The filament-forming protein Pil1 assembles linear eisosomes in fission yeast - PubMed (original) (raw)

The filament-forming protein Pil1 assembles linear eisosomes in fission yeast

Ruth Kabeche et al. Mol Biol Cell. 2011 Nov.

Abstract

The cortical cytoskeleton mediates a range of cellular activities such as endocytosis, cell motility, and the maintenance of cell rigidity. Traditional polymers, including actin, microtubules, and septins, contribute to the cortical cytoskeleton, but additional filament systems may also exist. In yeast cells, cortical structures called eisosomes generate specialized domains termed MCCs to cluster specific proteins at sites of membrane invaginations. Here we show that the core eisosome protein Pil1 forms linear cortical filaments in fission yeast cells and that purified Pil1 assembles into filaments in vitro. In cells, Pil1 cortical filaments are excluded from regions of cell growth and are independent of the actin and microtubule cytoskeletons. Pil1 filaments assemble slowly at the cell cortex and appear stable by time-lapse microscopy and fluorescence recovery after photobleaching. This stability does not require the cell wall, but Pil1 and the transmembrane protein Fhn1 colocalize and are interdependent for localization to cortical filaments. Increased Pil1 expression leads to cytoplasmic Pil1 rods that are stable and span the length of cylindrical fission yeast cells. We propose that Pil1 is a novel component of the yeast cytoskeleton, with implications for the role of filament assembly in the spatial organization of cells.

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Figures

FIGURE 1:

FIGURE 1:

Pil1 forms filaments in fission yeast cells. (A) Western blot showing that Pil1-3HA is expressed in both mitotic and meiotic cells, whereas Pil2-3HA is expressed in meiotic cells but not mitotic cells. (B) Localization of Pil1-mCherry in vegetative cells. Left, inverted maximum projection for Z-planes in the top half of the cell. Asterisk, septating cell. Right, single focal planes and maximum projection for a single cell. (C) Localization of Pil1-mCherry and Pil2-mEGFP in mating cells. Top, inverted maximum projection from Z-planes in the top half of the cell; bottom, the middle focal plane. (D) Budding yeast Pil1 (_Sc_Pil1) forms filaments when expressed in fission yeast cells. Images show inverted single focal planes and maximum projection. (E) Fission yeast Pil1 (_Sp_Pil1) forms puncta when expressed in budding yeast cells. Images show inverted single focal planes and maximum projection. Scale bars: B–E, 5 μm.

FIGURE 2:

FIGURE 2:

Pil1 filaments are independent of the actin and microtubule cytoskeletons. (A) Pil1 cortical filaments do not colocalize with or depend on the actin cytoskeleton. (B) Pil1 cortical filaments do not colocalize with or depend on microtubules. Images are maximum projections for Z-planes in the top half of the cell. Scale bars, 5 μm. Cells were treated with the indicated drug or DMSO control for 20 min before imaging.

FIGURE 3:

FIGURE 3:

Pil1 forms filaments in bacterial cells and in vitro. (A) Localization of Pil1-GFP in E. coli cells. Fluorescence images are inverted maximum-intensity projections, and differential interference contrast (DIC) is red in the merged image. Scale bar, 1 μm. (B) Coomassie-stained SDS–polyacrylamide gel of purified Pil1, which migrates as a doublet similar to budding yeast Pil1 purified from bacteria. (C) Coomassie-stained SDS–polyacrylamide gel of purified Pil1 following centrifugation at high speed or low speed, as indicated. Pellet and supernatant fractions were separated and analyzed by SDS–PAGE. (D, E) Electron micrographs of purified Pil1 filaments. Samples were negatively stained and visualized by electron microscopy. Scale bar: D, 200 nm; E, 100 nm.

FIGURE 4:

FIGURE 4:

Pil1 cortical filaments are static structures that assemble slowly. (A) Time-lapse microscopy of Pil1-mCherry during polarized cell growth. Images are inverted maximum projections for Z-planes in the top half of the cell. The green arrow highlights a newly assembled filament. (B) Time-lapse microscopy of Pil1-mCherry during cell division. The green arrow marks filament breakage. Scale bars, 5 μm. (C, D) Kymograph showing breakage and disassembly (C) or assembly (D) of single Pil1 filaments in cells. Images are inverted single focal planes from time-lapse microscopy.

FIGURE 5:

FIGURE 5:

Pil1 cortical filaments are fixed structures that are independent of the cell wall. (A) FRAP of Pil1-mCherry. The yellow box denotes the bleached region, and images are inverted maximum projections for Z-planes in the top half of the cell. (B) Quantification of fluorescence recovery in bleached or control regions of FRAP experiments. Data points are the mean of 10 regions; error bars, SD. (C) Kymograph of single Pil1-mCherry filament bleached in the middle; each time point is 20 s. (D) Localization of Pil1-mCherry in emerging spheroplast. Image is inverted maximum projection; arrow, cell wall ghost. (E) Pil1-mCherry localizes to cortical filaments in spheroplasts. Images show inverted single focal planes and maximum projection, as indicated. Scale bars: A, D, E, 5 μm.

FIGURE 6:

FIGURE 6:

Pil1 and Fhn1 are interdependent for localization. (A) Colocalization of Pil1-mCherry and Fhn1-mEGFP in cortical filaments; region boxed in white is magnified in the bottom row. (B) Localization of Fhn1-mEGFP in pil1Δ cells. (C) Localization of Pil1-mCherry in fhn1Δ cells. Note decreased abundance and length of cortical filaments. All images are inverted maximum projections from Z-planes in the top half of the cell; scale bars, 5 μm.

FIGURE 7:

FIGURE 7:

Pil1 overexpression impairs cell polarity and cytokinesis. (A) Tenfold serial dilutions of cells containing the indicated plasmids. Expression was induced by growth on media lacking thiamine. (B) DIC images of cells containing control plasmid or Pil1 overexpression plasmid. Expression was induced by removal of thiamine from growth media for 20 h at 32°C. Scale bar, 10 μm. (C) Pil1 overexpression disrupts the localization of mitosis/cytokinesis protein Cdr2 and cell polarity proteins Pom1 and Tea1. The indicated cells were grown in the presence of thiamine (repressed) or in the absence of thiamine for 36 h at 25°C to induce Pil1 overexpression. Pil1 overexpression disrupts the concentration of Cdr2 in the cell middle and leads to loss of Pom1 and Tea1 from cell ends. Images are inverted maximum projections from Z-planes in the top half of cells. Scale bars, 5 μm.

FIGURE 8:

FIGURE 8:

Formation of cytoplasmic rods by overexpressed Pil1. (A) Z-series and maximum projection of Pil1-GFP expressed by the medium-strength P41nmt1 promoter. (B) Pil1-GFP rods are stable in the absence of actin, microtubules, and septins. Cells were grown for 36 h at 32°C in the absence of thiamine and then treated with the indicated drug or DMSO control for 20 min. Images are maximum projections of Pil1-GFP overlaid on DIC image. Scale bars, 5 μm. (C) Time-lapse microscopy of cytoplasmic Pil1-GFP rods. Images are maximum projections; green arrow, mobile rod in cytoplasm. (D) FRAP analysis of cytoplasmic Pil1 rods. Kymograph shows time lapse for the rod outlined in gray. Yellow boxes in the prebleach image show sites of bleaching. Note the lack of movement for unbleached region in the center of the rod.

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