Role of Polo Kinase and Mid1p in Determining the Site of Cell Division in Fission Yeast

Abstract

The fission yeast Schizosaccharomyces pombe divides symmetrically using a medial F-actin– based contractile ring to produce equal-sized daughter cells. Mutants defective in two previously described genes, mid1 and pom1, frequently divide asymmetrically. Here we present the identification of three new temperature-sensitive mutants defective in localization of the division plane. All three mutants have mutations in the polo kinase gene, plo1, and show defects very similar to those of mid1 mutants in both the placement and organization of the medial ring. In both cases, ring formation is frequently initiated near the cell poles, indicating that Mid1p and Plo1p function in recruiting medial ring components to the cell center. It has been reported previously that during mitosis Mid1p becomes hyperphosphorylated and relocates from the nucleus to a medial ring. Here we show that Mid1p first forms a diffuse cortical band during spindle formation and then coalesces into a ring before anaphase. Plo1p is required for Mid1p to exit the nucleus and form a ring, and Pom1p is required for proper placement of the Mid1p ring. Upon overexpression of Plo1p, Mid1p exits the nucleus prematurely and displays a reduced mobility on gels similar to that of the hyperphosphorylated form observed previously in mitotic cells. Genetic and two-hybrid analyses suggest that Plo1p and Mid1p act in a common pathway distinct from that involving Pom1p. Plo1p localizes to the spindle pole bodies and spindles of mitotic cells and also to the medial ring at the time of its formation. Taken together, the data indicate that Plo1p plays a role in the positioning of division sites by regulating Mid1p. Given its previously known functions in mitosis and the timing of cytokinesis, Plo1p is thus implicated as a key molecule in the spatial and temporal coordination of cytokinesis with mitosis.

Roles of a Fimbrin and an α-Actinin-like Protein in Fission Yeast Cell Polarization and Cytokinesis

“…Medial-ring precursors start to accumulate at the cell center during the early stages of mitosis but only coalesce into a distinct ring during anaphase (Arai et al, 1998;Bähler et al, 1998a). Ain1p, Fim1p, and other actinbinding proteins such as Rng2p (Eng et al, 1998) may be involved in organizing the medial-ring precursors into a functional contractile ring (for review, see Bähler and Peter, 2000).…”

A ‘marker switch’ approach for targeted mutagenesis of genes in Schizosaccharomyces pombe

“…(B) The actin ring of plo1.ts18 mutant cells that have been incubated at the restrictive temperature for 3 h is misshapen and directs the deposition of a highly aberrant septum Targeted mutagenesis of a selected locus in Sz. pombe 593 in plo1.ts18 mutant cells was much larger than normal and appeared to randomly bisect the cell in a manner that was highly reminiscent of the plo1.1, plo1.24C and plo1.25 mutations reported by Bähler et al (1998a; Figure 3B). The ability of plo1.ts4 to use the F-actin ring to deposit septal material was severely compromised when the strain was grown in minimal medium (Tanaka et al, 2001); 4 h after the temperature was changed from 25 • C to 36 • C, 49% of cells contained multiple nuclei but had no septa (Tanaka et al, 2001).…”

“…Thus, cytokinesis was more sensitive than spindle formation to Plo1 levels. An additional Plo1 function was then identified in a screen for mutants that had defects in the location or structure of the actin ring that constricts to cleave the cell in two during cytokinesis (Bähler et al, 1998a). The positioning of the actin ring may therefore be more sensitive to Plo1 levels than the use of the ring to make a septum, and so this phenotype was not detected in the original analysis.…”

Roles of Pdk1p, a Fission Yeast Protein Related to Phosphoinositide-dependent Protein Kinase, in the Regulation of Mitosis and Cytokinesis

“…This is considerably higher than that seen in clp1⌬ (0.5%) and pdk1⌬ (7%) mutants. The additive phenotypic effect upon combination of pdk1⌬ and clp1⌬ suggested that pdk1⌬ cells were partially defective in the assembly and/or function of the cell division apparatus.The similarities between the localization of Pdk1p and Plo1p (which regulates Mid1p localization; Bähler et al, 1998) and the finding that mid1 mutants, as with pdk1⌬ mutants, are delayed for actomyosin ring assembly (Wu et al, 2003), suggested that pdk1⌬ cells might be defective for Mid1p localization. Alternatively, pdk1⌬ cells might be defective for proper assembly and function of other actomyosin ring components.…”

“…Gene products important for mitotic entry, mitotic progression, and cytokinesis have been identified from a number of genetic screens, as well as from reverse genetic approaches (Nurse et al, 1976;Hirano et al, 1986;Chang et al, 1996;Balasubramanian et al, 1998). Central to the regulation of mitosis and cytokinesis are the S. pombe polo-related protein kinase Plo1p (Ohkura et al, 1995;Bähler et al, 1998;Mulvihill et al, 1999) and members of the septation initiation network (SIN), a signaling pathway comprised of three protein kinases (Cdc7p, Sid1p, and Sid2p) and a GTPase (Spg1p) Rajagopalan et al, 2003). Members of the polo-kinase family are conserved across all eukaryotes (Nigg, 1998).…”