吳益群臺灣大學：分子與細胞生物學研究所王蕙瑜Wang, Huei-YuHuei-YuWang2007-11-252018-07-062007-11-252018-07-062007http://ntur.lib.ntu.edu.tw//handle/246246/49937calnexin (CNX) 和calreticulin (CRT) 一般廣為人知的功用，是位於內質網中扮演新生蛋白質的保護子，幫助蛋白質正確地進行折疊和糖基化。然而，有越來越多的研究顯示，CNX和CRT具有更多除了保護子之外的功能，例如，在細胞計畫性死亡方面，過度表現死亡細胞中特有的CNX片段在老鼠細胞中，會抑制其進行計畫性死亡；此外，CRT則被報導出現在哺乳類多種細胞的表面上，當細胞進行計畫性死亡時，這些CRT便做為可被吞噬細胞所辨視的訊號，促使死亡細胞被正常地吞噬而避免產生發炎反應。我們的實驗以線蟲為模式，進一步提供活體生物的證據，證明CNX和CRT在活體中亦同樣地對於細胞計畫性死亡的吞噬過程有所影響。線蟲的calnexin和calreticulin分別由cnx-1和crt-1兩基因所編碼。我們以分析此兩基因突變品種上出現之突起透鏡狀細胞屍體的數量和野生型品種N2比較為方法，來檢測它們在細胞死亡過程中的功能。我們發現，cnx-1; crt-1被雙重地突變而不再有功能的線蟲品系中，其細胞屍體數量和野生型品種比較，有顯著的提高，而cnx-1和crt-1各自的單一突變品種使細胞屍體提高的現象則相對之下輕微許多；這表示此兩基因可能分別作用在部份重複的途徑上。進一步以4D攝影分析線蟲AB世系細胞中最早死亡的13顆細胞之相對死亡時間和細胞屍體持續的時間長短的結果則顯示，此死亡細胞數量提高的現象應是源於吞噬步驟發生問題而非細胞死亡執行上的問題。由於在細胞屍體的吞噬機制上，先前已有兩條被確認的路徑，分別為ced-1, 6, 7 和 ced-2, 5, 10；為了釐清cnx-1和crt-1所作用的路徑為何，我們將此兩基因分別和ced-1及ced-5兩基因做雙重突變並分析這些雙重突變品系蟲體在L1幼蟲時期殘留的未被吞噬之細胞屍體數量。我們發現，cnx-1和crt-1的突變皆會進一步提高原本在ced-1或ced-5突變品系中L1幼蟲時期殘留未被吞噬之細胞屍體數量，這顯示CNX和CRT可能並不屬於這兩條路徑中的任何一條，而是定義了一條新的呑噬路徑。除此之外，我們亦分析了cnx-1突變下ced-1(e1735)品種蟲體之生殖母細胞的細胞死亡數量，發現cnx-1突變會額外地再提高ced-1突變下線蟲生殖母細胞的死亡數量，且此現象可被外加入之cnx-1::gfp 所消除；這兩現象顯示出cnx-1在線蟲生殖母細胞的細胞屍體吞噬過程亦具有功能。Calnexin and calreticulin are generally known as chaperons located in the ER. However, they are also involved in multiple functions other than chaperons. Overexpression of the cleavage product of calnexin in dying cells has been reported to inhibit apoptosis in mouse cells. On the other hand, calreticulin has been found on the cell surface in various mammalian cell cultures and acts as “eat-me signals” when cells undergo apoptosis. We provided the first in vivo evidence that the calnexin and calreticulin function in the engulfment of apoptotic cells in C. elegans. C. elegans calnexin and calreticulin are encoded by cnx-1 and crt-1, respectively. To examine their function in progressional cell death, we analyzed the cell-death phenotype in cnx-1 and crt-1 mutants. A significant increase of embryonic cell corpses were detected in the cnx-1; crt-1 double mutant strains while a milder increase of embryonic cell corpses were detected in their single mutant strains. This suggests that they may act on partially redundant pathways in the process. When cells undergo programmed cell death, cell corpses adopt a refractile and disc like structure. Using this morphological change as a cell death marker, we analyzed the death of the first 13 cells in the AB cell lineage of wild type and cnx-1; crt-1 mutant embryos. The 4D microscopic analysis revealed that cnx-1 and crt-1 mutations prolong cell-corpse duration time but do not affect the time when cells exhibit the death phenotype. This suggests that cnx-1 and crt-1 act in engulfment rather than in execution of apoptosis. Furthermore, from the result that cnx-1 and crt-1 can further increase the L1 persisting head cell corpse in both ced-1 and ced-5 mutants, we deduce that cnx-1 and crt-1 define a novel pathway parallel to the two classical ced-1, 6, 7 and ced-2, 5, 10 pathways. Finally, because cnx-1 can further increase the germ cell death in ced-1 mutant, and the phenotype can be rescued by introducing cnx-1::gfp into the cnx-1; ced-1 double mutants, we suggest that cnx-1 acts in the engulfing cells other than the dying cells during the engulfment process of germline apoptosis.Table of Contents……………………………………………… 1 Abstract…………………………………………………………5 中文摘要......................................... 7 Introduction……………………………………………… 9 Results……………………………………………………………… 13 cnx-1 RNAi shows higher embryonic cell corpse number than the wild type N2.............................. 13 cnx-1 RNAi in rrf-3 background shows higher embryonic cell corpse number than L4440 vector only control.…………………………………………………… 13 cnx-1 and its homologous gene crt-1 double knock-out mutants have dramatically increased embryonic cell corpse number, whereas only mild changes in cnx-1 or crt-1 single mutants………………………………………………… 14 The comparison between the first existing 13 embryonic cell corpses of N2 and cnx-1(nr2009); crt-1(jh101) has revealed that the cell corpses in the double mutant persist longer than in N2.……………………………15 The expression pattern of CNX-1 shows that CNX-1 is generally expressed in the ER and especially enriched in the cell corpses……………………………………. 17 cnx-1 and crt-1 seem to involve in the engulfing pathway independent of ced-1, -6, -7 and ced-2,-5,-10 ………………………………………………… 17 Loss of cnx-1 function further increases germ cell death in ced-1(e1735), and the phenotype can be rescued by introducing cnx-1::gfp into the cnx-1(nr2009); ced-1(e1735)………………………………………………………………………18 Discussion………………………………………………… 20 cnx-1 and crt-1 are involved in the engulfment of apoptosis in C. elegans………. 20 cnx-1 and crt-1 define a novel engulfing pathway other than the two classical ones... …………………………………………………………….. 20 cnx-1 is also involved in the engulfment of germline apoptosis………………….. 21 Materials and Methods……………………………………… 24 Strains and genetics……………………………….. 24 L1 head persisting cell corpses analysis……………… 24 4D analysis……………………………………………25 Generate double mutants………………………………… 25 Expression of gfp fusion constructs…………………… 26 References……………………………………………………27 Figures and Tables………………………………………30 Figure 1. Protein domains of calnexin and calreticulin…………………………30 Figure 2. The alignment of CRT among homologues in different species.……… 31 Figure 3. The alignment of CNX among homologues in different species.............. 32 Figure 4. The embryonic cell corpse assay of cnx-1(RNAi) in N2………………... 33 Figure 5. The embryonic cell corpse assay of cnx-1(RNAi) in the rrf-3 background...................... 34 Figure 6 Genomic organization of cnx-1(nr2009), cnx-1(nr2010), and crt-1(jh101) deletion mutants and protein analysis.…………………………………………… 35 Figure 7 Embryonic cell corpse assay of cnx-1(RNAi), cnx-1(nr2009), and cnx-1(nr2010)…………………………… 36 Figure 8. Embryonic cell corpse assay of crt-1(jh101)……………………………. 37 Figure 9. Embryonic cell corpse assay of cnx-1(nr2009), crt-1(jh101), and cnx-1(nr2009); crt-1(jh101)…………… 38 Figure 10. Embryonic cell corpse assay of cnx-1(nr2010) and cnx-1(nr2010); crt-1(jh101)………………………… 39 Table 1. 4D analysis of the first 13 embryonic cell corpses……………………40 Figure 11. Statistic analysis of the cell corpse duration.……………….………… 41 Figure 12. Statistic analysis of the duration of the cell corpses #2, #3, #5, and #9...42 Figure 13. Statistic analysis of the death time…………………………………..… 43 Figure 14. Statistic analysis of the death time of the cell corpse #2, #3, #5, and #9 ........................44 Table 2. Functional test of cnx-1::gfp.............. 45 Figure 15. The expression pattern of CNX1::GFP……… 46 Table 3. L1 persisting head cell corpse assay……… 47 Figure 16. Germ cell corpse assay of ced-1(e1735), cnx-1(nr2009); ced-1(e1735), and cnx-1(nr2009); ced-1(e1735)… 48876449 bytesapplication/pdfen-US細胞計畫性死亡吞噬Apoptosisengulfmentotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49937/1/ntu-96-R94b43010-1.pdf