鈉鉀ATP酶α次單元EAT-6調控線蟲計劃性細胞凋 亡中細胞外觀變化和磷脂絲氨酸的外翻

The alpha subunit of Na+/K+ ATPase/EAT-6 is involved in morphological changes and phosphatidylserine exposure during apoptosis in C. elegans

曾彥婷(Yen-Ting Tseng)

指導教授 ： 吳益群

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.10585

摘要

計畫性細胞凋亡的主要功能為清除個體中多餘的細胞並維持個體一定的細胞數，對於個體的成長及發育扮演極為重要的角色。細胞凋亡的過程主要可以分為：辨認死細胞、執行死亡、吞噬以及分解等四個步驟。在執行死亡的步驟進行過後，死細胞還會發生磷脂絲氨酸(phosphatidylserine/PS)外翻到細胞膜、細胞型態改變，以及DNA 斷裂降解等現象。過去的研究中發現在線蟲中調控細胞凋亡的基因有數十種，而其中執行死亡的步驟主要是由EGL-1(a BH3-containing protein)，CED-9(Bcl-2)，CED-4(Apaf-1)以及CED-3(Caspase)所調控；然而這四者又是如何被其他基因所調控仍有待研究。本實驗室在過去研究中發現，在eat-6 這個鈉鉀ATP 酶α 次單元異變種胚胎中，細胞屍體(cell corpse)數目有下降的趨勢。本篇論文主要探討EAT-6 是如何造成死細胞數目降低，而我利用死細胞的磷脂絲氨酸會外翻到細胞膜外並與分泌型的Annexin V 結合形成環狀外觀的特性進行分析。實驗結果指出eat-6 的異變種中，沒有死細胞型態但卻會與Annexin V 結合所形成環狀型態有增加的趨勢，這表示eat-6 會影響到死亡細胞型態的形成和脂絲氨酸的外翻。我的遺傳實驗結果也發現，eat-6 會與csk-1 這個非受體性酪氨酸磷酸酶一同參與在核心路徑中去調控細胞凋亡。而另一方面我也發現過度表現EAT-6 則會促進接觸性神經(touch neuron)的細胞凋亡，且eat-6 會作用在egl-1 的上游去促進細胞凋亡的功能且此種功能是需要其運輸離子的功能。以上實驗結果指出eat-6 除了擁有運送鈉鉀離子維持膜電位的功能外，也會影響到死細胞的屍體型態及其脂絲氨酸的外翻。
關鍵字：線蟲、計畫性細胞凋亡、鈉鈉鉀ATP 酶、細胞屍體、外觀型態變、脂絲
氨酸外翻

關鍵字

線蟲 ； 計畫性細胞凋亡 ； 鈉鈉鉀ATP 酶 ； 細胞屍體 ； 外觀型態變 ； 脂絲氨酸外翻

並列摘要

Programmed cell death (PCD) is important for development and homeostasis of multicellular organisms. The PCD process occurs via four steps: decision, execution,engulfment, and degradation. After execution step, the dying cells proceed phosphatidylserine (PS) translocation, refractile cell corpse morphology formation and DNA degradation during apoptosis. Previous studies have identified and characterized about 28 genes that regulate the PCD process in Caenorhabditis elegans. Among these genes, egl-l (encoding BH3-containing protein), ced-9 (Bcl-2), ced-4 (Apaf-1) and ced-3(Caspase) act in an inhibitory cascade to activate PCD. Our previous data have reported that loss of eat-6, which encodes the alpha subunit of sodium/potassium ion channel,results in significant reduction of cell corpses in the embryos. My studies are focused on how eat-6 affect the number of cell corpses. During apoptosis, the phosphatidylserine (PS)located in the inner leaflet of the plasma membrane is exposed on the surface of apoptotic cells. I used secreted Annexin V fusion GFP as a PS sensor to detect the dying cells. The results showed that Annexin V binding normally to the cell corpses in eat-6 mutant. However, the eat-6 mutant has more extra Annexin V-PS binding rings around the dying cells without cell corpse morphology than the wild type embryos. These results indicated that eat-6 may impact the formation of refractile cell corpse morphology and the translocation of PS. My genetic analysis also showed that eat-6 may work with csk-1, a non-receptor tyrosine kinase to promote cell death. In addition, overexpression of eat-6 can act upstream or in parallel to egl-1 to kill touch neurons and this killing function isdependent on its ATPase activity which is required for pumping. According to these data,eat-6 functions not only in maintaining membrane potential but also in affecting the morphology formation and PS translocation of dying cells.
Keywords: C. elegans, programmed cell death, Na+-K+ ATPase, cell corpse, morphology change, phosphatidylserine (PS) translocation

並列關鍵字

C. elegans ； programmed cell death ； Na+-K+ ATPase ； cell corpse ； morphology change ； phosphatidylserine (PS) translocation

參考文獻

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