Capulet和Slinghsot在果蠅複眼發育中所扮演的角色

Capulet and Slingshot act at distinct steps to modulate Twinstar-mediated actin remodeling during Drosophila eye morphogenesis

林巧敏(Chiao-Ming Lin)

指導教授 ： 徐瑞洲

國立清華大學/生命科學院/分子醫學研究所/博士(2012年)

https://doi.org/10.6843/NTHU.2012.00012

摘要

果蠅的成蟲複眼是由在幼蟲時期的單一層表皮細胞，經由廣泛地形態改變，進而形成正確地小眼排列方式所組成的。這些劇烈地細胞型態改變主要是由一群會與肌動蛋白相結合的蛋白質，透過重新調整細胞骨架蛋白的組成來達成。CAP/Capulet (Capt), Slingshot (Ssh) 和Cofilin/Twinstar (Tsr)都是屬於會利用與肌動蛋白相結合而限制肌動蛋白聚合成長鏈狀的相關蛋白質。在過去的研究中，透過低解析度的觀察下發現，當capt, ssh 和 tsr這三個基因產生突變時，會導致在果蠅不同組織內都會造成大量肌動蛋白的累積。在此篇論文中，我們利用果蠅幼蟲複眼的單層表皮細胞為模式，且在高解析度的情況下，分析比較這三個基因突變時所造成的影響。我們發現capt, ssh的突變在morphogenetic furrow (MF)上和之後會具有相似的表現型，但是tsr則不會有類似的情況產生。在capt, ssh這兩個基因突變的細胞內，我們都可以觀察到(1)具有六角形細胞外型且由不連續的adherens junctions(AJs)所包圍；(2)大量累積活化態的肌球蛋白和肌動蛋白，且兩者呈現互補的表現。我們進一步發現capt, ssh突變的表現型是與protein kinase A (PKA)的不活化以及Rho的活化有關。而肌動蛋白的累積則是和Hedgehog訊號傳遞下游分子Ci75的移除有關。相反地，capt, ssh卻又負向調控Ci155量的累積，並且作用於PKA對Ci155蛋白質水解過程的上游。與ssh不同的是，在capt突變的情況下可以觀察到大量活化態的Cofilin。雖然過去的研究都認為Capt的C端為主要的功能片段，我們發現當大量表現Capt的N端就可以使capt突變的現象恢復。因此我們認為Capt利用其N端來幫助Cofilin的再循環利用，而Ssh則是利用調控的Cofilin活化狀態，藉此共同影響藉由肌動蛋白的動態變化而改變細胞外型的MF的進行，如此才能在果蠅複眼發育中正確地篩選及排列小眼的相對位置，使其具有正確的功能。

關鍵字

果蠅複眼發育 ； 細胞骨架蛋白

並列摘要

The Drosophila compound eye is formed from a sheet of epithelial tissue that undergoes extensive morphogenesis to form a pattern of regularly spaced ommatidia. These dramatic cell shape changes are regulated by reorganization of cytoskeleton via various of actin-binding proteins (ABPs). CAP/Capulet (Capt), Slingshot (Ssh) and Cofilin/Twinstar (Tsr) are ABPs that restrict actin polymerization. Previously, it was shown that low resolution analyses of loss-of-function mutations in capt, ssh and tsr all show ectopic F-actin accumulation in various Drosophila tissues. Here, we compared their loss-of-function phenotype at single-cell resolution, using a sheet of epithelial cells in the Drosophila eye imaginal disc as a model system. Surprisingly, we found that capt and ssh, but not tsr, mutant cells within and posterior to the morphogenetic furrow (MF) shared similar phenotypes. The capt/ssh mutant cells possessed: (1) hexagonal cell packing with discontinuous adherens junctions(AJs); and (2) largely complementary accumulation of excessive phosphorylated myosin light chain (p-MLC) and F-actin rings at the apical cortex. We further showed that the capt/ssh mutant phenotypes depended on the inactivation of protein kinase A (PKA) and activation of Rho. We also found that the accumulation of F-actin is dependent on the removal of Ci75. Conversely, Capt/Ssh negatively regulated Ci155 levels within the MF at a step upstream of protein kinase A-mediated Ci155 proteolysis. Significantly, unlike ssh mutant cells, a marked reduction of phosphorylated cofilin (p-cofilin) was detected in capt mutant cells. Although most studies have focused on the role of the C-terminal actin-binding domain of Capt, we found that overexpressing the N-terminal region of Capt that recycles cofilin and a constitutively active form of cofilin rescued the capt and ssh mutant phenotypes. We conclude that Capt and Ssh act at distinct steps to recycle and dephosphorylate cofilin, respectively, and then alter the cell shape changes and progression of the MF that in turn precisely organize the pattern of ommatidia during Drosophila eye morphogenesis.

並列關鍵字

Drosophila ； capulet ； slinshot ； twinstar

參考文獻

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