Casein kinase 1γ ensures monopolar growth polarity under incomplete DNA replication downstream of Cds1 and calcineurin in fission yeast.

Cell polarity is essential for various cellular functions during both proliferative and developmental stages, and it displays dynamic alterations in response to intracellular and extracellular cues. However, the molecular mechanisms underlying spatiotemporal control of polarity transition are poorly understood. Here, we show that fission yeast Cki3 (a casein kinase 1γ homolog) is a critical regulator to ensure persistent monopolar growth during S phase. Unlike the wild type, cki3 mutant cells undergo bipolar growth when S phase is blocked, a condition known to delay transition from monopolar to bipolar growth (termed NETO [new end takeoff]). Consistent with this role, Cki3 kinase activity is substantially increased, and cells lose their viability in the absence of Cki3 upon an S-phase block. Cki3 acts downstream of the checkpoint kinase Cds1/Chk2 and calcineurin, and the latter physically interacts with Cki3. Autophosphorylation in the C terminus is inhibitory toward Cki3 kinase activity, and calcineurin is responsible for its dephosphorylation. Cki3 localizes to the plasma membrane, and this localization requires the palmitoyltransferase complex Erf2-Erf4. Membrane localization is needed not only for proper NETO timing but also for Cki3 kinase activity. We propose that Cki3 acts as a critical inhibitor of cell polarity transition under S-phase arrest.

Systematic localization study on novel proteins encoded by meiotically up-regulated ORFs in fission yeast.

We conducted a mitotic localization study on gene products encoded by 56 uncharacterized fission yeast ORFs that were transcriptionally up-regulated during meiotic division. Despite meiotic gene induction, these genes were expressed during mitosis as well. Seven gene products were localized in the nucleus and/or chromatin; another one was a mitosis-specific spindle pole body component and, intriguingly, its human homologue was also localized in the centrosome of cultured HeLa cells. Two products appeared to be localized in cytoplasmic microtubules, whereas four were mitochondrial proteins. Three other proteins were found in the medial ring upon cytokinesis and another was localized on the entire cell periphery. The remaining 38 proteins were detected in the cytoplasm and showed varied spatial patterns. This systematic study helps our integrated understanding of all the protein functions in the fission yeast as a eukaryotic model.

Search for kinases related to transition of growth polarity in fission yeast.

In eukaryotes, cell polarity is essential for cell proliferation, differentiation, and development. It is regulated in 3 steps: establishment, maintenance, and transition. Compared to current knowledge of establishment and maintenance, the mechanism regulating the transition of cell polarity is poorly understood. In fission yeast during the G2 phase, growth polarity undergoes a dramatic transition, from monopolar to bipolar growth (termed NETO: new end take off). In this study, we screened systematically for protein kinases related to NETO using a genome-wide kinase deletion library. Analysis of these deletions suggested that 35 and 2 kinases had a putative positive and a negative role, respectively, in NETO. Moreover, 5 kinases were required for NETO-delay in the G1-arrested cdc10 mutant. These results suggest that many signaling pathways are involved in the regulation of NETO.

Flagellar formation in C-ring-defective mutants by overproduction of FliI, the ATPase specific for flagellar type III secretion.

The flagellar cytoplasmic ring (C ring), which consists of three proteins, FliG, FliM, and FliN, is located on the cytoplasmic side of the flagellum. The C ring is a multifunctional structure necessary for flagellar protein secretion, torque generation, and switching of the rotational direction of the motor. The deletion of any one of the fliG, fliM, and fliN genes results in a Fla(-) phenotype. Here, we show that the overproduction of the flagellum-specific ATPase FliI overcomes the inability of basal bodies with partial C-ring structures to produce complete flagella. Flagella made upon FliI overproduction were paralyzed, indicating that an intact C ring is essential for motor function. In FliN- or FliM-deficient mutants, flagellum production was about 10% of the wild-type level, while it was only a few percent in FliG-deficient mutants, suggesting that the size of partial C rings affects the extent of flagellation. For flagella made in C-ring mutants, the hook length varied considerably, with many being markedly shorter or longer than that of the wild type. The broad distribution of hook lengths suggests that defective C rings cannot control the hook length as tightly as the wild type even though FliK and FlhB are both intact.