吳益群臺灣大學：分子與細胞生物學研究所曾睿英Tzeng, Ruei-YingRuei-YingTzeng2007-11-252018-07-062007-11-252018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/49910細胞計畫性的死亡是多細胞生物發生過程中的正常程序用來消滅不需要的細胞；而細胞吞噬則是必須的程序用以清除因細胞自我死亡所產生的細胞屍體。今年來的研究證明這些訊息傳遞路徑在物種間是有保守性的。Integrins 是一個很大的家族由異分子組成的細胞表面受器，參與細胞黏合、細胞遷移與細胞訊息傳遞過程。ina-1是線蟲integrin 的α次分子，主要影響神經及DTC細胞的遷移。我們發現當ina-1 缺失時，會導致線蟲細胞屍體數目增多，且我們證明此一增多的情況不是因為integrin 缺失導致細胞有anoikis 的性狀，並且這些細胞屍體都是由正常的細胞死亡產生的。這些結果顯示ina-1突變所產生的細胞屍體增加可能為影響了細胞吞噬的過程。我們進一步的去尋找ina-1與其他已知參與在細胞吞噬作用的其他成員之間的關係，發現ina-1可能可以定義一個新的細胞吞噬訊息傳遞路徑。 M9是人類肌肉相關蛋白。我們發現當cM9（線蟲的M9同源基因）突變時，在胚胎發育過程會產生比較少的細胞屍體。並且cM9突變種在腹節神經細胞裡有多餘的細胞逃過細胞死亡的命運而存活下來。因此，我們認為cM9應該為促進細胞死亡的基因。除此之外，我們在觸覺神經中過量表現ced-3(caspase)的情況下過量表現cM9，發現比起單獨ced-3所引發的觸覺神經細胞死亡，在ced-3和cM9一起過量表現有更多的神經細胞死亡。此一結果暗示cM9引發的細胞死亡可能是caspase independent。During the development of multicellular organisms, programmed cell death (apoptosis) is a normally occurring process used to eliminate unnecessary cells. Engulfment is an essential process for clearance of cell corpse generated by apoptosis. Recent studies demonstrate that the molecular control of these processes is evolutionally conserved in metazoans. Integrins are a large family of heterodimeric cell surface receptors involved in cell adhesion, cell migration and signal transduction. INA-1 is C. elegans integrin α subunit that plays roles in neural development and DTC migration. We found that ina-1 mutations increased number of cell corpses in embryos and that this ina-1-mediated enhancement in cell-corpse number is through programmed cell death. This implies that ina-1 might be important in the engulfment process to remove cell corpses. Interestingly, ina-1 acts synergistically with previously identified engulfment genes, suggesting that ina-1 may act in parallel to these genes during programmed cell death and may define a new engulfment pathway in C. elegans. M9 is human muscle specific protein. We found that animals bear a mutation in cM9 (which is C. elegans homolog of M9) decreased numbers of cell corpses throughout embryogenesis. We further showed that cM9 mutant animals had extra surviving cells in the ventral cord in the engulfment-defective ced-2 mutant background. Together these data suggested that cM9 function as a positive mediator during apoptosis in C. elegans. We also showed that cM9 acts synergistically with ced-3 (which is a caspase) to promote cell-killing, suggesting that cM9 may mediate apoptosis through a ced-3 independent pathway.Table of contents Table of contents…………………………………………………………………1 Abstract…………………………………………………………………………… 5 中文摘要…………………………………………………………………………….6 Introduction………………………………………………………………………..7 Experimental Procedures………………………………………………………16 Strains and Genetics………………………………………………………….16 Molecular Constructs……………………...................................................16 Transgenic Animals……………………………………………………..........17 Heat Shock Experiments……………………………………………………..18 Bacteria-mediated RNAi………………………………………………….......19 Results……………………………………………………………………………...20 ina-1 is likely a novel engulfment gene…………………………………..20 The ina-1 mutation and ina-1(RNAi) significantly increased numbers of cell corpses throughout embryogenesis………………………………………….20 The extra cell-corpses observed in ina-1 mutants are generated by programmed cell death………………………………………………………..20 ina-1 might define a new engulfment pathway……………………………...23 cM9 is a novel pro-apoptotic gene in C. elegans………………………..24 cM9 mutation decreased cell-corpse numbers throughout embryogenesis…………………………………………………………...24 cM9 mutations promote extra cell survived in ventral cord neuron cells…25 Overexpression of cM9 in the whole embryos does not increase embryonic lethality…………………………………………………………………………..26 cM9 acts cell autonomously to kill touch receptor neurons and the killing effect is enhanced by ced-3 overexpression………………………………...26 Intermediate filament may have a minor role in cell-killing…………………27 Discussion………………………………………………………………………....28 ina-1 may function in the engulfment process………………………………28 Caspase-independent apoptosis – more than one way to kill a cell………28 References…………………………………………………………………………31 Figures and tables………………………………………………………………..37 Figure 1. The genetic pathway of apoptosis in C. elegans………………37 Figure 2. The ina-1 mutation and ina-1(RNAi) significantly increased numbers of cell corpses throughout embryogenesis……………………….38 Figure 3. Cell-corpse numbers in ina-1 mutants in the presence or absence of dcDAPK mutation…………………………………………………39 Figure 4. Cell-corpse numbers in ina-1 mutants in the presence or absence of p53 (cep-1) mutation……………………………………………..40 Figure 5. The extra cell corpses in ina-1(RNAi) are generated by central programmed cell death………………………………………………………..41 Figure 6. Cell-corpse numbers in ina-1 mutants in the presence or absence of ced-4(n2273) mutation…………………………………………...42 Figure 7. Cell-corpse numbers in ina-1 mutants in the presence or absence of dcM9(gk126) mutation…………………………………………...43 Figure 8. Cell-corpse numbers in ina-1 mutants in the presence or absence of psr-1(tm469) mutation……………………………………………44 Figure 9. Two parallel and partially redundant signaling pathways control the engulfment of apoptotic cells in C. elegans……………………………..45 Figure 10. The cm9 mutation decreased numbers of cell corpses throughout embryogenesis……………………………………………………46 Figure 11. Engulfment or apoptosis mutations lead to the survival of cells programmed to die in the ventral cord……………………………………….47 Figure 12. cM9 acts cell autonomously to kill touch receptor neurons and the killing effect is enhanced by ced-3 overexpression…………………….48 Table 1. ina-1(RNAi) enhances numbers of persistent cell corpses in ced-1, ced-2, and ced-5 mutant backgrounds………………………………49 Table 2. The dcM9(gk126) mutation results in the survival of cells that normally die in the ventral cord……………………………………………….50 Table 3. Overexpression of cm9 under the control of heat-shock promoters does not enhance embryonic lethality in wild type or in ced-3 overexpression backgrounds…………………………………………………51 Table 4. Intermediate filament a-2 and c-1 may have a minor role in cell-killing in a ced-2(n1994) background……………………………………52 Appendix………...…………………………………………………………………53 Appendix 1. The evolutionary tree of integrin α subunits and β subunits……………………………………………………………………..…..53 Appendix 2. The 11 intermediate filaments in C. elegans……………….54 Appendix 3. ina-1 does not involve in the ced-2, 5, 10, 12 engulfment pathway…………………………………………………………………………55 Appendix 4. ina-1 does not involve in the ced-2, 5, 10, 12 engulfment pathway…………………………………………………………………………56 Appendix 5. ina-1 does not involve in the ced-2, 5, 10, 12 engulfment pathway…………………………………………………………………………57 Appendix 6. ina-1 does not involve in the ced-1, 6, 7 engulfment pathway…………………………………………………………………………58 Appendix 7. ina-1 does not involve in the ced-1, 6, 7 engulfment pathway…………………………………………………………………………59 Appendix 8. ced-10 Mutations Reduced Numbers of Persistent Cell Corpses in Strong ced-2, ced-5, and ced-12 Mutants……………………..60 Appendix 9. ced-10 mutations reduced numbers of persistent cell corpses in strong ced-5 mutants throughout embryogenesis……………………….61 Appendix 10. A. Persistance of Pn.aap cell corpse in ced-5 and ced-10ced-5 mutants. B. The Pn.aap surviving rate in ced-10 double mutants is higher than ced-2 ced-5 mutants alone…………………………62771793 bytesapplication/pdfen-US線蟲細胞凋亡C. elegansapoptosisengulfmentotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49910/1/ntu-94-R92b43010-1.pdf