Etd1p is a novel protein that links the SIN cascade with cytokinesis

Abstract

In animal cells, cytokinesis occurs by constriction of an actomyosin ring. In fission yeast cells, ring constriction is triggered by the septum initiation network (SIN), an SPB-associated GTPase-regulated kinase cascade that coordinates exit from mitosis with cytokinesis. We have identified a novel protein, Etd1p, required to trigger actomyosin ring constriction in fission yeasts. This protein is localised at the cell tips during interphase. In mitosis, it relocates to the medial cortex region and, coincident with cytokinesis, it assembles into the actomyosin ring by association to Cdc15p. Relocation of Etd1p from the plasma membrane to the medial ring is triggered by SIN signalling and, reciprocally, relocation of the Sid2p-Mob1p kinase complex from the SPB to the division site, a late step in the execution of the SIN, requires Etd1p. These results suggest that Etd1p coordinates the mitotic activation of SIN with the initiation of actomyosin ring constriction. Etd1p peaks during cytokinesis and is degraded by the ubiquitin-dependent 26S-proteasome pathway at the end of septation, providing a mechanism to couple inactivation of SIN to completion of cytokinesis.

Figure 1

etd1 is an essential gene required for cytokinesis. (A) etd1-1 is a conditional mutant that requires ethanol for growth. Wild-type and etd1-1 cells were grown at 25°C in YE medium containing 6% ethanol (permissive condition) and then replica-plated to medium without ethanol (restrictive condition). (B) etd1-1 cells grown under permissive (+ethanol) and restrictive conditions (−ethanol) for 4, 8 and 12 h were fixed and stained with DAPI to visualise DNA and calcofluor to visualise cell wall material. (C) One copy of the etd1 open reading frame (ORF) was replaced by ura4 (scheme) to construct a deletion allele (etd1Δ) in a diploid strain. Tetrad dissection of asci from this etd1Δ/etd1⁺ diploid strain is shown (left panel). etd1Δ spores were germinated in medium lacking uracil (middle panel) and the etd1Δ spores fixed and stained with DAPI (right panel). (D) Strong expression of etd1 also impairs cytokinesis. etd1-1 mutant cells expressing etd1 (cDNA) under the nmt1 thiamine-repressible promoter were grown in minimal medium containing thiamine. To determine the phenotype associated with overexpression of etd1, these cells were washed three times with thiamine-free medium and finally resuspended in medium with or without thiamine for 18 h at 25°C. Cells were fixed and stained with DAPI and calcofluor. Bars: 3 μm.

Figure 2

Etd1p localises to the division site at the end of mitosis. (A) Low-level expression of Etd1p-GFP (driven by the nmt41x promoter) was used to analyse the in vivo localisation of Etd1p. Cell 1 is in interphase, cell 2 is in early anaphase, cell 3 in late anaphase, cell 4 is undergoing septation, cell 5 formed the primary septum and cell 6 completed cytokinesis. GFP images (upper panels) and phase contrast (lower panels) are shown. Asterisks indicate localisations of Etd1p in cells 1 and 2. (B) Cell wall digested with novozyme and cell wall-free spheroplasts observed by fluorescence microscopy. Etd1p-GFP was associated with the plasma membrane. (C, D) cdc25-22 cells expressing Etd1p-GFP were synchronised by a cdc25-22 block–release protocol after 4 h of incubation at 36°C. Time-lapse images of living cells were collected every 5 min for 2 h after release at 25°C, using a confocal microscope. Two stacks of images were captured, one with a step size of 1 μm between focal planes (C) and the other of 0.3 μm serial sections to reconstruct three-dimensional images of the cell (D). (E) Time-lapse images in Etd1p-GFP Cdc7p-GFP living cells were used to determine precisely the transition of Etd1p from the cell tip to the cell centre during interphase (cell 1), entry into mitosis (cell 2)—as determined by Cdc7p association to the SPB—and initiation of anaphase (cell 3)—according to SPB segregation. (F) Localisation (asterisks) of Etd1p-GFP or Cdc15p-GFP—actomyosin ring marker—in nda3-arrested cells in metaphase (nda3-KM311 mutant background, incubated for 3 h at the restrictive temperature of 20°C). Cells incubated at the permissive temperature are shown as a control (32°C). In arrested cells, Cdc15p was always found in the medial ring while Etd1p was mainly in the cell tips (about 82%, cell 1) and a small fraction was at the middle of the cell (about 18%, cell 2). (G) Analysis of the relocation of Etd1p-GFP from a broad band (cell 1) to a compact ring (cell 2), in relation to Cdc7p-GFPU as described in panel E.

Figure 3

Assembly of Etd1p-GFP into the actomyosin ring. (A) Localisation of Etd1p-GFP in mid1-deleted cells (mid1Δ) and wild-type cells (wt). (B) Medial ring cdc8-110 and cdc15-140 thermo-sensitive mutant cells expressing Etd1p-GFP were grown to mid-exponential phase at 25°C; half of the culture was shifted to 36°C for 4 h, and living cells were photographed. (C) Protein extracts prepared from cells expressing Etd1p-GFP, Cdc15p-13Myc or both were immunoprecipitated with anti-Myc antibodies; the immunoprecipitates were run on SDS–PAGE gels and probed with anti-Myc and anti-GFP antibodies. Western blots of total extracts were also probed with anti-Myc and anti-GFP antibodies to check the levels of tagged proteins.

Figure 4

Actomyosin ring is assembled in the etd1-1 mutant but fails to contract. (A) Assembly of Cdc15p-GFP—a key component of the actomyosin ring—was imaged in living wild-type (upper panels) or etd1-1 mutant cells under the restrictive condition (lower panels) by time-lapse microscopy at 5 min intervals on a single focal plane with a step size of 0.3 μm. (B) Rhodamine-conjugated phalloidin was used to stain F-actin structures in etd1-1 mutant cells under the restrictive condition. Actin cables and patches are indicated. (C) The S. pombe coronin homologue crn1 tagged with GFP was used as a marker of F-actin patches. Living cells were imaged as described in panel A. (D) The myosin regulatory light-chain Rlc1p tagged with GFP was used as a marker of the actomyosin ring. Living cells were imaged as described in panel A.

Figure 5

Effects of Etd1p on SIN signalling. (A) Localisation of Mob1p-GFP to the division site depends on Etd1p. Mob1p-GFP was imaged in live wild-type cells (upper panels) or etd1-1 mutant cells (lower panels) under restrictive conditions (−ethanol) by time-lapse microscopy at 5 min intervals on a single focal plane. (B) Etd1p is required to maintain SIN active. Cdc7p-GFP was imaged by time-lapse microscopy at 5 min intervals on a single focal plane in living wild-type cells (upper panels) and etd1-1 mutant cells (lower panels) under restrictive conditions (−ethanol). Fluorescence intensity was quantified (arbitrary units) and represented for each SPB (uSPB, up; dSPB, down). (C) Hyperactivation of SIN does not bypass the requirement of Etd1p for Sid2p-GFP localisation. Sid2p-GFP was localised in cells with (−thiamine) or without Etd1p (+thiamine) under a normal (25°C) or hyperactive (34°C) SIN cascade. Cells of the etd1Δ nmt81x:etd1 sid2-GFP cdc16-116 strain expressing etd1 were grown to mid-exponential phase at 25°C. Thiamine was added to half of the culture, and after 2 h, half of each subculture was shifted to 34°C. Sid2p-GFP was observed in living cells after 4 h. Bar: 3 μm.

Figure 6

(A) Effects of SIN on Etd1p localisation. sid2-250 and cdc16-116 thermo-sensitive mutant cells expressing Etd1p-GFP were grown to mid-exponential phase at 25°C; half of the culture was shifted to 36°C for 2 h, and live cells were imaged. Asterisks indicate the main cellular location of Etd1p-GFP. In sid2-250 cells at 36°C in which SIN is inactive, Etd1-GFP accumulates at the cell membrane overlying the division site, and relocalises to the cell tips, but it is not localised in the medial ring. In cdc16-116 cells at 36°C, Etd1p localises to the multiple septa originated by SIN hyperactivation. (B) Effects of the etd1-1 mutation on the localisation of sterol-rich membrane domains in comparison to the effects of sid2-250 (sin mutant) and cdc8-110 (actomyosin ring mutant) thermo-sensitive mutants. Cells were incubated at their respective restrictive conditions for 2 h, stained with filipin and imaged (wild-type cells were also used).

Figure 7

The abundance of etd1 mRNA and protein is cell cycle regulated. etd1 mRNA and protein levels were followed by respective Northern and Western blot analyses in a cdc25-22 block–release experiment as described in Figure 2. RNA and protein samples were collected every 15 min along two consecutive cell cycles after the release to 25°C. (A) Samples were scored for separation of the nuclei and the appearance of the division septum to verify the synchrony of the culture. (B) Proteins were separated by SDS–PAGE and the Western blot was probed with either anti-HA (12CA5) or anti-Cdc2p (C2) antibodies (upper panels). A Northern blot was probed for etd1; rRNA was stained with bromophenol blue as a loading control prior to hybridisation (lower panels). (C) Etd1p is polyubiquitinated and stabilised in the mts3-1 mutant defective in the 26S proteasome. etd1-HA or etd1-HA mts3-1 strains were grown at 25°C to mid-exponential phase in YE and then shifted to 36°C for 4 h. Protein extracts were collected at times of 0, 1, 2 and 4 h after the shift and the levels of Etd1p and Cdc2p (as a loading control) were determined by Western blot using anti-HA (12CA5) or anti-Cdc2p (C2) antibodies (upper panels). To determine Etd1p ubiquitination, His₆-ubiquitin was expressed from the nmt1 promoter in mts3-1 etd1-HA mutant cells for 16 h at 25°C and then shifted to 36°C for 4 h. The His₆-ubiquitin conjugates were purified in Ni²⁺-NTA columns and analysed by Western blot using 12CA5 antibodies.

Figure 8

A model for the function of Etd1p in the coordination of SIN activity and actomyosin ring constriction. (A) During interphase, Etd1p localises to the cell cortex, mainly at the cell tips. (B) Early in anaphase, Etd1p relocalises to the middle of the cell following actomyosin ring assembly (which occurs in metaphase). (C) Late in anaphase, SIN activity triggers the recruitment of Etd1p to the actomyosin ring, possibly by association with Cdc15p. The actomyosin ring initiates constriction coupled to septum formation by the septum synthesis machinery (SSM). (D) Etd1p levels increase during ring constriction, and the protein accumulates behind the constricting ring. The accumulation of Etd1p maintains the SIN active during the progression of ring constriction and septation. (E) Etd1p is degraded by the proteasome pathway at the end of septation and contributes to coordinate exit from cytokinesis and inactivation of SIN.