(a) EcoRI restriction analysis of PCR products from an inner fragment of the cdc15-lyt1 allele (lanes 1, 2, and 3 represent three different amplificates) and the CDC15 gene (lane 4), showing the disappearance of an EcoRI cleavage site in the mutant allele. (b) Sequence comparison of the CDC15-encoded peptide in the area where the lyt1 mutation maps from the mutant, the wild-type, and the putative homologue in the fission yeast S. pombe (Sp), cdc7. Underline, EcoRI site in the corresponding DNA sequence; boldface; residues affected by the mutation; asterisks, conserved residues; dots, structurally similar residues in the S. cerevisiae and S. pombe sequences.

Microscopic observations by phase–contrast (a–d and h–q), fluorescence (e and f), scanning electron (g), and transmission electron (r–t) microscopy of haploid and diploid cdc15 mutant strains. (a) Strain L2C24d (cdc15-lyt1) transformed with the pBS9 plasmid bearing the CDC15 gene after 6 h of incubation at 37°C, thus displaying wild-type behavior. (b) Diploid MY1 strain (cdc15-1/cdc15-lyt1) under the same conditions showing abundant chained cells. (c) L2C24d (cdc15-lyt1) strain under the same restrictive conditions. (d) The same strain incubated in an osmotically stabilized medium (supplemented with 1 M sorbitol) under identical conditions, showing an exacerbated expression of its characteristic apical growth phenotype. (e) Nucleus staining in fixed RNase-treated cells from strain L2C24d (cdc15-lyt1) after 6 h of incubation at 37°C. (f) Cells from the diploid strain MY1 (cdc15-1/cdc15-lyt1) after the same treatment. (g) A characteristic asymmetric doublet from the L2C24d (cdc15-lyt1) strain incubated at the restrictive temperature for 6 h. (h–l) Series showing the development of a cell from the L2C24d (cdc15-lyt1) strain at 37°C. Pictures were taken at 0 h (h), 1 h (i), 2.5 h (j), 3.5 h (k), and 4.5 h (l). (m–q) Series showing the development of a cell from the MY1 (cdc15-1/cdc15-lyt1) strain in the same conditions. Pictures were taken at 0 (m), 0.5 h (n), 1.5 (o), 2.5 (p), and 3.5 h (q). (r and s) Section of cells from the L2C24d (cdc15-lyt1) strain after 6 h of incubation at 37°C, showing that septation has not been initiated. (t) A chain of cytokinetic-defective cells from the diploid MY1 (cdc15-1/ cdc15-lyt1) strain under the same conditions. Bars, 8 μm.

Evolution of actin polarization in the L2C24d (cdc15-lyt1) strain at the restrictive temperature. Actin was stained with rhodamine-conjugated phalloidine. The plot represents the percentage of each cell type within the population (y axis) versus time of incubation at 37°C (x axis). Only live cells can be stained for the visualization of actin, so no lysed cells were included in this assay. (▴) Single unbudded cells with a random distribution of actin patches. (♦) Doublets with a depolarized actin cytoskeleton, as corresponds to anaphase-arrested cells. (▪) Doublets with a polarization of the actin cytoskeleton to the neck area for the promotion of septum morphogenesis. (•) Cells showing a pattern of actin polarization towards the incipient, emergent or developing bud. (✖) Doublets or asymmetric doublets that polarize the actin cytoskeleton towards the distal pole of one of the cells. Bars, 5 μm.

Visualization of the septin ring by fluorescence (a–d) and confocal (e–g) microscopy of the L2C24d strain (cdc15-lyt1) transformed with the pLA10 plasmid bearing a CDC10–GFP fusion (Cid et al., 1998a) (a, b, and e–g) and MY3 strain (cdc15-lyt1/cdc15-1 CDC10–GFP) (c and d). Cells were kept at 37°C for 4.5 h to allow expression of the characteristic morphogenetic phenotype. (a and b) Fluorescence microscopy shows that the septins remain at the mother–daughter neck in asymmetric doublets. (c and d) The formation of chains of cells in diploid cdc15/cdc15 mutants is supported by the formation of novel septin rings, but the first ring in the neck between the oldest cells persists. (e–g) Simultaneous staining with rhodamine-conjugated phalloidin allows the visualization of both the actin and septin cytoskeletons. Red, actin-rich areas; green, corresponds to the septin–GFP fusion. In e and g, the fluorescence image is overlapped to a phase contrast micrograph for better definition of cell shape. Bars, 5 μm.

Flow cytometry graphics of RNase-treated propidium iodide-stained cells of the L2C24d strain. Cells were incubated at 37°C in osmotically stabilized media and samples were taken every 30 min. Peaks correspond to cell populations containing different amounts of DNA. The peak corresponding to a DNA content of 1 nucleus per cell (G1 peak) appears at ∼15 fluorescence units (see scale at the bottom), the G2/M peak (2 nuclei per unit) stands at ∼30 units, and cells displaying a DNA content equivalent to 4 nuclei appear at 60 U. Upon expression of the cdc15 phenotype (right), the G1 peak progressively disappears due to the accumulation of cells in anaphase. After 150 min, a new well-defined peak reveals a new population of cells with a larger amount of DNA. None of these phenomena occurred when the same strain was transformed with a CDC15-containing plasmid (left).

(a–f) Morphological observations by phase contrast microscopy of mutants in genes involved in late mitotic events after 6 h of incubation at 37°C: (a) L119-7d (dbf2-1); (b) RH1779 (cdc14-1); (c) EO156 (tem1-3); (d) 4965-3a (cdc16); (e) 4086-23-2a (cdc23); (f) 9002 (cdc27). (g) L2C24d (cdc15-lyt1) strain transformed with the pGAL–CLB2 plasmid after 6 h of incubation at 37°C with galactose as a carbon source. (h) The same strain transformed with a pRS316 control plasmid under the same conditions. Arrows, distal projections. Bars, 5 μm.

Flow cytometry plots of RNase-treated propidium iodide-stained cells of the VCY242d strain both untransformed (left) and transformed with a CDC10-bearing plasmid (right). Cells were incubated at 37°C in osmotically stabilized media. Samples were taken at the times indicated. Peaks that appear at 15, 30, and 60 arbitrary fluorescence units (see the scale at the bottom) correspond respectively to cells with DNA contents equivalent to 1, 2, and 4 nuclei. Comparison of the two series reveals an earlier (∼30 min) and larger amount of cells included in the third peak when both cdc10 and cdc15 mutations are coexpressed than when the cdc15 mutation is expressed alone.

Scheme of bud development and its estimated timing, as deduced from budding time assays, in a wild-type strain, haploid and diploid cdc15 mutants. Haploid cdc15 mutants fail to localize actin to the bud-daughter constriction at the end of mitosis and display an arrest in the transition from anaphase to telophase. The mother cell fails to accomplish any morphogenetic response in a haploid background, but the daughter cell polarizes growth with a delay of 45 min with respect to a wild-type daughter. Such polarization follows an incorrect distal pattern. This behavior leads to immediate cell lysis. In contrast, diploid cdc15/ cdc15 mutants are frequently able to develop a new bud at the distal pole after overcoming mitotic arrest and the mother cell also eventually starts a new round of the cell cycle.

Hypotheses for the mechanisms involved in the coordination of morphogenetic events at the end of mitosis. Hypothesis (a) implies that the Cdc15p-mediated pathway is directly or indirectly involved in B cyclin degradation by the APC and that the consequent inactivation of the Cdc28p–B cyclin complex accounts for the polarization of the morphogenetic apparatus to the neck area for septum development. Hypothesis (b) argues that the Cdc15p pathway, similar to the cdc7 pathway in the fission yeast, would directly trigger the morphogenetic response for septum formation, perhaps activated by signals derived from Cdc28p–B cyclin inactivation or coordinated with it via feedback mechanisms (broken line). The arrest in anaphase observed in cdc15 and related mutants would be due to a cell cycle checkpoint that would inactivate Cdc28p until morphogenetic events at the neck are settled. The fact that a septin (cdc10) mutation prevents the delay in budding observed in a cdc15 mutant suggest that the components of such putative checkpoint rely on the integrity of the septin ring for their functionality.