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標題: | 分子與遺傳分析線蟲psr-1在細胞屍體吞噬過程中的角色 Molecular and Genetic Analysis of C. elegans psr-1 in the Cell-Corpse Engulfment Process |
作者: | Pei-Ken Hsu 徐培根 |
指導教授: | 吳益群(Yi-Chun Wu) |
關鍵字: | 線蟲,細胞屍體,吞噬, psr,cell-corpse,engulfment, |
出版年 : | 2005 |
學位: | 碩士 |
摘要: | 在多細胞生物中,正確的清除細胞自伐所造成的死亡細胞對於個體的生存十分重要。在線蟲死亡細胞的清除過程中,PSR-1被認為是作用在CED-2、CED-5、CED-10及CED-12上游的受器,調控這個過程。我們證實PSR-1會直接與CED-5及CED-12有直接的交互作用(Wang et al., 2003),並且找出PSR-1中參與作用的區域。PSR-1中另外可能有2個NLS,所以我們產生α-GST-PSR-1-IN的多株抗體,希望能分析PSR-1在細胞內的分佈。但是沒有抗體可以辨識線蟲lysate中自源性或是過量表現的PSR-1蛋白質。我們同時也在psr-1突變種的線蟲中表現不同片段的PSR-1蛋白質(dNLS1, dNLS-2, dNLS1&2, dTM),發現只有PSR-1-dTM不能復原psr-1突變所造成的細胞死亡清除的缺失。因此PSR-1是作用在細胞膜上來促進死亡細胞的吞噬,並且可能是扮演受器的角色。
為了了解psr-1和其他參與吞噬作用的基因之間的關係,我們觀察雙重或三重突變種中的細胞屍體數目。我們也發現了psr-1及mer-1可能共同作用在吞噬的路徑並且兩者間無直接的交互作用。另外,我們發現unc-73及mig-2可能共同作用在一個psr-1沒有參與的吞噬路徑。根據我們的實驗,我們推測PSR-1是可能扮演吞噬受器的角色來調控細胞屍體的吞噬,並且可能經由與MER-1共同作用來達成。 In multicellular organisms, the proper removal of apoptotic cell corpses prevents the possible dispersal of harmful cellular contents from dying cells. In C. elegans, PSR-1 is proposed to function as a receptor and act upstream of the pathway mediated by CED-2, CED-5, CED-10, and CED-12, possibly through physical interaction with CED-5 and CED-12 to control the engulfment process (Wang et al., 2003). We further mapped the binding region in PSR-1 for CED-5 and CED-12 interaction. To examine the expression pattern of PSR-1, we generated mouse and rabbit polyclonal antibodies against GST-PSR-1-IN fusion protein in order to test the subcellular localization of PSR-1. However, these antibodies and α-human PSR antibodies fail to recognize either bacterially-expressed GST-PSR-1-IN or endogenous or overexpressed PSR-1 in worm lysates. In addition to a transmembrane domain (TM), PSR-1 is predicted to possess two nuclear localization signals (NLSs). To test if any of these motifs is important for PSR-1 function, we performed deletion assays. We found that TM but not NLS is important for the engulfment function of PSR-1. These results support the previous hypothesis that PSR-1 functions on the cell membrane and possibly as a receptor to promote cell corpses uptake. In order to investigate the relationship between psr-1 and other possible engulfment genes, mer-1, unc-73, and mig-2, we analyzed the numbers of corpse in double and triple mutants. We showed that psr-1 and another engulfment receptor, mer-1, act in the same genetic pathway, and that there is no direct interaction between PSR-1 and MER-1 intracellular or extracellular domains. Besides, unc-73 and mig-2 may play minor roles and act in the same genetic pathway which does not involve psr-1 to control cell-corpse engulfment. Together our experiments suggested that PSR-1 may act as a membrane receptor to regulate cell-corpse engulfment and that it may function with MER-1 to achieve this process. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35872 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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