研究位於果蠅胚胎背部閉鎖時期之上皮細胞黏著帶一新型孔隙之經由微管進行細胞間胞內體運輸之機制與區域限定細胞質連通現象

於果蠅胚胎背部閉鎖時期，上皮細胞中的微管配置會大幅度地由巢狀構造重新排列違反向平行的配置。除了在背部頂端細胞中促進偽足形成的功能之外(Jankovics and Brunner, 2006)，微管也被指出參與了含有黏著帶蛋白分子的胞內體於側邊上皮細胞之間的移動(Li et al., 2015)。然而，其間的運作機制仍幾乎為未知。透過活體影像與免疫組織染色，合成中的微管與乙醯化的微管皆被發現能經由類似孔隙的結構穿過在黏著帶的細胞膜。結合了位於黏著帶與間壁帶的訊號所形成的綠色螢光蛋白區帶，揭露了於側邊上皮細胞的細胞膜上有著一新型黏著帶附著孔隙的存在，且此孔隙能容許細胞間傳遞胞內體。利用以可激發綠色螢光蛋白進行的細胞質連通分析與細胞系譜標定實驗，發現了側邊上皮細胞可透過與環橋及系譜無關的方式連通其它相鄰細胞之細胞質。以光遺傳二聚法連接微管驅動蛋白與位於黏著帶之免疫球蛋白黏著分子的實驗結果中，也支持了微管於細胞間胞內體運輸一事扮演著軌道的角色。穿透式電子顯微鏡影像結果，暗示了於背部閉鎖期間，與微管的架皆可能導致黏著帶細胞膜的不穩定；此外也得到了內有貼覆著許多胞內體的微管、可能為黏著帶附著孔隙之結構。與在哺乳類細胞得到之成果一致，支撐與重新排列微管配置需要鈣黏著分子複合體與許多微管相連蛋白。這些蛋白也因而被認為是「捕捉者」，用來抓住並穩定在細胞邊緣近黏著帶的微管。根據結果，我們提出了一個假說模型：於閉鎖時期，微管被重新配置到黏著帶一層。此一與黏著帶之相互作用造成了局部脂質的不穩定，進而有助於初生孔隙的形成。經過後續的擴張與穩固之後，穩定的乙醯化微管可成為新型成為管之生長軌道，有意於成長與穿越細胞膜上的孔隙、不被黏著帶複合體抓住。這同時也只能相鄰細胞能夠連通細胞質，並容許細胞間胞內體穿越其中。

關鍵字

細胞間溝通；細胞間胞內體；黏著帶附著孔隙；背部閉鎖；微管；黏著帶；鈣黏著分子；免疫球蛋白黏著分子

並列摘要

Microtubule (MT) arrays are dramatically reorganized from nest‐like structures into anti‐parallel ones during dorsal closure (DC) in Drosophila embryos. In addition to its function in dorsal most epithelial (DME) cells for promoting fillopodia formation (Jankovics and Brunner, 2006), MTs are reported to be involved in the intercellular transport (IT) of adherens junction (AJ) protein-containing intercellular endosomes (IEs) in those lateral epithelial (LE) cells (Li et al., 2015); however, the underlying mechanism is poorly understood. By live imaging and immuno-histochemical (IHC) staining, both the synthesizing MTs (EB1) and acetylated MTs (Ac-MTs) were found to be able to pierce through "pore-like" structures on plasma membrane (PM) at AJs. The "GFP belt" resulted from signal combination from AJ and SJ markers revealed the existence of novel AJ-associated pores on the PM of LE cells, which allowed the IT. Analysis with photo-activatable GFP (C3PA-GFP) and cell lineages labeling experiments suggested cytoplasmic connections among neighboring LE cells that were independent of ring canals or cell lineages. Optogenetic dimerization between the Kinesin and Echinoid (Ed) suggested the role of MTs as tracks in the transport of IEs. TEM images implied the instability of AJ membrane during DC upon conjugating with MTs and also the possible structure for AJ-associated pores with vesicle-decorated MTs in the lumen. Consistent with what have been suggested in mammalian cells, cadherin complexes and several MT‐associated proteins were also required for the embracement and rearrangement of MT arrays, and therefore were concerned as the “capturers” for MTs by stabilizing them at the cell cortex near AJs. According to the results, we suggested a hypothetic model for the AJ-associated pores and the MT-dependent IT : MT arrays are rearranged and relocated to the AJs during DC. The association among MTs and AJs leads to local lipid instability, which might benefit the formation of nascent AJ-associated pores. After further expansion and stabilization, acetylated MT arrays may act as the tracks for the synthesizing MTs to grow along and across the "pores" without being captured, and thereby allow the IT of IEs and the cytoplasmic connections.

並列關鍵字

Intercellular communication ； Intercellular endosomes ； Adherens junctions-associated pores ； Dorsal closure ； Microtubules ； Adherens junctions ； DE-cadherin ； Echinoid

參考文獻

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研究位於果蠅胚胎背部閉鎖時期之上皮細胞黏著帶一新型孔隙之經由微管進行細胞間胞內體運輸之機制與區域限定細胞質連通現象

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