Mitosis and cytokinesis defects in sfi1-M46 cells. (A) S. pombe cell cycle. Red dots, SPBs; blue circles, nuclei; green line, spindle; purple circles, contractile rings; black, cell wall and septum. Red open circles represent new SPBs that are being assembled; different stages of assembly are not distinguished here. In the rightmost cell, the duplicated SPBs are connected by a bridge before spindle formation and are not resolvable by light microscopy at this stage. (B) DIC images of wt (strain JW729) and M46 (JW3887) cells grown in YE5S medium at 25ºC. The various morphological defects are indicated by numbers: 1) multiple septa; 2) aberrant septa; 3) septum in a short cell; 4) cell lysis; 5) lack of septum in an elongated cell. Arrowheads mark septa. (C, D) Percentages of septating cells with more than one septum (C) and of septating cells <9 μm (D) in wt (JW729) and M46 (JW3887) cells. Cells were grown in YE5S at 25°C or shifted to 36°C for 4 h before imaging. Means ± SDs from three independent experiments. n > 40 septating cells for each experiment. (E) Schematic representation of Sfi1 domains. W, the Trp-containing repeat; the repeat with a mutated Trp in sfi1-M46 is shown in red. (F) Time courses of localization of Cdc7-EGFP and Rlc1-tdTomato in sfi1⁺ (JW4558) or sfi1-M46 (JW4557) cells. Cdc7 signal is indicated by arrowheads. Asterisks, the abnormal septum. In this and other figures, dashed lines mark cell boundaries. (G) Mitotic defects revealed by Hoechst 33258 staining of sfi1-M46 cells; the dye stains both DNA and newly formed septa. Green box, cells with one septum and defective DNA separation; blue box, a cell with abnormal DNA but no septum; red box, cells with multiple or abnormal septa. Most such cells also show mitotic defects, but in ∼15%, no mitotic defect is evident by DNA staining. Bars, 5 μm.

Sfi1 is essential for bipolar spindle formation. (A) Dissected tetrads from sfi1∆/sfi1⁺ diploid strain (JW5364) after 7 d on YE5S plate at 25°C. Colonies in each row are from the same tetrad. (B) Cell numbers in sfi1∆ microcolonies after tetrads were incubated on YE5S plates at 25°C for 12 d. (C, D) Cells expressing both Pcp1-GFP (green) and mRFP-Atb2 (red). (C) The first mitotic division of the four germinating spores from one sfi1∆/sfi1⁺ tetrad imaged with an interval of 10 min. Time zero indicates SPB separation. See Supplemental Video S1 for the complete time series. (D) Monopolar spindle formation in sfi1∆ segregants starting from the second mitotic division. Colonies in the same row are from the same sfi1∆/sfi1⁺ tetrads, and three tetrads imaged at different times are shown. DIC images of one wt and one sfi1∆ colony in each tetrad are shown. sfi1⁺ colonies at 2 d had grown bigger than the imaged field, so that only the colony edges were imaged. Arrowheads, monopolar spindles. See Supplemental Video S2 for the complete time series. Bars, 5 μm.

Localization and recruitment of Sfi1 to SPBs. (A) Approximate colocalization of Pcp1-GFP and Sfi1-tdTomato (strain JW4685). (B) Spatial relationships between Pcp1-GFP and Sfi1-tdTomato in a cell entering mitosis (JW4685). The first time point with two resolvable Pcp1 foci was defined as time zero. Left, maximum projections of stacks covering the whole cell; right, twofold blow-ups of the images on the left. Bar on the right, 1 μm. (C) Numbers of Sfi1 molecules on each SPB at different cell cycle stages in an asynchronous cell population. For cells in mitosis or septation, each SPB was measured separately for this and other figures. The mean numbers of Sfi1 molecules on each SPB in septating (n = 56) and late interphase (length ≥12 μm; n = 23) cells were 220 and 360, respectively, and are indicated by horizontal lines. (D) Numbers of Sfi1 molecules on SPBs over time in individual wt cells (JW4361). Each gray line represents one SPB. Time zero is the first time point with two resolvable SPBs in that cell. Mean molecules (±SEM) of 20 separated SPBs are plotted in red (before) and purple (after cell separation). Bars, 5 μm (except where noted).

Regulation of SPB assembly and mitosis by Sfi1. (A) Localization of Sad1 and Sfi1 in sfi1⁺ (JW4841) and sfi1-M46 (JW4842) cells. Percentages of cells with detectable Sfi1-mEGFP signal at the Sad1-tdTomato foci (SPBs) are shown on the right. (B) Monopolar-spindle formation by some sfi1-M46 cells (asterisks). Arrowheads mark bipolar spindles. Strains were JW5285 and JW5377. The percentages of spindles that were bipolar spindles are shown below the images. (C, D) Localization of Sfi1-mEGFP in sfi1⁺ (JW4829) and sfi1-M46 (JW4831) cells. Spindles are marked by mRFP-Atb2. (C) Apparent absence of Sfi1-M46 from SPBs in cells with monopolar spindles. (D) Absence of detectable Sfi1-M46 at the poles of some bipolar spindles. Percentages of cells with Sfi1-M46 on 2, 1, or 0 SPBs are shown at the bottom. Bars, 5 μm (A–D). (E–H) Electron micrographs of sfi1⁺ cdc10-V50 (JW5132; E and F) and sfi1-M46 cdc10-V50 (JW5130-3; G and H) cells grown at 36°C for 4.5 h. Graphic illustrations are shown below or on the right of micrographs. (F–H) Thin sections spaced at 70 nm of the same cell are shown in each panel. B, the bridge; C, cytoplasm; CP, central plaque, N, nucleus; SPB, spindle pole body. Arrows indicate nuclear envelopes; black arrowhead, invaginated nuclear envelope; white arrowhead, bulged nuclear envelope; black asterisks, dark-stained material deposited close to invaginated nuclear envelope; white asterisks, dark-stained material in the cytoplasm. Bars, 100 nm (E–H).

Preferential localization of Sfi1-M46 to the SPB that does not retain Cdc7 during cell division. (A–C) Quantification of fluorescence intensity differences between the two SPBs in individual cells of strains JW4841 (sfi1⁺) and JW4842 (sfi1-M46). (A) Representative images of Sfi1 and Sad1 and method of calculation. (B, C) Measured differences in Sfi1 (B) and Sad1 (C) intensities. Asynchronous cells with SPBs at a variety of distances from each other were scored. (D, E) Three patterns of Sfi1partitioning in sfi1⁺ (JW5203) and sfi1-M46 (JW5215) cells during division. (D) Images were collected at 10-min intervals; the first time with two distinct Sad1-marked SPBs is set as 0. Pattern I, Sfi1 detectable at both SPBs; pattern II, Sfi1 detectable only at the SPB that does not retain Cdc7 signal; pattern III, Sfi1 detectable only at the SPB that retains Cdc7 signal. Bars, 5 μm. (E) Quantification of cells displaying pattern I, II, or III (or no detectable Sfi1 signal) at the 30-min time point (D).

Unequal partitioning of Sfi1-M46 underlies the mitotic defects. (A) Illustration of SPB-assembly cycle in sfi1⁺ and sfi1-M46 cells (see the text for details). (B) Illustration of imaging cells for two cell cycles. SPB color code as used in E and F. (C, D) Representative images from a time-lapse series with sfi1⁺ (C; strain JW4829) and sfi1-M46 (D; strain JW4831) cells. See Supplemental Videos S3 and S4 for the full series. Arrowheads, bipolar spindles; asterisks, monopolar spindles; arrow, the appearance of Sfi1-M46 in a cell that had no detectable Sfi1 signal on the SPB after the previous mitosis. Bars, 5 μm. (E, F) Sfi1 levels on SPBs through two cell division cycles in sfi1⁺ (E) and sfi1-M46 (F) cells. Each line represents one SPB. Time zero is when the first SPB separation occurs. Dashed lines, SPBs without detectable signals at those time points. Asterisk, monopolar spindle formation; a.u., arbitrary units. (G) Quantification of numbers of Sfi1 foci in sfi1⁺ and sfi1-M46 cells during two consecutive mitoses. Daughter cells generated from the first mitosis were analyzed separately in the second mitosis.

The repeats in Sfi1 are all important but are not identical. (A) The positions of the sfi1 W-to-R mutations examined in this study. (B, C) Phenotypes of the mutant strains (JW5308–JW5311) grown at 25°C or 4 h after a shift to 36°C. (B) DIC and DNA staining; arrowheads, cells with mitosis and/or cytokinesis defects. (C) Quantification of cytokinesis defects as indicated by the presence of more than one septum. Means and SDs from three independent experiments are shown. n > 50 septating cells for each experiment. (D) SPBs (Pcp1) and spindles (Atb2) in the mutants (JW5285, JW5379, JW5380, JW5381, and JW5475) at 25 or 36ºC. White dashed lines outline mitotic cells; solid white line, a cell apparently without an SPB; arrowheads, bipolar spindles; asterisks, monopolar spindles. (E) Localization of Sad1 and mutant Sfi1 proteins at 25°C (strains JW4841, JW5409, JW5527, JW5410, and JW5474). Arrowheads, SPBs with Sad1 but not Sfi1; asterisk, an elongated cell with a single SPB focus containing Sad1 and Sfi1(W763R). Bars, 5 μm.

Summary of the apparent patterns of Sfi1 recruitment and SPB assembly in wt cells and in mutants that inherit different amounts of Sfi1 at the preceding mitosis.