DPY-24蛋白質層次在Distal Tip Cells遷移中的調控機制

Abstract

在線蟲的hermaphrodites發育過程中，兩個distal tip cells (DTCs)會進行細胞遷移並使生殖腺發育為前後各一U字型的形狀，這個U字型的細胞遷移路徑分為三個階段：階段一，兩個DTC沿著腹側向線蟲身體兩側遷移至咽喉及肛門旁；階段二，DTC轉90度進行背向轉彎；階段三，DTC再轉90度並沿著背側遷移回到身體中央並停止。dpy-24基因表現出的蛋白質DPY-24具有一個PR domain及一個zinc finger，而這個protein藉由轉錄調控unc-5的表現來控制DTC進行dorsal turn的時間。dpy-24突變株會出現DTC提早進行dorsal turn的性狀。利用anti-DPY-24抗體進行免疫染色的結果顯示DPY-24蛋白質可以在DTC進行dorsal turn之前被偵測到，但是在轉彎之後就再也無法偵測到DPY-24信號。為了研究DPY-24 protein量在轉彎之後下降的原因，我們製造了許多轉錄與轉譯的GFP接合表現質體，發現由dpy-24 promoter驅動表現的GFP (Pdpy-24::gfp::unc-54 3’UTR)及與GFP接合的dpy-24 3’un-translated region (3’UTR) (Plag-2::gfp::dpy-24 3’UTR) 在線蟲DTC中都會持續表現到階段3。我們接下去測試這個調控是在轉譯或是後轉譯層次進行，將Plag-2::dpy-24(cDNA)::unc-54 3’UTR的表現質體微注射到wild-type的線蟲之後發現只要有dpy-24 cDNA表現就可以調控DTC的遷移，因此我們可以知道dpy-24在DTC中的調控層次是在轉譯、後轉譯或是在蛋白質穩定性的層次。

During hermaphrodite development two distal tip cells (DTCs) migrate to generate U-shaped gonadal arms. These cells undergo three phases of migration, they first move along the ventral side to the ends of the body (phase I), then make a dorsal turn (phase II) and finally migrate back to the center of body along dorsal side (phase III). The dpy-24 gene encodes a protein with a PR domain and zinc-finger and likely controls the timing of DTC dorsal migration by transcriptional repression of unc-5. Mutations in dpy-24 resulted in the precocious dorsal turn of DTCs. Immunostaining using anti-DPY-24 antibodies detected DPY-24 protein in DTCs during migration phase I but not II or III. Interestingly, the constitutive expression of dpy-24 partially blocked DTC dorsal turn. These results together indicate that the drop of DPY-24 level is crucial for switching the migration phase of DTCs from I to II. To investigate the cause for the decrease of DPY-24 level in migration phase II, we generated various transcriptional and translational gfp reporter gene fusions. The gfp reporter construct Pdpy-24::gfp::unc-54 3’UTR carrying the dpy-24 promoter and unc-54 3’ un-translational region (UTR) and the plasmid Plag-2::gfp::dpy-24 3’UTR containing the lag-2 promoter and dpy-24 3’ UTR showed GFP expression in all migration phases I, II and III. These results indicate that the drop of DPY-24 level is likely not regulated at the transcriptional level. We then test if the regulation may be at the post-transcriptional, translational or post-translational level. We inject the construct Plag-2::dpy-24(cDNA)::unc-54 3’UTR into N2, and the results show that most of these transgenic animals are wild-type. Therefore, the regulation level of DPY-24 in DTCs is translational level, post-translational level or protein stability level.