吳益群Wu, Yi-Chun臺灣大學:分子與細胞生物學研究所黃騰緯Huang, Teng-WeiTeng-WeiHuang2010-06-022018-07-062010-06-022018-07-062008U0001-2207200811443800http://ntur.lib.ntu.edu.tw//handle/246246/184691在C. elegans中，兩條平行且部分重複的訊息傳遞途徑作用於凋亡細胞的吞噬機制中，至少有八個基因（ced-1, ced-6, ced-7, dyn-1 and ced-2, ced-5, ced-10, ced-12）參與其中。這些基因一旦發生突變，就會造成吞噬功能的缺陷，並產生存留的細胞屍體。CED-2是一個帶有SH2 domain和 SH3 domain的adapter protein，它可能會與CED-5/CED-12形成的heterodimeric guanine nucleotide exchange factor結合，共同活化CED-10（small Rac GTPase），造成細胞骨架的重組，使吞噬細胞能夠吞噬凋亡細胞。我們意外的發現ced-10(n3246) mutation顯著降低ced-5 mutants中的細胞屍體數目。在使用4D顯微攝影，分析AB cell lineage和MS cell lineage中最早的14個細胞死亡過程後，結果發現ced-10(n3246)部分抑制了ced-5 mutant中的吞噬缺陷。這些結果顯示在吞噬凋亡細胞的過程中，ced-10可能具有負向調節的作用。有趣的是，ced-10(n3246)並不會減低ced-12 single mutant或ced-12; ced-5 double mutants中的細胞屍體數目，顯示ced-10的負向調節作用需要ced-12。我們也發現ced-2(n1994)可以減少ced-10(n3246)中的細胞屍體數目，而ced-2(e1752)則否；細胞屍體數目減少的原因是吞噬缺陷受到部分抑制。這些發現意味著CED-2可能透過N-terminal SH3 domain進行對吞噬機制的抑制作用。進一步的遺傳分析顯示ced-2的負向調節作用需要ced-12而不需ced-5。總括來說，透過遺傳和性狀分析，我們的研究顯示ced-2和ced-10在細胞凋亡過程中，對吞噬機制具有先前未知的負向調節作用。In Caenorhabditis elegans, two partially redundant pathways containing at least eight genes (ced-1, ced-6, ced-7, dyn-1 and ced-2, ced-5, ced-10, ced-12) function in the engulfment of apoptotic cells. Mutations in any of these genes cause engulfment defects and hence result in the persistence of cell corpses. CED-2, a SH2 and SH3 containing adapter protein, may bind to the CED-5/CED-12 heterodimeric guanine nucleotide exchange factor complex to activate CED-10, a small Rac GTPase, leading to cytoskeletal reorganization during engulfment of apoptotic cells. Surprisingly, we found ced-10(n3246) significantly reduced cell corpse number in ced-5 mutants. Using four-dimensional microscopic recording, we analyzed the first 14 cell deaths in the AB and MS cell lineages and found that ced-10(n3246) partially suppressed the engulfment defect of ced-5 mutants. These results reveal a possible negative role of ced-10 in cell-corpse engulfment. Interestingly, ced-10(n3246) did not reduce cell corpse number of ced-12 single or ced-12; ced-5 double mutants, indicating that the negative role of ced-10 may require ced-12. We also found that ced-2(n1994), but not ced-2(e1752), mutation reduced cell corpse number in the ced-10(n3246) background; the reduction was caused by a partial suppression of the Ced-10 engulfment defect. These findings suggest that CED-2 may exert an inhibitory function through its N-terminal SH3 domain. Further genetic analyses showed that the negative role of ced-2 required ced-12 but not ced-5. In summary, our genetic and phenotypic analyses reveal novel roles of ced-2 and ced-10 in negative regulation of cell-corpse engulfment during programmed cell death.Table of contents……………………………………………………1ntroduction..............................................6aterials and Methods....................................10. elegans strains, alleles and genetic analysis..........10nalysis of embryonic cell corpses........................11icroscopy and 4D microscopic time-lapse recording........11esults...................................................13ed-10 mutation reduced cell corpse number of strong ced-5 mutants during embryogenesis..............................13ed-10(n3246) mutation partially suppressed the engulfment defects in ced-5(n1812)...................................14he negative role of ced-10 in the ced-5(n1812) background depends on ced-12. .......................................15ed-2(n1994) mutation reduced numbers of cell corpses in ced-10(n3246) mutant in mid-embryogenesis. ...............16ed-2(n1994) mutation reduced the duration of cell corpses in ced-10(n3246) mutant after comma stage. ...............17he N-SH3 domain of CED-2 may participate in a negative role on CED-10. .....18ed-2(n1994) and ced-2(e1752) mutations did not reduce cell corpse number in ced-5(n1812) mutant. ced-2(n1994) increased cell corpse number in ced-12(k149) mutant to the level similar to that of ced-5(n1812). ...............................................19ecrease of cell corpses in ced-10(n3246) mutant by ced-2(n1994) mutation depends on ced-12 but not ced-5. ...........................................................................20iscussion................................................21ed-2 and ced-10 have roles in negative regulation of cell-corpse engulfment during programmed cell death. ...............................................................................21he short isoform of CED-10 may responsible for the negative role. ...................22ED-2 may have a negative role different to mammalian CrkII. ...........................23eference................................................25igures and Tables........................................29igure 1. Protein domains and the mutation sites of CED-2, CED-5, and CED-12....................................................................................................................29igure 2. Protein Sequence and the mutation site of CED-10.................................31igure 3. ced-2, ced-5, ced-10, and ced-12 mutant stains had more embryonic cell corpses than wildtype during embryogenesis. ........................................................32igure 4. ced-10(n3246) mutation reduced cell corpses in ced-5 mutants through embryogenesis. .......................................................................................................33igure 5. Comparison of the duration of first 14 cell corpses shows that ced-10(n3246) mutation reduced the duration of cell corpses in ced-5(n1812) mutant. ....................................................................................................................34igure 6. ced-10(n3246) mutation did not reduce cell corpse number in ced-12(k149) mutant during embryogenesis. .........................................................35igure 7. ced-12(k149) mutation did not enhance the engulfment defect in ced-5(n1812) mutant. ..............................................................................................36igure 8. ced-12(k149) mutation prevented ced-10(n3246) mutation from decreasing the cell corpse number in ced-5(n1812) mutant. ....................................................................................................................37igure 9. ced-2(n1994) mutation reduced cell corpses in ced-10(n3246) mutant in mid-embryogenesis. ................................................................................................38igure 10. Comparison of the duration of first 14 cell corpses shows that ced-2(n1994) mutation did not reduce the duration of cell corpses in ced-10(n3246) mutant. ....................................................................................................................39igure 11. Comparison of the duration of cell corpses precenting at comma stage shows that ced-2(n1994) mutation reduced the duration of cell corpses in ced-10(n3246) mutant. ............................................................................................40igure 12. ced-2(e1752) mutation increased cell corpses in ced-10(n3246) mutant during embryogenesis. ............................................................................................41igure 13. ced-2(e1752) mutant had more cell corpses than ced-2(n1994) mutant during embryogenesis. ............................................................................................42igure 14. ced-2(n1994) and ced-2(e1752) mutations did not reduce cell corpse number in ced-5(n1812) mutant. ............................................................................43igure 15. ced-2(n1994) mutation increased cell corpse number in ced-12(k149) mutant to the level similar to that of ced-5(n1812). ...............................................44igure 16. ced-2(n1994) reduced cell corpse number in ced-10(n3246)ced-5(n1812). ..................................................................................45igure 17. ced-12(k149) mutation prevented ced-2(n1994) mutation from decreasing the cell corpse number in ced-10(n3246) mutant. ................................46igure 18. Model of the negative regulatory mechanisms of ced-2 and ced-10.....47igure 19. The alignment of CED-2 among homologs in different species............48able 1. There is no difference between death time of first 13 cell corpses in AB lineage in ced-10(n3246)ced-5(n1812) double mutant and that in ced-5(n1812) mutant. ....................................................................................................................49able 2. More cell corpses of the first 14 cells that die were engulfed in ced-10(n3246)ced-5(n1812) than in ced-5(n1812) before 2 fold stage. .................50able 3. There is no difference between death time of first 13 cell corpses in AB lineage in ced-2(n1994)ced-10(n3246) double mutant and that in ced-2(n1994) mutant. ....................................................................................................................51able 4. Engulfed and unengulfed cell corpses of the first 14 cells that die were both increased in ced-10(n3246)ced-2(n1994) compared with in ced-10(n3246) .........................................................................................................52able 5. More cell corpses presenting at comma stage in ced-2(n1994)ced-10(n3246) than those in ced-10(n3246) were engulfed before 2 fold stage. ................................................................................................................53ppendix.........................................................................................................................54ppendix 1. Comparison of death time of the 14 cells between eif3.k(gk126) and wild type..................................................................................................................54ppendix 2. Comparison of cell corpse duration of the 14 cell corpses between eif3.k(gk126) and wild type.....................................................................................55ppendix 3. Comparison of time from division to death of the 3 cells between eif3.k(gk126) and wild type.............................................................................................56application/pdf1390643 bytesapplication/pdfen-US線蟲凋亡細胞吞噬機制ced-2ced-10engulfmenthttp://ntur.lib.ntu.edu.tw/bitstream/246246/184691/1/ntu-97-R94b43033-1.pdf