遺傳分析C01F4.2/Arhgap6 基因於線蟲細胞吞噬作用中所扮演之角色

Abstract

清除凋亡細胞於計畫性細胞凋亡中是重要過程。在線蟲的研究中發現，於凋亡細胞吞噬過程中，有兩條訊息傳導路徑可活化CED-10/Rac GTPase，其一為ced-1, ced-6, ced-7;另一為ced-2, ced-5, ced-12，造成細胞骨架之重組以吞噬凋亡細胞。於哺乳類細胞株的研究中發現另外兩個同屬Rho-family GTPase的蛋白可調控細胞吞噬作用：RhoA與Cdc42。近年研究認為，特定Rho-family GTPase之活化與去活化在凋亡細胞吞噬過程中均伴演重要角色。這些調控具有時間性與空間性，且需要完善控制。為了尋找參與凋亡細胞吞噬過程中新的Rho-family GTPase之負向調控分子，我們篩檢了十六個於線蟲中會轉錄具有RhoGAP domain蛋白之基因突變體。我們發現，於C01F4.2(ok1316)線蟲中，胚胎時期之凋亡細胞屍體數較野生種高。將C01F4.2a蛋白進行序列比對結果發現其與人類之ARHGAP6蛋白具高度相似性，此蛋白具有特定調控RhoA之活性。藉由分析細胞屍體之殘留時間，我們推測於C01F4.2(ok1316)突變株中所增加之細胞屍體數乃導因於細胞屍體清除機能的缺失。於進一步的遺傳分析中，我們發現C01F4.2基因的缺失並不會增強吞噬基因突變株之性狀。由此我們推測C01F4.2應與兩條訊息傳導路徑之吞噬基因共同作用以調控凋亡細胞之吞噬過程。 重要名詞：計畫性細胞凋亡、細胞吞噬作用、Rho-family GTPases、GAP。

Clearance of apoptotic cells is an ultimate step of programmed cell death. In C. elegans, two partially redundant signaling pathways, ced-1, ced-6, ced-7 in one, and ced-2, ced-5, ced-12 in the other, converge at CED-10, a Rho-family small GTPase, leading to actin cytoskeleton rearrangement during the engulfment of apoptotic cells. Studies in mammalian cell lines also linked the function of two additional Rho-family GTPases (RhoA and Cdc42) to phagocytosis. Recent studies suggested that the activation and inactivation of specific Rho-family proteins is important during the engulfment of apoptotic cells. These regulations should be tightly controlled spatially and temporally. In order to identify novel negative regulator for Rho-family GTPases during engulfment process, we examined 16 potential gap (GTPase-activating protein) genes that encode protein with RhoGAP domain in C. elegans and identified C01F4.2. The C01F4.2(ok1316) mutant and C01F4.2(RNAi) have increased embryonic cell corpse number compared with that of wild-type. The C01F4.2a protein is similar to human ARHGAP6 protein, which has a GTPase-activating activity specific for RhoA. The cell corpse duration analysis showed that the elevated cell corpses number in C01F4.2(ok1316) embryos was due to the defect in the clearance of apoptotic cells. Our double mutant analysis showed that the C01F4.2(ok1316) mutation did not enhance the cell-corpse number of mutants defective in either of the two previously identified engulfment pathways. Therefore C01F4.2 appear to be required functions in both pathways to regulate cell corpses engulfment. Keywords: Programmed cell death, Engulfment, Rho-family GTPases, GAP