Cdk1 promotes cytokinesis in fission yeast through activation of the septation initiation network

Abstract

In Schizosaccharomyces pombe, late mitotic events are coordinated with cytokinesis by the septation initiation network (SIN), an essential spindle pole body (SPB)-associated kinase cascade, which controls the formation, maintenance, and constriction of the cytokinetic ring. It is not fully understood how SIN initiation is temporally regulated, but it depends on the activation of the GTPase Spg1, which is inhibited during interphase by the essential bipartite GTPase-activating protein Byr4-Cdc16. Cells are particularly sensitive to the modulation of Byr4, which undergoes cell cycle-dependent phosphorylation presumed to regulate its function. Polo-like kinase, which promotes SIN activation, is partially responsible for Byr4 phosphorylation. Here we show that Byr4 is also controlled by cyclin-dependent kinase (Cdk1)-mediated phosphorylation. A Cdk1 nonphosphorylatable Byr4 phosphomutant displays severe cell division defects, including the formation of elongated, multinucleate cells, failure to maintain the cytokinetic ring, and compromised SPB association of the SIN kinase Cdc7. Our analyses show that Cdk1-mediated phosphoregulation of Byr4 facilitates complete removal of Byr4 from metaphase SPBs in concert with Plo1, revealing an unexpected role for Cdk1 in promoting cytokinesis through activation of the SIN pathway.

FIGURE 1

Byr4 is a Cdk1 substrate. (A) Top, recombinant MBP-purified proteins were treated in vitro with kinase-active (KA) or kinase-dead (KD) Cdk1 complex (Cdc2-Cdc13) and subjected to SDS–PAGE, followed by autoradiography. Bottom, Coomassie blue (CB) gel of purified recombinant MBP, MBP-Byr4, and MBP-Byr4(7A) proteins. (B) Byr4 and Byr4(7A) were immunoprecipitated from mitotically arrested cells of the indicated backgrounds. Gel shifts were visualized by anti-Byr4 immunoblotting of Phos-tag gels (2 μM) and incubation of immunoprecipitates with (+) or without (−) λ-phosphatase (λ-PPase). Anti-PSTAIRE antibody served as loading control for lysates used in immunoprecipitations.

FIGURE 2

Preventing Cdk1 phosphorylation of Byr4 causes cytokinetic defects. (A) Wild type, byr4(7A), and a strain deleted for byr4 (byr4Δ mob1-R4) were grown to early-mid log phase at 25ºC. Cells were fixed and stained with DAPI and methyl blue. Representative images. Scale bar, 3 μm. Cells displaying an abnormal septation phenotype are quantified (n = 800). (B) Representative image of time-lapse microscopy (20-min intervals) of byr4(7A) cells. Arrow highlights protrusion at division site. Scale bar, 5 μm. (C) Cells of the indicated genotypes were spotted on YE media in 10-fold serial dilutions, and plates were imaged after incubation for 3 d at the indicated temperatures.

FIGURE 3

Preventing Cdk1-mediated phosphorylation of Byr4 compromises SIN signaling. (A) Representative montages of time-lapse microscopy (5-min intervals) in the indicated genetic backgrounds (Supplemental Videos S2 and S3). Cdc15-GFP was used as ring marker and Nog1-mCherry as nucleolar marker. Scale bar, 3 μm. (B) Representative montages of time-lapse microscopy (5-min intervals) illustrating cytokinesis in a binucleate cell of the byr4(7A) background (Supplemental Video S4). Scale bar, 3 μm. (C) The dynamics of GFP-tagged Cdc7 SPB localization was monitored by time-lapse microscopy (5-min intervals) in the indicated genetic backgrounds (Supplemental Videos S5 and S6). The time point just before spindle pole body separation was set to 0 min. Sid4-RFP served as a constitutive SPB marker. Representative montages. Scale bar, 3 μm.

FIGURE 4

Cdk1 and Plo1 independently and additively mediate Byr4 removal from mitotic SPBs. (A) Byr4 localization was detected by indirect immunofluorescence or 3xGFP-tag (indicated by *, plo1-24c byr4-3xGFP). Wild-type and byr4(7A) cells were grown at 32ºC. Strains containing the temperature-sensitive plo1-24c allele were grown at the permissive temperature (25ºC) and, where indicated, shifted to the semipermissive temperature (32ºC) for 3 h before fixation. The distribution of Byr4 on mitotic SPBs in the respective strains at the indicated temperatures is illustrated. (B) Serial 10-fold dilutions of cells of the indicated genotypes were plated on YE media. Plates were imaged after incubation for 3 d at the indicated temperatures.

FIGURE 5

Byr4 phosphorylation by Cdk1 is important for cytokinesis. Cells of the indicated genetic backgrounds were grown at the permissive temperature (25ºC) for plo1-24c and arrested in S phase (HU) before a shift to the restrictive temperature (36ºC) for 3 h before fixation. Respective phenotypes were assessed by septa and nuclei staining and quantified (n = 500). Representative micrographs of the plo1-24c and plo1-24c byr4(7A) strains. Scale bar, 5 μm.

FIGURE 6

Schematic of Byr4 phosphoregulation. During mitosis, Cdk1 and Plo1 phosphorylate Byr4 independently, leading to the removal of Byr4 from SPBs and the inhibition of the GAP Byr4-Cdc16. Consequently, SIN activation and cytokinesis are induced.