吳益群Wu, Yi-Chun臺灣大學:分子與細胞生物學研究所謝曉涵Hsieh, Hsiao-HanHsiao-HanHsieh2010-06-022018-07-062010-06-022018-07-062008U0001-3007200814433900http://ntur.lib.ntu.edu.tw//handle/246246/184709Integrins 是參與細胞黏合、細胞遷移、細胞週期、細胞分化與細胞凋亡等等細胞活動的由異分子組成的細胞表面受器。Integrins 在細胞吞噬過程中的功能已在細胞實驗中發現，但仍缺乏in vivo的證據。PAT-2是線蟲兩種α次分子中的一種，前人的研究發現PAT-2參與線蟲體壁肌肉肌節的形成，pat-2的功能喪失會造成胚胎體軸的延長停留在2-fold時期。我們發現在pat-2 loss-of-function (lf)的胚胎中，細胞屍體的數量比起野生型胚胎有顯著上升的情形。利用time-lapse microscopic recording進行分析，我們發現細胞屍體的數量上升是由於移除細胞屍體的過程受到影響所導致。目前已知在線蟲中有兩條細胞屍體的吞噬路徑，分別由ced-1、 ced-6、ced-7 與ced-2、ced-5、ced-12 組成。我們的研究發現pat-2並不作用在這兩條路徑中，而是與參與肌肉肌節的形成的基因成員一起作用以促進細胞屍體的吞噬。PAT-2在肌肉細胞專一性的表現能消除 pat-2(lf)的胚胎中細胞屍體數量上升的情形，而PAT-2在表皮細胞中的功能也在我們的研究中發現。以上結果顯示，PAT-2 integrin定義了一條新的細胞吞噬路徑，此路徑在肌肉細胞中扮演重要功能，並可能也作用在其他細胞種類。Integrins are heterodimeric transmembrane receptors that function in cell adhesion, migration, cell cycle progression, differentiation and apoptosis. Its role in the phagocytosis of apoptotic cells has been shown in cultured mammalian cells but has not yet been characterized in vivo. There are two α subunits, PAT-2 and INA-1, and a sole β subunit, PAT-3, in C. elegans. PAT-2 is known to be essential for sarcomere assembly and pat-2 mutants arrested at the 2-fold stage when active muscle movements start. We found that in pat-2 loss-of -function embryos the number of cell corpses significantly increased when compared with that of wild type. A time-lapse microscopic recording showed that the increase of cell corpse number in the pat-2(st567) embryos was due to the failure of cell-corpse removal. C. elegans cell-corpse engulfment was previously shown to be mediated by two partially redundant pathways, ced-1, ced-6, ced-7 in one and ced-2, ced-5 and ced-12 in the other. Clustering of PAT-2 around cell corpses was not affected by ced-1 or ced-5 mutation. In addition, the pat-2 mutation further enhanced the single mutants or double mutants between the two pathways, suggesting that pat-2 may function in a third engulfment pathway. Like pat-2, other genes involved in the body wall muscle assembly also function of cell-corpse engulfment, likely in the same pathway with pat-2. Muscle-specific expression of pat-2 rescued the corpse-engulf-defect of pat-2 embryos. This result indicates that muscle cells, at least primarily, act as engulfing cells in the PAT-2-mediated engulfment process. We conclude that PAT-2 integrin defines a novel engulfment pathway in muscle cells.口試委員會審定書 ..................................................................................................2文摘要 ..................................................................................................................3bstract ...................................................................................................................4. Introduction ...............................................................................................8. Material and method …………………………………………………14.1 C. elegans strains used ……………………………………………….……14.2 To maintain C. elegans ……………………………………….……………14.3 To identify cell corpse under DIC microscope ……………………….……14.4 RNAi experiments ………………………………………………….…...…14.5 Time-lapse recording with DIC microscopy ………….…………….……..15.6 Plasmid construction ………………………………………………….…...15.7 Transgenic animals ………………………………………………………...16. Results………………………………………………………………………17.1 The number of cell corpses increased in pat-2 embryos …………….......…17.2 Ced phenotype of pat-2 embryos was due to the failure of cell corpse removal ………………………………………………………………….…18.3 PAT-2 functioned in engulfing cells and surrounded cell corpses …………18.4 pat-2 may function in a novel engulfment pathway ……………………..…19.5 Genes involved in the muscle assembly pathway functioned together with pat-2 ………………………………………………………………………..20.6 Muscle cells acted as engulfing cells in the PAT-2-mediated engulfment process ……………………………………………………………….……..20.7 The major engulfment pathway in muscle differed from that in hypodermal cells …………………………………………………………………………21. Discussion ………………………………………………………………22.1 pat-2 acts in engulfing cells and defines a novel engulfment pathway ……………………………………………………………….…..22.2 pat-2 acts predominantly in muscle cells and also has minor roles in hypodermal cells during cell-corpse removal …………………………………….……...23.3 How does pat-2 pathway work during engulfment? …………………...……24. Reference …………………………………………………………………28. Figures and Tables ………………………………………………….….37igure 1. Embryos at different developmental stages and the morphology of cell corpses under Nomarski (DIC) optics …………………………….….37igure 2. A time-lapse microscopic recording showed that the timing and numbers of cell death events were not affected by pat-2(st567) ……………….…38igure 3. A time-lapse microscopic recording showed that the duration of cell corpses increased compared with that of wild-type embryos ……………....…39igure 4. A pat-2 cell corpse persisted longer with an abnormal morphological appearance ………………………………………………………...….40igure 5. PAT-2 co-localized with CED-1 around cell corpses ……..…….....…42igure 6. PAT-2 surrounded cell corpses ………………………………..…...…44igure 7. Localization of PAT-2 around cell corpses was not disrupted by ced-1 and ced-5 mutations . ……………………………………………….….…46igure 8. pat-2 was strongly expressed in body wall muscles (BWM) ………....47igure 9. MLC-3, the sole essential myosin light chain in C elegans, co-localized with PAT-2 around cell corpse ……………………………….……………49igure 10. Muscle cells were competent engulfing cells .………………………51igure 11. PAT-2 was expressed in hypodermal cells .…………………………52igure 12. ced-1 was expressed in muscles cells .………………………………53able 1. The number of cell corpses increased in pat-2(st567) embryos ………54able 2. Genomic DNA of pat-2 rescued both Pat and Ced phenotype of pat-2(st567) embryos ………………………………………………………………55able 3. The pat-2 mutation further enhanced the mutants in the two pathways ……………………………………………………………..56able 4. The pat-2 mutation further enhanced the double mutants between the two pathways ………………………………………………………….….57able 5. The number of cell corpses increased in mutants of genes involving in body wall muscle assembly ……………………………………………..…58able 6. Genes involved in the body wall muscle assembly pathway functioned together with pat-2 ……………………………………………..……59able 7. pat-2 primarily functioned in muscle cells ………………………...…60able 8. Duration of corpses of C1, C2, C3 cells ………………………...……61able 9. Muscle cell phenotype in Pat mutants …………………………......…62application/pdf4759937 bytesapplication/pdfen-USintegrinPAT-2細胞凋亡細胞屍體細胞吞噬線蟲programmed cell deathcell corpseengulfmentC. eleganshttp://ntur.lib.ntu.edu.tw/bitstream/246246/184709/1/ntu-97-R95b43026-1.pdf