Sid2p, a spindle pole body kinase that regulates the onset of cytokinesis

Abstract

The fission yeast Schizosaccharomyces pombe divides by medial fission through the use of an actomyosin contractile ring. Precisely at the end of anaphase, the ring begins to constrict and the septum forms. Proper coordination of cell division with mitosis is crucial to ensure proper segregation of chromosomes to daughter cells. The Sid2p kinase is one of several proteins that function as part of a novel signaling pathway required for initiation of medial ring constriction and septation. Here, we show that Sid2p is a component of the spindle pole body at all stages of the cell cycle and localizes transiently to the cell division site during medial ring constriction and septation. A medial ring and an intact microtubule cytoskeleton are required for the localization of Sid2p to the division site. We have established an in vitro assay for measuring Sid2p kinase activity, and found that Sid2p kinase activity peaks during medial ring constriction and septation. Both Sid2p localization to the division site and activity depend on the function of all of the other septation initiation genes: cdc7, cdc11, cdc14, sid1, spg1, and sid4. Thus, Sid2p, a component of the spindle pole body, by virtue of its transient localization to the division site, appears to determine the timing of ring constriction and septum delivery in response to activating signals from other Sid gene products.

Figure 1

Sid2p-GFP localizes to the SPB and the site of cell division. Sid2p-GFP–expressing cells (YDM415) were fixed and photographed at various stages as described in the text (A), or were fixed and stained for DNA (DAPI), and Cdc4p (B) or tubulin (C). (B) DAPI, Cdc4p, Sid2p-GFP, and merged images are shown. Mitotic cells that had not yet completed anaphase show Cdc4p medial rings but no medial Sid2p-GFP signal (top panel). Postanaphase cells show colocalization between Cdc4p and Sid2p-GFP (bottom panel). (C) Sid2p-GFP (green/yellow) images were merged with the tubulin images (red). Metaphase (left panel), late anaphase (middle panel), or telophase (right panel) cells are shown. (D) Sid2p-GFP localizes to the SPB by immunoelectron microscopy. Cells were grown at 32°C to mid-log phase and prepared for immunoelectron microscopy with antibodies against GFP and with gold-labeled secondary antibodies. Three serially sectioned images are shown. Arrowheads indicate gold particles labeling the SPB, and the position of the nuclear matrix (Nu) and the SPB is indicated.

Figure 2

Localization of Sid2p-GFP to the cell division site depends on the presence of an actin ring and Cdc15p. Both cdc3-124 (YDM427) and cdc15-140 (YDM425) cells expressing Sid2p-GFP were grown to mid-log phase at 25°C, shifted to 36°C for 2.5 h, then fixed and photographed. Arrow indicates structure described in the text.

Figure 4

Sid2p-GFP localization depends on the other Sid proteins. (A) sid1-239 (YDM420) and cdc11-123 (YDM421) cells expressing Sidp2-GFP were grown to mid-log phase at 25°C and then shifted to 36°C for 2.5 h, and fixed and stained for DNA. (B) Sid2p-GFP protein is stable in the sid mutants. Cells expressing no Sid2p-GFP (Wild-type; YDM105), or expressing Sid2p-GFP in a wild-type background (sid2-GFP; YDM415) or in sid4-A1 (sid2-GFP, sid4-A1; YDM419), sid1-239 (sid2-GFP, sid1-239; YDM420), cdc11-123 (sid2-GFP, cdc11-123; YDM421), or cdc7-24 (sid2-GFP, cdc11-123; YDM422) mutant background were grown as described in A, and then Sid2p-GFP levels were analyzed by Western blotting with antibodies against GFP.

Figure 3

Sid2p-GFP requires microtubules to efficiently localize to the division site. Sid2p-GFP–expressing cells, or Sid2p-GFP cells carrying either the nda3-km311 mutation (YDM426), or the nda3-km311 and mad2Δ (YDM541) mutations were grown to mid-log phase at 30°C and then shifted to 19°C for 4 h and fixed in the cold, and either stained with calcofluor, or stained for tubulin. Images were captured for each strain showing the GFP signal (Sid2p-GFP) and calcofluor staining (Calcofluor), or the tubulin staining. A representative montage of cells is shown. Arrows and arrowheads indicate structures described in the text.

Figure 5

Sid2p functions downstream from Cdc7p and Spg1p. (A) sid2-250 cdc7-HA cells (YDM533) were grown to mid-log phase at 25°C and then shifted to 36°C for 2.5 h, and fixed and double stained for Cdc7p using antibodies against the HA epitope or for DNA. (B) sid2-250 nmt1-spg1 cells (YDM554) were grown to mid-log phase at 25°C in medium containing thiamine, and then the culture was shifted into medium lacking thiamine and grown at 25°C for an additional 23 h. The culture was then split two ways into medium lacking thiamine, with one culture placed at 25°C and the other at 36°C for 5 h, and then cells were fixed and stained with calcofluor.

Figure 6

Sid2p has protein kinase activity which is essential for its function. (A) Immune complex kinase assays were performed using myc antibodies (see Materials and Methods) on lysates from wild-type cells, or cells expressing Sid2p-13Myc, HA-Sid2, or HA-Sid2-K238R. Assays were carried out with (+) or without (−) addition of MBP as an artificial substrate. (B) sid2-250 cells (YDM71) transformed with plasmids pRep42, pRep42-GFPsid2, or pRep42-GFPsid2-K238 were plated at 25°C or 36°C on plates containing thiamine.

Figure 7

Sid2p kinase activity is cell cycle regulated. cdc25-22 sid2-13Myc cells (YDM500) were blocked at 36°C for 3 h then returned to 25°C, allowing them to synchronously enter the cell cycle. Every 15 min, samples were collected for measurement of kinase activity as well as the percentage of cells that were in anaphase/telophase (circles) and the percentage undergoing septation (open diamonds). Sid2p kinase activity was plotted (filled diamonds) by first normalizing the amount of MBP phosphorylation (bottom panel) to the amount of Sid2-13Myc protein present as determined by Western blotting.

Figure 8

Sid2p kinase activity depends on the other Sid proteins. Cells expressing Sid2p-13Myc in an otherwise wild-type background (YDM497; wt), or in cdc7-24 (YDM508), cdc11-123 (YDM511), cdc14-118 (YDM509), sid1-239 (YDM512), sid3-106 (YDM515), sid4-A1 (YDM513), or cdc15-140 (YDM668) mutant backgrounds were grown to mid-log phase at 25°C then split into two cultures, one at 25°C and one at 36°C. Each culture was then incubated for 3 h at its respective temperature and assayed for kinase activity. Relative kinase activities are depicted graphically.