| dc.description.abstract | Cell cycle phase and cellular calcium play key roles in determining cell fate during differentiation and development in Dictyostelium discoideum. Earlier studies have shown that these two cellular functions are correlated such that the early phase of the cell cycle (G1, S, and early G2) correlates with high cellular Ca² and a prestalk tendency, whereas the late phase of the cell cycle (mid to late G2 and M) correlates with relatively low cellular Ca² levels and a prespore tendency. The cell cycle mediates its effect on cell fate in conjunction with cellular Ca².
The present study aims at understanding the molecular players involved in the possible mechanism of cell fate determination as mediated by cell cycle phase and calcium. The approach is to (a) identify “cell cycle genes” (including genes that interact with them) by functional complementation of Saccharomyces cerevisiae cdc mutations that have an associated “calcium phenotype” and (b) analyze the role of these complementing genes in cellular Ca² regulation and cell fate determination.
Towards this, a complementation screen for the S. cerevisiae cdc24-4 temperature-sensitive mutant was carried out. The cdc24-1 mutant of S. cerevisiae arrests in the cell cycle post Start. Cdc24 functions as a guanine nucleotide exchange factor for Cdc42 Rho-GTPase. In addition, the protein seems to contain calcium-binding EF-hand domains. The cdc24 mutant shows a 50% increase in calcium influx rate under conditions of cell cycle arrest. These features indicate that it is a candidate cell cycle gene with a “calcium phenotype.”
The complementing cDNA obtained in the screen was found to encode the previously identified ribosomal protein S4 of D. discoideum. S4 is a specific suppressor of cdc24-4. Among the known roles played by S4 in the cell is that of a component of the ribosome where it functions to maintain translational fidelity. Screening eight different missense and nonsense mutations for translational errors under conditions of S4 overexpression shows that the rescue of cdc24-4 is not due to non-specific translational errors.
Under conditions of arrest, the cdc24-4 mutant has 7.7-fold more sequestered Ca² than the wild type. Transformation with S4 reduces the sequestered calcium level in the cdc24-4 mutant by 3.1-fold, implying that S4 in some way is able to partially restore the cellular sequestered Ca² level. There is no significant alteration in cytoplasmic Ca² levels in the cdc24-4 mutant as compared to the wild type. The results suggest that the documented increased influx of Ca² in the cdc24-4 mutant is probably counteracted by increased sequestration, thereby maintaining physiological levels of cytoplasmic Ca². The rescue of cdc24-4 mutation by S4 is not achieved by a mere lowering of sequestered Ca² levels.
Thus, the D. discoideum S4 seems to possess an as-yet-unidentified function apart from its well-documented role in ensuring translational fidelity in the ribosome. However, the molecular mechanism underlying rescue remains uncharacterized. The results imply a role for S4 wherein it interacts with the cell cycle and can substitute for the loss of a cell cycle gene in S. cerevisiae.
In D. discoideum, S4 is encoded by a single-copy gene. The S4 transcript is developmentally regulated. In situ hybridization reveals that it is predominantly expressed in prespore cells. To analyze the role of S4 during D. discoideum development, an S4 antisense mRNA driven by the discoidin I promoter was used. Cells expressing the antisense mRNA are normal in terms of growth and cell division but show abnormal development. They arrest at the tipped mound stage with the generation of a multiple-tip structure.
During development, the antisense mRNA-expressing cells cannot be distinguished from the wild type cells until the tight aggregate to mound stage, after which the phenotype manifests. This is not the terminal stage in these cells. Rarely, tips manage to migrate and induce elongated structures. These culminate and form fruiting bodies with stalks and viable spores. The severity of the antisense phenotype is variable; some clones are able to overcome the block in development partly, while others form morphologically abnormal fruiting bodies.
Analysis of cell patterning in the antisense mRNA-expressing cells reveals that prestalk and prespore cells sort out but not completely. The aberrant prestalk and prespore cell localization in the antisense cells suggests a defect in morphogenesis. Due to this, cells do not sort out to their defined territories and development halts. The defect in S4 antisense cells is not cell-autonomous. It can be rescued by as few as 10% wild-type cells in chimeras. Rescue by the wild type is probably mediated through small diffusible molecules; cell-cell contact does not seem to be necessary. Pulsatile cAMP is able to phenotypically suppress the multiple-tip phenotype. The outcome is consistent with a cAMP-based tip inhibition model.
These results point to an as-yet-unidentified extra-ribosomal role for S4. | |
