Cooperation between the septins and the actomyosin ring and role of a cell-integrity pathway during cell division in fission yeast

Abstract

A major question about cytokinesis concerns the role of the septin proteins, which localize to the division site in all animal and fungal cells but are essential for cytokinesis only in some cell types. For example, in Schizosaccharomyces pombe, four septins localize to the division site, but deletion of the four genes produces only a modest delay in cell separation. To ask if the S. pombe septins function redundantly in cytokinesis, we conducted a synthetic-lethal screen in a septin-deficient strain and identified seven mutations. One mutation affects Cdc4, a myosin light chain that is an essential component of the cytokinetic actomyosin ring. Five others cause frequent cell lysis during cell separation and map to two loci. These mutations and their dosage suppressors define a signaling pathway (including Rho1 and a novel arrestin) for repairing cell-wall damage. The seventh mutation affects the poorly understood RNA-binding protein Scw1 and severely delays cell separation when combined either with a septin mutation or with a mutation affecting the septin-interacting, anillin-like protein Mid2, suggesting that Scw1 functions in a pathway parallel to that of the septins. Taken together, our results suggest that the S. pombe septins participate redundantly in one or more pathways that cooperate with the actomyosin ring during cytokinesis and that a septin defect causes septum defects that can be repaired effectively only when the cell-integrity pathway is intact.

Figure 1.—

Mutations showing synthetic effects with septin mutations. Cells were observed directly in liquid culture medium by DIC microscopy (A–D and I–P) or streaked on plates to observe overall growth rates (E–H). (A–D) Wild-type strain JW81 and the septin-mutant strains JW267, JW289, and JW182. (I–L) Strains carrying the new mutations in otherwise wild-type backgrounds (JW399, JW403, JW406, and JW330). (M–P) The double or triple mutants (JW401, JW402, JW405, and JW321-1). Plates show the same strains. spn1 spn4 designates 81nmt1-spn1⁺ 81nmt1-spn4⁺. (B, J, K, N, and O) Cells were grown in EMM5S without thiamine and then shifted to EMM5S + thiamine at 30° for 24 hr to repress the nmt1 promoters. (A, C, L, and P) Cells were grown in YE5S at 30°. (D, I, and M) Cells were grown overnight in EMM5S at 32° and then shifted to 23° for 8 hr. (E) Cells were grown on YE5S at 23° for 5 days. (F and G) Cells were grown on EMM5S + thiamine at 36° for 3 days. (H) Cells were grown on YE5S at 30° for 2 days. Arrowheads in C, D, M, and P indicate cells that appear to have lysed; such cells are numerous in J, K, N, and O. Bars, 10 μm.

Figure 2.—

Genetic interactions among AMR and septin mutations. (A–D) Synthetic lethality between cdc4-s16 and myp2Δ. (A) Strains JW400 (cdc4-s16) and TP5 (myp2Δ) were crossed, and tetrads were dissected and incubated on YE5S medium at 32° for 6 days. Nine tetrads were tetratypes (i.e., contained one wild-type spore, two single-mutant spores, and one inviable or very slow-growing double-mutant spore) and one was a parental ditype (i.e., each spore was a single mutant and thus grew well). (B–D) DIC micrographs of aberrant cytokinesis in strains JW400, TP5, and JW345 (cdc4-s16 myp2Δ). Cells were grown overnight in YE5S liquid medium at 30°. Bar (B–D), 10 μm. (E and F) Absence of synthetic interaction between myo2-E1 myp2Δ and spn1Δ. (E) Strains TP90 (myo2-E1 myp2Δ) and JW380 (myp2Δ spn1Δ) were crossed, and tetrads were dissected and incubated on a YE5S plate at 25° for 7 days. Circles, myo2-E1 myp2Δ segregants; squares, myo2-E1 myp2Δ spn1Δ segregants. (F) Cells of strains TP90 and JW2560 were grown exponentially in YE5S liquid medium at 25°, shifted to 36° for 4 hr, and examined. Bar, 10 μm.

Figure 3.—

Cell-lysis phenotype of the s34 spn1 double mutant and normal septin localization in the s34 and s44 mutants. (A) Lysis during cell separation in s34 spn1Δ strain JW374. Cells were grown to exponential phase in EMM5S + 1 m sorbitol at 30°, washed twice in EMM5S, and observed by time-lapse DIC photomicroscopy on EMM5S + 25% gelatin at 24° (see materials and methods). Selected images are shown from a series recorded at 30-sec intervals; times are indicated in hours, minutes, and seconds. Cells are labeled for reference in the text. The entire series can be viewed in File S1. (B) Localization of Spn1 in s34 spn1-mEGFP strain JW1126 (left panels) and s44 spn1-mEGFP strain JW1125 (right panels). Cells were grown in YE5S at 25° and observed by DIC and fluorescence microscopy. Arrowheads in B, lysed cells. Bars, 10 μm.

Figure 4.—

Independence of septin and Rho-GEF localization to the division site. (A) Relationships among Rho-GEFs from S. pombe (http://www.genedb.org/genedb/pombe/) and S. cerevisiae (http://www.yeastgenome.org/), as well as a Rho-GEF from Drosophila (Pebble) and one from humans (ECT2) that have also been implicated in cytokinesis (Miki et al. 1993; Prokopenko et al. 1999; Tatsumoto et al. 1999; O'Keefe et al. 2001; Giansanti et al. 2004; Shandala et al. 2004). Clustal W was used to align the Rho-GEF domains and then derive a phylogenetic tree based on this alignment. (B and C) Localization and dynamic behavior of Rgf1 and Rgf3 at the division site in relation to the septins and other cell-division markers in wild-type cells. Strains JW1113 (spn1-mEGFP sad1-mRFP1), JW1124 (rgf1-GFP sad1-mRFP1), and JW1131 (rgf3-mEGFP sad1-mRFP1) were filmed in EMM5S at 25°. (B) Kymographs constructed using Image J and the movements of the spindle-pole bodies (SPBs), as marked by Sad1-mRFP1, to allow alignment at the time of SPB separation (vertical line). For the SPB, a 13.7-μm slit parallel to the long axis of the cell was used; for the other proteins, a 4.1-μm slit across the midplane of the cell was used. (C) Timeline showing the initial appearance of Spn1 (n = 8 cells) and Rgf1 (n = 8) at the division site. SPB separation is defined as time zero, and the mean time of appearance of each protein (±1 standard deviation) is plotted. The beginning of septation was scored by DIC. The data for Cdc4 are from Wu et al. (2003). (D and E) Tests of possible septin dependence of Rho-GEF localization. (D) Strains JW1124 (rgf1-GFP), JW1139 (rgf1-GFP spn1Δ), JW1131 (rgf3-mEGFP), and JW1128 (rgf3-GFP spn1Δ) were grown in EMM5S (JW1124 and JW1131) or YE5S (JW1139 and JW1128) medium at 25° and examined by DIC and/or fluorescence microscopy. (E) Strain JW1128 was examined by time-lapse microscopy. Selected DIC and GFP images (elapsed times given in minutes) are shown from a series recorded at 1-min intervals. The entire series can be viewed in File S2. (F) Cell morphology and septin localization in cells overexpressing Rgf1 or Rgf3. Strains JW1123 (spn1-mEGFP 3nmt1-rgf1) and JW1122 (spn1-mEGFP 3nmt1-rgf3) were grown under inducing conditions (EMM5S medium) for 24 hr at 25°; paired DIC and fluorescence images are shown. Arrowheads indicate a cell with a region of thickened cell wall. Bars (B and D–F), 10 μm.

Figure 5.—

Characterization of mutant ng124 and its identification as an allele of scw1. (A and B) Selected DIC and fluorescence images are shown from time-lapse-microscopy series recorded at 1- or 2-min intervals; times are indicated in hours and minutes. The entire series can be viewed in File S3 and File S4. (A) Cell-separation defect in an ng124 spn1Δ double mutant. Strain JW321-1 was grown in YE5S medium at 30° and observed on YE5S at 24°. Arrows indicate abnormal-shaped septa; arrowheads indicate cells that appear to have lysed. (B) Apparently normal septin localization and variable cell-separation delay in an ng124 single mutant. Strain JW320 (ng124 spn4-GFP) was grown in EMM5S at 23° and observed on EMM5S at 24°. Cells are numbered for reference in the text. (C) Noncomplementation of ng124 and scw1Δ in diploid cells. Diploid strains JW2613 (scw1⁺/ng124) and JW2614 (scw1Δ/ng124) were observed by DIC microscopy after growth on YE5S-Ade at 25°. (D) Rescue of ng124 by plasmids containing scw1⁺. Strain JW2155 (ng124) was transformed with empty vector or with chromosome III plasmids that contain only scw1 (nucleotides 673,476–675,161) in their region of overlap (nucleotides 672,482–675,308), grown in EMM5S-Leu medium at 25°, and observed by DIC microscopy. Bars, 10 μm.

Figure 6.—

Functional relationships among the septins, Mid2, and Scw1. Cells were observed by spinning-disk confocal microscopy (A–F) or by wide-field fluorescence microscopy (G–J) after growth in YE5S at 25° (A–G) or EMM5S at 30° (H–J). In each case, the cells shown are representative of large numbers of cells examined. For the fluorescence images in A–F, stacks of 70 z-sections (0.07-μm) were captured using the same laser power and acquisition settings and projected into three-dimensional images using Image J (see materials and methods). Selected views of the three-dimensional images are shown with corresponding DIC images; the entire series can be viewed in File S5, File S6, File S7, File S8, and File S9. (A and B) Localization of Spn1-GFP in wild-type strain JW306. The images in B are a stereo pair. (C) Localization of Spn4-GFP in scw1-ng124 strain JW320. (D) Localization of Mid2-GFP in wild-type strain JW326. (E) Localization of Spn1-GFP in mid2Δ strain JW318. (F) Localization of Mid2-GFP in scw1-ng124 strain JW1203. (G) Localization of Mid2-GFP in spn4Δ strain JW332. (H–J) Mutant phenotypes of strains (H) JW430 (mid2Δ), (I) JW315 (scw1-ng124 mid2Δ), and (J) JW314 (spn4Δ mid2Δ). Cells were stained with bisbenzimide to view nuclei and septa. Bars, 10 μm.

Figure 7.—

Possible organization of parallel pathways that cooperate to allow successful cytokinesis, septation, and cell separation in fission yeast, as suggested by the results of this study. Boldface type indicates the products of genes identified in this study. The cell-wall-integrity pathway headed by Wsc1 contains additional proteins beyond those shown here; the degree to which this pathway participates in unperturbed cytokinesis is not known. See text for details.