Specific detection of fission yeast primary septum reveals septum and cleavage furrow ingression during early anaphase independent of mitosis completion

<div><p>It is widely accepted in eukaryotes that the cleavage furrow only initiates after mitosis completion. In fission yeast, cytokinesis requires the synthesis of a septum tightly coupled to cleavage furrow ingression. The current cytokinesis model establishes that simultaneous septation and furrow ingression only initiate after spindle breakage and mitosis exit. Thus, this model considers that although Cdk1 is inactivated at early-anaphase, septation onset requires the long elapsed time until mitosis completion and full activation of the Hippo-like SIN pathway. Here, we studied the precise timing of septation onset regarding mitosis by exploiting both the septum-specific detection with the fluorochrome calcofluor and the high-resolution electron microscopy during anaphase and telophase. Contrarily to the existing model, we found that both septum and cleavage furrow start to ingress at early anaphase B, long before spindle breakage, with a slow ingression rate during anaphase B, and greatly increasing after telophase onset. This shows that mitosis and cleavage furrow ingression are not concatenated but simultaneous events in fission yeast. We found that the timing of septation during early anaphase correlates with the cell size and is regulated by the corresponding levels of SIN Etd1 and Rho1. Cdk1 inactivation was directly required for timely septation in early anaphase. Strikingly the reduced SIN activity present after Cdk1 loss was enough to trigger septation by immediately inducing the medial recruitment of the SIN kinase complex Sid2-Mob1. On the other hand, septation onset did not depend on the SIN asymmetry establishment, which is considered a hallmark for SIN activation. These results recalibrate the timing of key cytokinetic events in fission yeast; and unveil a size-dependent control mechanism that synchronizes simultaneous nuclei separation with septum and cleavage furrow ingression to safeguard the proper chromosome segregation during cell division.</p></div

The onset of septation depends on the inactivation of Cdc2/Cdk1 kinase in fission yeast.

<p><b>(A)</b> Septation start in wild-type cells coincides with the decline of Cdk1-associated cyclin B Cdc13. <b>(B)</b> Short cells display premature septation onset that coincides with the anaphase B onset and the decline of nuclear Cdc13. <b>(C)</b> The initiation of septum deposition is significantly delayed in long cells but coincides with the decline of nuclear Cdc13. Cells were grown in YES at 25°C (A and B) or released at 25°C after 3.5 h of cell cycle arrest at 36°C (C), and imaged as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.g001" target="_blank">Fig 1</a>. Graphs to the right show the total fluorescence of Cdc13-GFP in the nuclei in the series to the left. Dashed lines in graphs correspond to the arrowheads in the series. A.U., arbitrary units. Nuclear Cdc13-GFP was quantified as described in the Material and Methods section. <b>(D)</b> The septation onset coincides with the decline of nuclear Cdc13 during early anaphase B. Graph showing the levels of nuclear Cdc13 fluorescence in the population of wild-type (n = 2, 14 cells), long (n = 2, 8 cells) and short cells (n = 2, 15 cells) in the indicated mitotic stages in the cells of A, B and C. Cells were grown and imaged as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.g001" target="_blank">Fig 1</a>, and Cdc13 was quantified at the indicated stages as in A. Blue arrow indicates the levels of Cdc13 at septation onset. Error bars indicate standard deviation (SD). <b>(E, F)</b> Inactivation of Cdc2 kinase in early mitosis induces a very premature septation onset. (E) ATP-analogue sensitive <i>cdc2-asM17</i> mutant cells carrying Cdc13-GFP were G2-arrested by growth in the presence of 1 μM 1-NP-PP1 for 3.5 h at 32°C. Then, the cells were G2-released by transfer to a fresh medium and imaged to detect the entry into mitosis. Cdc2 was inactivated during early mitosis transferring the cells to a fresh medium containing either DMSO (control cells; n = 2, 14 cells) or 10 μM 1-NP-PP1 (cells with inactive Cdk1; n = 4, 37 cells). Cells were imaged at 32°C as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.g001" target="_blank">Fig 1</a>. (F) Graph showing the elapsed time in the indicated mitotic intervals of cells in E. The asterisks indicate the significant statistical difference between paired strains analyzed by the Student’s test: * p <0.05; ** p <0.01; *** p <0.001; NS: not significant (p >0.05). Error bars indicate standard deviation (SD). Symbols are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.g001" target="_blank">Fig 1</a>. Bars, 5 μm.</p

Time intervals of the different mitotic and cytokinetic events at 25°C.

<p>Time intervals of the different mitotic and cytokinetic events at 25°C.</p

Septum and cleavage furrow initiation overlaps with the early stages of anaphase B in fission yeast.

<p><b>(A)</b> Scheme of the mitotic phases analyzed in this study. Prophase begins with the duplication of the spindle pole body (SPB) and continues with the elongation of the mitotic spindle (blue). In metaphase the spindle does not change in length. The cyclin B Cdc13 (green) localizes to the spindle pole bodies (SPB) and mitotic spindle during prophase and metaphase. In anaphase A, Cdc13 relocates to the nuclear envelope (red), and is then degraded. Anaphase B starts with spindle and chromosome mass elongation. Finally, spindle disassembly and retraction of the nuclei from the poles indicate the exit from mitosis and the start of telophase. During telophase the nuclei are stably located away from the cell middle by the postanaphase array (PAA) of microtubules (MT). <b>(B, C)</b> Septum deposition starts at early anaphase B, long before the spindle disassembly. Wild-type cells carrying Hht1-RFP (histone H3) and GFP-Atb2 (MT) or Cdc13-GFP were grown in YES with Calcofluor white (CW, 5 μg ml^-1) at 28°C and imaged by time-lapse fluorescence microscopy (1 medial z slice, 1 min elapsed time). The data of this figure are developed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.t001" target="_blank">Table 1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.t003" target="_blank">Table 3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.s009" target="_blank">S1 Table</a>. White arrow: first CW-stained septum synthesis. Arrowheads: black, prophase onset; green, anaphase B onset (time 0); blue, septum deposition onset (time immediately before septum detection with CW); red, telophase onset. <b>(D-F)</b> Septum formation during early anaphase B implies concomitant plasma membrane invagination (cleavage furrow formation) and closure of a contractile actomyosin ring (AR). Transmission electron microscopy images of wild-type cells as in B (D) and magnifications of the division site (E, F), showing the initial primary septum (PS) emergence in anaphase B and the septum (S) in telophase, were examined. A higher magnification of the PS of early anaphase B cells of D and E is shown (F). The distance between nucleus middle (N) and septum is shorter in cells with a nascent PS (arrows to the left, 1.9 to 2.4 μm, early anaphase B, nuclei close to the cell middle) than in cells with larger septum (brackets, 3.3 to 4.3 μm, telophase, nuclei stably located away from the cell middle). The spindle size, calculated as the distance between nuclei ends (arrows to the right), is shown. <b>(G)</b> The septum exhibits two different ingression rates, very slow during anaphase B and much faster during telophase. Top, kymographs and schemes showing the progression of septum and GFP-Bgs1 ring ingression during both anaphase B and telophase. Wild-type cells carrying Hht1-RFP and GFP-Atb2 (left; n = 6, ≥25 cells) or GFP-Bgs1 (right; n = 3, ≥22 cells) were grown and imaged as in B. Arrowheads are as in B. Bottom, graph showing the rate of septum and GFP-Bgs1 ring ingression during the indicated mitotic intervals. Error bars indicate standard deviation (SD). Bars, 5 μm (B, C) and 2 μm (D-G).</p

The levels of Etd1 and Rho1 regulate the timing of septation start.

<p><b>(A)</b> The timing of septum deposition onset correlates with the start of increase of Etd1 in the cell middle. Cells were grown in MM without thiamine (<i>GFP-etd1</i>^<i>+</i> induced) at 25°C for 24 h and imaged as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.g001" target="_blank">Fig 1</a>. <b>(B)</b> The increase of Etd1 in the cell middle and concomitant initiation of septation are delayed in long cells. Cells were analyzed as in A after 3.5 h of cell cycle arrest at 36°C. Graphs to the right show the total fluorescence of GFP-Etd1 at the cell poles and middle in the series to the left. A.U., arbitrary units. Arrow, cortical localization of Etd1 in the cell middle. Dashed outlines indicate the ROIs used to measure the total fluorescence of GFP-Etd1 in the corresponding regions of the cell. <b>(C)</b> The timing of septation onset is dependent on the level of <i>etd1</i>^<i>+</i>. Cells expressing endogenous <i>etd1</i>^<i>+</i> and 41X<i>-GFP-etd1</i>^<i>+</i> grown at 32°C for 24 h either in the absence (ON, high <i>etd1</i>^<i>+</i> level; n = 4, 34 cells) or in the presence of thiamine (OFF, wild-type <i>etd1</i>^<i>+</i> level; n = 2, 11 cells), and cells expressing <i>etd1</i>Δ 81X<i>-etd1</i>^<i>+</i> grown for 15 h with thiamine (OFF, very low <i>etd1</i>^<i>+</i> level; n = 3, 23 cells), just before the emergence of SIN phenotype were analyzed as in A. <b>(D, E)</b> The start of septum synthesis does not dependent on cortical Etd1. Cells expressing a functional Etd1 version that is absent from the cortex from <i>etd1</i>Δ 41X-<i>GFP-etd1</i>-∆<i>9</i> strain grown at 32°C either for 6 to 9 h without thiamine (ON; n = 2, 37 cells) or with thiamine (OFF; n = 2, 17 cells) were analyzed as in A. <b>(F)</b> The start of septum deposition is dependent on the level of <i>rho1</i>^<i>+</i>. Cells expressing endogenous <i>rho1</i>^<i>+</i> and 3X<i>-rho1</i>^<i>+</i> grown at 32°C for 16 h either without (ON, high <i>rho1</i>^<i>+</i> level; n = 2, 14 cells) or with thiamine (OFF, wild-type <i>rho1</i>^<i>+</i> level; n = 2, 16 cells) were analyzed as in A. The asterisks in C, E and F indicate the significant statistical difference between paired strains analyzed by the Student’s test: * p <0.05; ** p <0.01; *** p <0.001; NS: not significant (p >0.05). Symbols are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.g001" target="_blank">Fig 1</a>. Bars, 5 μm.</p

The elapsed time from telophase onset to septum closure correlates inversely with the cell size.

<p>Wild-type (n = 2, 10 cells) <b>(A)</b>, cell cycle-short <i>wee1-50</i> (n = 3, 10 cells) <b>(B)</b>, and cell cycle-long <i>cdc25-22</i> (n = 3, 10 cells) and <i>cdc10-119</i> (n = 2, 10 cells) <b>(C)</b> cells were grown as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.g002" target="_blank">Fig 2</a> and imaged as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.g001" target="_blank">Fig 1</a>. <b>(D)</b> The elapsed time from septation onset to septum closure is similar in wild-type and long cells, and increased in short cells. <b>(E)</b> The elapsed time from telophase onset to septum closure is reduced in long cells and increased in short cells with respect to that of wild-type cells. Graphs show the elapsed time for the indicated intervals in the cells of A, B and C. Error bars indicate standard deviation (SD). <b>(F)</b> Scheme of the percentages of elapsed times shown in E. The cell width (μm ± SD) and rate of septum ingression (nm/min ± SD) for each strain are shown. Brown arrowhead, septum closure. Other symbols are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.g001" target="_blank">Fig 1</a>. Bars, 5 μm.</p

The activation of septum synthesis in early anaphase B depends on SIN function, but not on the asymmetric SIN in the SPB.

<p><b>(A)</b> Sid2-Mob1 kinase complex localizes to the division site just after Bgs1 localization and coincident with the septation onset. Cells were grown and imaged as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.g001" target="_blank">Fig 1</a>. <b>(B)</b> Septation start is delayed when the function of Sid2 is compromised. Cells were grown in YES at 25°C, shifted to 28°C for 4 h and imaged as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.g001" target="_blank">Fig 1</a>. The data of cells of B are developed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.t001" target="_blank">Table 1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.t003" target="_blank">Table 3</a>. <b>(C, D)</b> Septation start coincides with the asymmetric disappearance of Cdc7 from one SPB. Cells were grown in YES at 25°C (wild-type, C) or released at 25°C after 3.5 h of cell cycle arrest at 36°C (<i>cdc25-22</i>, D), and imaged by time-lapse fluorescence microscopy (maximum-intensity projections of 7 z slices at 0.4 μm intervals for Cdc7-GFP and 1 medial z slice for CW-staining, 1 min elapsed time). <b>(E)</b> Timely activation of septum synthesis does not depend on SIN asymmetry. Defective SIN-Inhibitory Phosphatase (SIP) complex <i>csc2</i>Δ cells were examined as in C. The data of cells of C, D and E are developed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.s010" target="_blank">S2 Table</a>. Graphs to the right show the total fluorescence of Cdc7-GFP in each SPB in the series to the left. Dashed lines in graphs correspond to the arrowheads in the series. A.U., arbitrary units. Symbols are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.g001" target="_blank">Fig 1</a>. Bars, 5 μm.</p

The premature and uncoupled septation start caused by the absence of spindle MT is dependent on the SIN pathway but not on the spindle assembly checkpoint.

<p><b>(A)</b> The absence of spindle MT induces a dramatically premature and uncoupled septum synthesis start and ingression onset even in the presence of the spindle assembly checkpoint. Wild-type cells containing <i>mad2</i>⁺ gene were processed with MBC as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.g007" target="_blank">Fig 7C</a> to avoid spindle assembly and nuclear separation. <b>(B)</b> The premature septum synthesis initiation of A (also compare with values in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.g007" target="_blank">Fig 7F</a>) depends on the SIN. <i>sid2-250</i> cells grown at 28°C for 3 h were treated with MBC as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.g007" target="_blank">Fig 7C</a>. <b>(C)</b> Uncoupled timings of septum synthesis and septum ingression in the presence of MBC are restored to wild-type levels when the SIN activity is affected. Table showing the time between anaphase A (green) and septum synthesis start (dark blue) or septum ingression onset (light blue) in the indicated cells. Symbols and values are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.g007" target="_blank">Fig 7</a>. A.U., arbitrary units. Bars, 5 μm. <b>(D)</b> Model for activation of septum and cleavage furrow start during early anaphase B. At early anaphase B, inactivation of Cdk1 (red, cyclin Cdc13) allows increase of SIN signaling (green, SIN Cdc7 of SPB) by enrichment of the SIN activator Etd1 (blue) in the cell middle. Initial SIN activation triggers Sid2 relocation to the division site and activation PS synthesis start by Bgs1 GS. Septum ingression is slow during anaphase B, and greatly increases at telophase onset coinciding with the spindle disassembly, the full SIN activation and the stable localization of Ags1 and Bgs4 GSs to the septum edge membrane.</p

The spindle and the proximity of the nucleus to the division site are required for proper septum synthesis activation in fission yeast.

<p><b>(A)</b> Scheme of the steps required to prevent nuclear separation and to maintain or separate the undivided nucleus from the cell middle and/or from the division site. (A-1 and <b>C</b>) Nucleus and division site are maintained in the cell middle; cells were treated for 90 min and imaged with methyl 2-benzimidazolecarbamate (or carbendazim, MBC, 50 μg ml^-1) to avoid spindle assembly and nuclear separation. (A-2 and <b>D</b>) Nucleus and division site are relocated to a cell end; cells were treated for 45 min, centrifuged to displace the nucleus, treated 45 more min and visualized with MBC. (A-3 and <b>E</b>) The nucleus is relocated and separated from the division plane; cells were treated for 90 min, centrifuged and examined with MBC. <b>(B)</b> <i>mad2</i>Δ cells were grown and imaged without MBC as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.g001" target="_blank">Fig 1</a>. The <i>mad2</i>Δ cells were used to avoid a delay caused by the activation of the spindle assembly checkpoint. <b>(C-E)</b> The premature and uncoupled septation start caused by the absence of the spindle depends on the position of the nucleus. <i>mad2</i>Δ cells were processed as in A to prevent nuclear separation and to maintain or separate the undivided nucleus from the cell middle and/or the division site. (C) The nucleus and division site are maintained in the cell middle. (F) The nucleus and division site are relocated to a cell end. (E) The nucleus is relocated and separated from the division plane. MBC-treated cells were imaged as in B. Anaphase A onset was considered as time zero. Graphs to the right are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007388#pgen.1007388.g004" target="_blank">Fig 4</a>. Dashed lines and arrowheads: green, anaphase A onset; dark blue, septum synthesis start; light blue, septum ingression onset. White arrowhead: first CW-stained septum synthesis detection. White arrow: first CW-staining increase showing septum ingression. A.U., arbitrary units. <b>(F)</b> Uncoupled septum synthesis and ingression timing with MBC is restored to wild-type levels when the undivided nucleus is separated from the division site. Table showing the time between anaphase A (green) and septum synthesis start (dark blue) or septum ingression onset (light blue) in the indicated cells. Parenthesis: n, number of experiments and cells; T, delay in septum synthesis and ingression start with respect to control cells with MBC as in C. Bars, 5 μm.</p