Cytokinesis failure and aberrant DNA content in cells with ESCRT-III defects.
(A) High-pressure frozen and freeze-substituted cells of strain MWY24 were embedded in plastic, thin-sectioned and visualized by TEM. The red arrows indicate sites of impending or aborted cytokinesis. (B) Approximately 10 000 PI-stained cells of each indicate genotype were analyzed by flow cytometry to measure DNA content. Histograms of the resulting distributions were manually offset rightward on the x-axis to facilitate comparison of the profiles. The arrows indicate the approximate peak values corresponding to IC, 2C, or 4C DNA content. The strains were: SEY6210 (WT haploid), GOY65 (brolΔ), BWY102 (vps24Δ), MWY24 (snf7Δ), SEY6210 carrying plasmid pG0216 (Brol overexpression), SEY6210 carrying plasmid pMWM3 [Brol (1–387) overexpression], and SEY6210 diploid (WT diploid). (C) As in (B), for strains SEY6210 (WT haploid), MBY3 (vps46Δ), MBY30 (vps36Δ), EEY6-2 (vps23Δ), EEY2-1 (vps20Δ), and MBY28 (vps2Δ).

Synthetic effects on colony growth and/or number of nuclei in snf7Δ cla4Δ, snf7Δ elm1Δ, and snf7Δ cdc10-1 mutants.
(A–C) Fivefold serial dilutions of cells cultured at room temperature were spotted on agar plates and incubated at the indicated temperature for 2–3 d before plates were photographed. The growth medium was rich (YPD) (A, C), or synthetic drop-out medium selective for the indicated plasmids (B). All strains were, as indicated, the SNF7⁺ or snf7Δ meiotic progeny of diploid cells carrying a mutation at another locus, and were made by crossing the snf7Δ haploid JTY4935 with JTY4003 (hsl1Δ), JTY4007 (nap1), JTY4005 (kcc4Δ), JTY4008 (siz1Δ), YMYB12 (cla4Δ), JTY4000 (elm1Δ), or JTY3986 (cdc10-1), Plasmids FD44 ([CLA4]), pRS416-SNF7-FLAG ([SNF7-FLAG]), pRS416-SNF7 (L121D)-FLAG ([SNF7 (L121D)-FLAG]), pRS416-CUP-ELM1 ([YCpELM1]) or YEp13-ELM1 ([YEp-ELM1]) were introduced by transformation into the haploid spore clones. (D) The strains in (C) were grown overnight in YPD medium at the indicated temperature prior to DAPI staining and visualization of nuclei in single focal planes, At 26°C, for cdc10-1 SNF7⁺, 14/475 were multinucleate at 26°C, 223/373 at 30°C; for cdc10-1 snf7Δ, 6/746 were multinucleate at 26°C, 86/382 at 30°C.

ESCRT-III and septin localization patterns and effects on morphogenesis during normal and defective cell division.
Cells from exponentially-growing cultures were visualized by transmitted light microscopy of single focal planes (A–E) and either epifluorescence microscopy at regular intervals in the z direction combined with deconvolution of the resulting single focal plane images to eliminate out-of-plane fluorescence, followed by projection of each focal plane’s image onto a single image (B, E), or standard epifluorescence microscopy of single focal planes (D, G). (A) Strains were SEY6210 (wild-type), MWY24 (snf7Δ), MBY30 (vps36Δ), or MBY3 (vps4Δ) and were cultured at 30°C. (B) mCherry-tagged Cdc10, expressed from the endogenous CDCIO locus, was visualized with a rhodamine filter set in SNF7⁺ (JTY3992) or snf7Δ (YCS639) cells cultured at 30°C. (C) Cells of strain JTY4957 (snf7Δ ELM1), JTY4000 (SNF7⁺ elm1Δ), or JTY4973 (snf7Δ elm1Δ) pre-grown in YPD at room temperature were shifted to 37°C for 3 h. The arrowheads indicate constrictions along the lengths of snf7Δ elm1Δ buds. (D) Cells of strain JTY3986 (cdc10-1-GFP SNF7⁺) or JTY4000 (cdc1O-1-GFP snf7Δ), pre-grown in YPD at room temperature, were shifted to 30°C for 3 h. Cell morphology was visualized by differential interference contrast (DIC). Cdc10-1-GFP localization was visualized with an eGFP filter set (GFP). The arrowheads indicate cells with multiple attached buds. The arrow indicates an elongated, lysed cell. (E) Snf7-GFP was visualized with a TRITC filter set in cells of strain BY4741 (CDC10⁺) or YCS640 (cdc10Δ) cultured at 30°C and carrying plasmid pRS416-SNF7-GFP. (F) Snf7-GFP (green) and Cdc10-mCherry (red) were visualized in cells of strain JTY3992 cultured at 30°C using a TRITC or a rhodamine filter set, respectively. (G) Snf7-mRFP (red) and Myol-GFP (green) were visualized using a TRITC or an eGFP filter set, respectively, in diploid cells made by mating strain JTY3562 with strain JTY4510 and cultured at 30°C. Left column: cells with Snf7-mRFP at the bud neck (arrowheads); right column: cells in which no Snf7-mRFP was observed at the neck, imaged in the same fields as those in the left column.

ESCRT-I, -II, and -III mutations in elm1Δ cells exacerbate cytokinesis defects, elevate Chs2 levels, and cause ectopic chitin deposition.
(A) Fivefold serial dilutions of cells cultured at room temperature were spotted on a YPD agar plate and incubated at 37°C for 3 d before colonies were photographed. (B) Top: Immunoblot of Chs2 and Clb2 from protein extracts of the indicated strains. Bottom: the ratio of intensity of Chs2 to another loading control (Pgk1; not shown) for each lane above is plotted and normalized to the value for the elm1Δ strain JTY4000. (C) YPD cultures of the indicated strains were grown to saturation at room temperature and stained with the chitin-binding dye Calcofluor White. Chains of elongated cells were selected for imaging. Arrowheads: regions of intense staining. Strains were, as indicated, JTY4000 (elm1Δ), JTY5595 (vps23Δ), JTY5596 (vps27Δ), JTY5597 (vps36Δ), JTY4973 (elmlΔ Snf7Δ), JTY5598 (elm1Δ vps23Δ), JTYSS99 (elm1Δ vps27Δ), JTY5600 (elm1Δ vps36Δ).