cdc3 and cdc12 exhibit a synthetic-lethal genetic interaction. Siblings from a tetratype tetrad in a cross between cdc3-313 and cdc12-112 mutants were streaked on YE5S plates and incubated at 25°, 29°, or 36°C. Growth was assayed by colony formation. Note that the cdc3 cdc12 double mutant exhibited markedly reduced growth at 25° and 29°C where the single mutants are viable. Strains were FC322 (cdc12-112), FC323 (wt), FC324 (cdc3313), and FC325 (cdc3-313 cdc12-112).

Localization of cdc12p in mid1 mutant cells during early mitosis. mid1-366 mutant cells were grown in YE5S at permissive temperature (25°C), and then shifted to restrictive temperature (35.5°C) for 2 h. Cells were processed for immunofluorescence with anti-cdc12 antibody, anti-tubulin antibody or anti-actin antibody, and DAPI. On the basis of spindle and nuclear morphology, cells pictured are in early mitosis, the cell cycle period around ring formation. Note different configurations of cdc12p dot and strand (arrows). Bar, 10 μm.

The positions of the cdc12p spot and the ring coincide. FC565 (pnmt-cdc12) cells were processed as described in Fig. 4. Shown are four representative cells in early mitosis, around the period of ring formation. Top three panels show three different focal planes of the same cell, and the bottom two panels show tubulin and DAPI staining as labeled. (A) Cell with a cdc12p spot. (B) Cell with a cdc12p associated with a complete ring. (C) Cell with a cdc12p spot with an incomplete ring. Note the discontinuity of the ring adjacent to the spot in the top panel. (D) Cell with a cdc12p ring but no spot. Bar, 10 μm.

A model for the establishment of the division site in S. pombe. (A and B) In interphase, a marker spot is established at the cell middle, possibly by a signal from the nucleus. This spot may contain cdc12p; alternatively, upon overexpression, cdc12p may accumulate at such a structure. (C) In early mitosis, the spot remains on the cortex. (D) In early mitosis, the ring forms at the site marked by the spot by extension of a strand from the spot around the circumference of the cell. (E) During anaphase, the ring becomes more robust. (F) After nuclear division, the ring closes during septation and cell division. Roles of the actin ring genes are inferred from cdc12p localization in mutants. Aspects of this model are inferred from cdc12p overexpression and mutant data. Alternate models are discussed in the text.

Upon overexpression, cdc12p localizes to a medial spot. FC565 (pnmtcdc12) cells were induced in derepressing conditions for 20 h at 30°C, fixed in cold methanol, and processed for immunofluorescence with anti-cdc12 antibody (left panels), anti-tubulin antibody (middle panels), and DAPI (right panels). The tubulin and DAPI staining patterns identify the cell cycle stage. (A–C) All panels show three representative fields from the same population of cells. (Cell 1) Interphase cell with a medial cdc12p spot. (Cell 2) Early mitotic cell with a medial spot. (Cell 3) Cell in postanaphase with a cdc12p ring. (Cell 4) Cell in postanaphase with a closed ring. (Cell 5) Cell after cell division with a cdc12p spot at the presumed midbody on the cell tip. (Cell 6) Cell with two spots, one at the cell tip and one at the cell middle. Bar, 10 μm.

cdc12p fragment binds to profilin cdc3p in vitro. (A) Purified proteins used in these studies. (Lane 1) Purified GST-cdc3. (Lane 2) Purified GST. (Lane 3) Purified His-tagged cdc12p fragment. (B and C) cdc12p binds to GST-cdc3 but not to GST. In each 180-μl reaction, cdc12p (1.6 μM final concentration) and 20-μl 50% slurries of glutathione agarose beads containing indicated concentrations of GST-cdc3 or GST were added to each reaction. Reactions were incubated at room temperature for 1 h, and the beads were pelleted. (B) Coomassie-stained gel of total proteins before pelleting (lane 1) and pellet fractions (lanes 2–10) at equivalent volumes. Bands were quantitated by densitometry relative to total protein in lane 1 and plotted in C. (D and E) Poly-l-proline inhibits binding. Varying amounts of poly-l-proline were added to the binding reactions similar to ones described above except cdc12p was 0.8 μM. (D) Coomassie-stained gel of the pellet fractions. (E) cdc12p in the pellets was quantitated by densitometry relative to supernatant fractions (not shown).

Localization of cdc12p in different actin ring mutants during mitosis. (A) cdc12-299 (FC127); (B) cdc3313 (FC114); (C) cdc8-346 (FC131); (D) cdc15-287 (FC55); (E and F) cdc4-377 (FC361). Mutant cells were grown in YE5S at permissive temperature (25°C), and then shifted to restrictive temperature (35.5°C) for 4 h. Cells were fixed and processed for immunofluorescence with anti-cdc12 antibody, anti-tubulin antibody or anti-actin antibody, and DAPI. On the basis of spindle and nuclear morphology, cells pictured are in anaphase, the cell cycle period of maximum cdc12p ring staining in wild-type cells. (A–C) Cells are in mitosis in which two nuclei are dividing into four and thus exhibit double spindles; (E–F) cells exhibit single spindles. (A–C) Mutants exhibit no specific cdc12p staining; the cytoplasmic staining is nonspecific background staining. (E and F) Note the medial spot of cdc12p (arrow) in the cdc4 mutant, which localizes to the origin of an actin aster (F, middle panel, labeled a). Bar, 10 μm.

cdc12 encodes a protein similar to the dia/BNI1 family of proteins. (A) Localization of the cdc12 gene. Restriction map of the original 6-kb genomic clone insert (pFC103) that rescues cdc12-112 mutant cells. (Arrow) Extent and orientation of the single large open reading frame in the region. Truncated versions of the clone and their ability to complement a cdc12-112 mutant; ++, full rescue; +, partial rescue. cdc12::ura4 deletion construct used in deleting cdc12. Much of the cdc12 open reading frame is replaced with a 1.6-kb ura4 insert. (B) cdc12 open reading frame. Regions of predicted coiled-coil are underlined (Lupas et al., 1991), and the polyproline regions are boxed. (C) Similarity in gene domain structure. Proteins share a central FH1 polyproline rich domain (black box), an FH2 domain (stripe box), and a larger COOH-terminal area of homology (gray) also diagrammed in D. (D) Similarity of the proteins in a COOH-terminal region. Proteins were aligned using Megalign in DNA Star. Residues identical to a calculated consensus are colored in black, and conserved residues are colored in gray. These sequence data are available from GenBank/EMBL/DDBJ under accession number Q10059.

Phenotype of cdc12⁺ and cdc12::ura4 deletion cells. (A and B). Wild-type cells stained with rhodamine-phalloidin, showing normal interphase (A) and mitotic (B) actin patterns. (C–L) cdc12::ura4 cells stained with rhodamine-phallodin (C, E, G, and I), DAPI (D, F, H, and J), or calcofluor (K and L). Diploid ura4/ ura4 leu1/leu1 ade6-M16/ade6-M22 cdc12⁺/cdc12::ura4 cells were sporulated, and the spores were germinated in media containing no uracil, so only cdc12::ura4 spores could germinate. Spores were grown for 15 h at 30°C, fixed, and stained. (C–F) cdc12::ura4 cells in interphase, showing normal interphase polarized actin distribution at the cell tips. Cell in D has two nuclei, and cell in F has proceeded through another generation and contains four nuclei. (G–J) cdc12::ura4 mitotic cells with actin dots instead of an actin ring. Cell in G is focused on the middle of the cell, while the cell in I is focused on the cell surface. Nuclear patterns in H and I are characteristic of cells in mitosis (see Fig. 6 A for comparison). Calcofluor-stained cells show no septum cell wall deposition even after multiple nuclear divisions. The rounded bulge that stains with Calcofluor (K and L) in cdc12::ura4 cells represents the site of the original spore wall. Bar, 10 μm.

cdc12p localizes to the cell division ring in wild-type cells. (A) Specificity of the anti-cdc12 affinity-purified antibody. 5 μg of crude whole yeast cell extracts were analyzed by Western blotting using affinity-purified anti-cdc12 antibody. (Lane 1) Extract from wild-type cells. (Lane 2) Extract from cells overexpressing cdc12p. Cells transformed with the plasmid pnmt-cdc12 (FC291) were shifted to derepressing conditions (minimal media without thiamine) for 20 h at 30°C. Molecular weight markers (kD) are indicated. Serial dilutions showed that the pnmt-cdc12 extract contains 10–20-fold more cdc12p than the wild-type extracts (data not shown). (B–K) cdc12p localization in wild-type cells. Wild-type cells were grown in YE5S at 30°C in exponential phase, fixed, and processed for immunofluorescence using anti-cdc12 antibody and DAPI (see Materials and Methods for details). (B, D, F, H, and J) Anti-cdc12 staining. (C, E, G, I, and K) DAPI staining. (B and C) A typical field of cells containing cells from different phases of the cell cycle. Note examples of ring staining. (e) Cell in early mitosis in which the ring is beginning to form in an asymmetrical fashion. (i) Interphase with no specific cdc12 staining. (B, inset) Cross-section of a cdc12p ring. (D and E) Cell in early mitosis; (F and G) cell in early anaphase; (H and I) cell in midanaphase; (J and K) cell in postanaphase during ring closure. Patchy cytoplasmic staining is also seen in cells lacking cdc12p, and thus probably represents background staining (data not shown). Bar, 10 μm.