Calcium And Calcium Calmodulin Role In The Cell Cycle In Schizosaccharomyces Pombe (from Schizosaccharomyces Pombe)

Studying the regulation mechanism of Ca2+ and calmodulin in cell cycle has important theoretical and practical significance. We used fission yeast Schizosaccharomyces pombe (S.pombe) , an unicellular eukaryotic organism, as research material. Electroporation was adopted to load Ca2+ fluorescent indicator into yeast cell and under the laser scanning confocal microscopy (LSCM) , we observed cytosolic Ca2+ distribution and relative content as well as fluorescence intensity of GFP-CaM in different phases of cell cycle of yeast cell. Flow cytometry provided a way of determining the relative DNA content of populations of fission yeast.We used an auxotrophic strain, MFP7 (h, adel-D25, ade6-M210, S65T-GFP::caml+, leul-32, ura4-D18), to observe the change of fluorescence intensity of GFP-CaM during the course of yeast cell cycle. We found that exogenous Ca2+ and low concentration of TFP could stimulate the proliferation of S.pombe, and it was the same with MFP7. In order to study the mechanism of the effect of low concentration TFP on the proliferation of S.pombe, we watch yeast cells loaded with Fluo-3 under laser scanning confocal microscope (LSCM). The fluorescence intensity reflected the cytosolic free calcium concentration. The result showed that, the cytosolic free Ca2+ concentration in S.pombe cultured in Ca2+-free medium was 2~3 times lower than that in S.pombe cultured in medium containing 10umol/L Ca24, while Ca2+ concentration in S.pombe treated with 50umol/L TFP was 4-5 times higher. We also found that inorganic sulfate which can change the membrane potential of yeast cell restrained the growth of S.pombe, and the cytosolic free Ca2+ concentration was decreased. Therefore, we conclude that TFP stimulate the proliferation of S.pombe cell by changing membrane potential to increase Ca2+ influx.We also observed the influence of different concentrations of exogenous Ca2+,5mmol/L EGTA, 100 u mol/L TFP and synchronization on the fluorescence intensity in MFP7 cell. The results indicated that the content of cytosolic CaM in cells treated with exogenous Ca2+ has increased indistinctively, while fluorescence intensity in cells treated with TFP decreased. So we believed that exogenous Ca2+ has little effect on the expression of CaM. High concentration of TFP can enter yeast cells and combine to CaM to make it inactive, which is the reason that TFP restrain the growth of yeast cells. 5mmol/L EGTA could completely arrest the cell proliferation of MFP7 after 28h, when the fluorescence intensity in cells wasobviously increased with flow cytometry and LSCM. It shows that the expression level of CaM begins to increase at late Gl. After adding lOmmol/L Ca2+ to medium for about 24h, the cells resumed growth. At that time, cytosolic fluorescence intensity decreased to normal level, which shows that most of cells get through the Gl/S point and enter the log phase. When cultured in medium that NEUCl was omitted, most of the cells were synchronized at Gl stage of cell cycle. With flow cytometry, we found that cytosolic CaM content of Gl cells was higher than that of normal cells at log stage. The results under LSCM showed the change of cytosolic CaM in different phases of cell cycle. During late Gl/early S, CaM levels begin to increase and reach the maximum level in the starting M population. GFP-CaM was observed in the region of the septum as it formed, then at only one end of newly formed cells. Later, fluorescence was seen at both ends of the growing cell. These behaviors are similar to those of known components of the polarized fission yeast actin cytoskeleton. The result shows that CaM concentrates at the new growing point and has closely relation with the dividing and elongation of cells.