genes in Pil1-Sle1 genetic interaction screens. (C) Tetrad analysis confirms that pil1Δ, sle1Δ, syj1Δ, and tax4Δ are all synthetically lethal with inp53Δ. Inviable spores represented by dashed black squares; replica plating showed that inviable spores are the double mutants.
- Fig. 2.
Eisosomes function in a genetic pathway for PI(4,5)P2 regulation. (A) Regulation of PI(4,5)P2 synthesis and hydrolysis in fission yeast. (B) Mutations in the Pil1–Sle1–Syj1–Tax4 pathway suppress temperature-sensitive growth defects of its3-1. (C) its3-1 suppresses the synthetic lethality of inp53Δ pil1Δ and inp53Δ syj1Δ mutants. Panels B and C are 10-fold serial dilutions grown at the indicated temperatures on rich media.
- Fig. 3.
Eisosome defects in syj1 and its3 mutants. (A) Pil1 cortical filaments are dependent on Syj1 but not Inp53 for proper organization. Images are inverted maximum projections for Z-planes in the top half of the cell. Scale bar: 5 µm. (B) Eisosome organization is dependent on Its3. Cells were grown at 25°C and then switched to 32°C for 10 minutes. Images are inverted maximum projections from Z-planes in the top half of the cell. Scale bar, 5 µm. (C) Quantification of Pil1 cytoplasmic concentration in the indicated strain and temperature. Levels are presented as arbitrary fluorescence units (AFU) and represent mean ± s.d. for 25 cells.
- Fig. 4.
Pil1 generates cortical pits in syj1Δ its3-1 double-mutant cells. (A) Thick Pil1 filaments at the cortex of syj1Δ its3-1 double-mutant cells. Images are inverted maximum projections from Z-planes in the top half of the cell. Scale bar, 5 µm. (B) Thin section electron microscopy of syj1Δ its3-1 double-mutant cells. Cross-section view displays exaggerated pit-like invagination. The white arrow highlights a pit-like invagination that is magnified in right panel. Scale bars: 500 nm (left panel); 100 nm (right panel).
- Fig. 5.
Pil1 binds and tubulates liposomes in vitro. (A) Liposome pelleting assays. Pil1 was incubated with the indicated liposomes, and supernatant (S) and pellet (P) fractions were analyzed by SDS-PAGE followed by Coomassie staining. (B) Electron microscopy of negative-stained lipososomes containing PC/PS/PE/PI(4,5)2 in the presence or absence of purified Pil1. (C) Liposome pelleting assays. Pil1 was incubated with PC liposomes in the presence or absence of 1.5% PI(4,5)P2, and samples were analyzed as in panel A. (D) Electron microscopy of negative-stained samples from panel C.
- Fig. 6.
Syj1 and Inp53 are spatially separated in cells. (A) Localization of endogenously tagged Syj1–mEGFP and Inp53–mEGFP. Images are inverted single focal planes from the cell middle. (B) Inp53–mEGFP does not colocalize with eisosomes, marked by Pil1–mCherry. (C) Most Syj1–mEGFP does not colocalize with eisosomes, marked by Pil1–mCherry. Arrows mark cortical puncta of Syj1–mEGFP. Images in B and C are inverted maximum projections from Z-planes in the top half of the cell, and also single focal planes from the cell middle. Scale bars: 5 µm.
- Fig. 7.
Inp53 localizes to endocytic actin patches. (A) Colocalization of Inp53–mEGFP and Fim1–mCherry in actin patches; the region boxed in white is magnified in the bottom row. Images are inverted single focal planes. (B) Syj1–mEGFP does not colocalize with actin patches, marked by Fim1–mCherry; the region boxed in white is magnified in the bottom row. Images are inverted single focal planes. Scale bars, 5 µm.
- Fig. 8.
Pil1–Sle1–Syj1–Tax4 pathway regulates TORC2 signaling. (A) 10-fold serial dilutions of the indicated tor1-ts single- and double-mutant cells at different temperatures. Note that temperature-sensitive growth of tor1-ts is suppressed by mutations in components of the Pil1–Sle1–Syj1–Tax4 pathway. (B) Digital interference contrast (DIC) images of tor1-ts single- and double-mutant cells. Cells were grown for 13 hours at 34°C. Scale bar, 10 µm.
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