The mammalian actin elongation factor ENAH/MENA contributes to autophagosome formation via its actin regulatory function

Macroautophagy/autophagy is a catabolic process crucial for degrading cytosolic components and damaged organelles to maintain cellular homeostasis, enabling cells to survive in extreme extracellular environments. ENAH/MENA, a member of the Ena/VASP protein family, functions as a highly efficient actin elongation factor. In this study, our objective was to explore the role of ENAH in the autophagy process. Initially, we demonstrated that depleting ENAH in cancer cells inhibits autophagosome formation. Subsequently, we observed ENAH’s colocalization with MAP1LC3/LC3 during tumor cell starvation, dependent on actin cytoskeleton polymerization and the interaction between ENAH and BECN1 (beclin 1). Additionally, mammalian ATG9A formed a ring-like structure around ENAH-LC3 puncta during starvation, relying on actin cytoskeleton polymerization. Furthermore, ENAH’s EVH1 and EVH2 domains were found to be indispensable for its colocalization with LC3 and BECN1, while the PRD domain played a crucial role in the formation of the ATG9A ring. Finally, our study revealed ENAH-led actin comet tails in autophagosome trafficking. In conclusion, our findings provide initial insights into the regulatory role of the mammalian actin elongation factor ENAH in autophagy. Abbreviations: 3-MA 3-methyladenine; ABPs actin-binding proteins; ATG autophagy related; ATG9A autophagy related 9A; Baf A1 bafilomycin A1; CM complete medium; CytERM endoplasmic reticulum signal-anchor membrane protein; Cyto D cytochalasin D; EBSS Earl’s balanced salt solution; ENAH/MENA ENAH actin regulator; EVH1 Ena/VASP homology 1 domain; EVH2 Ena/VASP homology 2 domain; GAPDH glyceraldehyde-3-phosphate dehydrogenase; Lat B latrunculin B; LC3-I unlipidated form of LC3; LC3-II phosphatidylethanolamine-conjugated form of LC3; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; mEGFP monomeric enhanced green fluorescent protein; mTagBFP2 monomeric Tag blue fluorescent protein 2; OSER organized smooth endoplasmic reticulum; PRD proline-rich domain; PtdIns3K class III phosphatidylinositol 3-kinase; WM wortmannin.

巨自噬（Macroautophagy/autophagy）是一类至关重要的分解代谢过程，其功能为降解胞质组分与受损细胞器，以维持细胞稳态，使细胞能够在极端胞外环境中存活。ENAH/MENA（ENAH肌动蛋白调控因子）作为Ena/VASP蛋白家族的成员，是一类高效的肌动蛋白延伸因子。本研究旨在探究ENAH在自噬进程中的调控作用。首先，我们证实了在癌细胞中敲低ENAH可抑制自噬体的形成。随后，我们观察到在肿瘤细胞饥饿状态下，ENAH与微管相关蛋白1轻链3（MAP1LC3/LC3，以下简称LC3）发生共定位，该过程依赖于肌动蛋白细胞骨架的聚合以及ENAH与BECN1（beclin 1）的相互作用。此外，在饥饿条件下，哺乳动物自噬相关蛋白9A（ATG9A）会在ENAH-LC3斑点周围形成环状结构，这一过程同样依赖肌动蛋白细胞骨架的聚合。进一步研究发现，ENAH的Ena/VASP同源1结构域（EVH1）与Ena/VASP同源2结构域（EVH2）对于其与LC3、BECN1的共定位不可或缺，而富脯氨酸结构域（PRD）则在ATG9A环状结构的形成中发挥关键作用。最后，本研究揭示了ENAH介导的肌动蛋白彗星尾参与自噬体的运输过程。综上，本研究的发现首次阐明了哺乳动物肌动蛋白延伸因子ENAH在自噬过程中的调控作用。
缩写说明：3-MA：3-甲基腺嘌呤（3-methyladenine）；ABPs：肌动蛋白结合蛋白（actin-binding proteins）；ATG：自噬相关（autophagy related）；ATG9A：自噬相关蛋白9A（autophagy related 9A）；Baf A1：巴弗洛霉素A1（bafilomycin A1）；CM：完全培养基（complete medium）；CytERM：内质网信号锚定膜蛋白（endoplasmic reticulum signal-anchor membrane protein）；Cyto D：细胞松弛素D（cytochalasin D）；EBSS：伊格尔平衡盐溶液（Earl’s balanced salt solution）；ENAH/MENA：ENAH肌动蛋白调控因子（ENAH actin regulator）；EVH1：Ena/VASP同源1结构域（Ena/VASP homology 1 domain）；EVH2：Ena/VASP同源2结构域（Ena/VASP homology 2 domain）；GAPDH：甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase）；Lat B：拉曲库林B（latrunculin B）；LC3-I：LC3的未脂酰化形式（unlipidated form of LC3）；LC3-II：LC3的磷脂酰乙醇胺结合形式（phosphatidylethanolamine-conjugated form of LC3）；MAP1LC3/LC3：微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3）；mEGFP：单体增强绿色荧光蛋白（monomeric enhanced green fluorescent protein）；mTagBFP2：单体Tag蓝色荧光蛋白2（monomeric Tag blue fluorescent protein 2）；OSER：有序光滑内质网（organized smooth endoplasmic reticulum）；PRD：富脯氨酸结构域（proline-rich domain）；PtdIns3K：III类磷脂酰肌醇3-激酶（class III phosphatidylinositol 3-kinase）；WM：渥曼青霉素（wortmannin）。