細胞週期的調控對於生物體的增生過程非常重要。本研究利用單細胞綠藻 (衣藻, Chlamydomonas reinhardtii) 為模式生物探討細胞大小的調控機制。研究中使用之smt15-1為細胞大小調控異常的小細胞突變株；由蛋白質功能性區域分析結果，推測SMT15為硫酸鹽運輸蛋白 (sulfate transporter)。本實驗利用qRT-PCR分析，得知缺硫逆境下，smt15-1無法正常調節缺硫反應機制 (sulfur acclimation response, SAC response)。進一步發現smt15-1細胞體內，累積較多硫化物代謝途徑的終產物-榖胱甘肽 (glutathione)。文獻資料指出真核生物中，榖胱甘肽會調節細胞內的氧化還原狀態 (reduction/oxidation state) ，且與細胞週期有關。本研究也觀察到榖胱甘肽含量隨衣藻細胞週期而變動。此外，還發現G1後期和S/M階段，smt15-1會累積較野生株多的榖胱甘肽。本研究因而從文獻資料及初步實驗結果，提出假說：細胞內榖胱甘肽所主導之氧化還原狀態，對衣藻細胞週期的調控非常重要。本實驗分別利用乙醯半胱胺酸 (N-acetyl-L-cysteine, 還原劑) 和順丁烯二酸二乙酯 (Diethylmaleate, 榖胱甘肽的抑制劑) 處理，進而改變細胞體內的氧化還原狀態，結果皆會影響細胞進入S/M 階段。此外，過量表達榖胱甘肽生合成基因 (glutathione synthetase) 的轉殖株，可提高榖胱甘肽含量，造成細胞分裂數增加，導致子細胞大小 (daughter cell size) 變小，此結果與smt15-1性狀相似。另外，於缺硫逆境下使用順丁烯二酸二乙酯抑制榖胱甘肽的功能基，會稍微回復芳基硫酸酯酶 (Arylsulfatase, 為SAC相關基因) 的誘導表現量，即表示smt15-1細胞內於缺硫逆境下，累積較多的榖胱甘肽會降低SAC基因的誘導表現。綜合以上結果，本研究證實SMT15可調節衣藻細胞內之榖胱甘肽含量，進一步影響缺硫反應機制及細胞分裂次數繼而改變細胞大小。

Regulation of the cell cycle is essential to drive cell proliferation. Chlamydomonas reinhardtii is a unicellular green alga that utilizes a specialized cell cycle program, multiple-fission, for cell division. In this study, smt15-1 mutant was isolated as a cell size mutant that had defective putative sulfate transporter and aberrant cell cycle program. smt15-1 had increased amount of total glutathione and failed to fully acclimate to sulfur starvation condition. Previous studies have suggested that the GSH-mediated sub-cellular reduction/oxidation (redox) homeostasis is important for the cell cycle control in eukaryotic cells. We also observed the total glutathione content oscillated during the mitotic cell cycle in Chlamydomonas. In addition, smt15-1 mutant accumulated more glutathione than wild-type strain at late G1 and S/M phases in synchronized culture. Base on these result, we hypothesized that GSH-mediated cellular redox regulation is important for cell-cycle control in Chlamydomonas. Indeed, increased cellular reducing state by adding N-acetyl-L-cysteine (NAC) caused a delay in entry into mitosis in synchronized cultures. Additionally, depleted GSH using diethylmaleate (Et2Mal, glutathione-depleting reagent) resulted in aberrant entry into the cell cycle. Decreasing GSH levels in the smt15-1 mutant using Et2Mal under sulfur-depleted condition led to a slight increase in induction of an arylsulfatase (ARS) mRNA, indicating that accumulation of GSH in smt15-1 under sulfur-depleted condition attenuated sulfur acclimation (SAC) response. Furthermore, increasing total glutathione content by overexpressing the glutathione synthetase (GSH2) led to increase in cell division number and decrease in daughter cell size, which was reminiscent to smt15-1 mutant. In conclusion, SMT15 modulates intracellular glutathione that affects SAC response and cell division in Chlamydomonas.

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