The autophagic protein LC3 translocates to the nucleus and localizes in the nucleolus associated to NUFIP1 in response to cyclic mechanical stress

The trabecular meshwork (TM) is a key regulatory tissue of intraocular pressure (IOP) in the anterior chamber of eye. Dysfunction of the TM causes resistance to outflow of aqueous humor, which in turn leads to elevated IOP, a main risk factor of glaucomatous neurodegeneration. Due to variations in IOP, TM cells are continuously exposed to mechanical deformations. We previously reported activation of macroautophagy/autophagy, as one of the physiological responses elicited in TM cells following mechanical strain application. By using biochemical fractionation analysis and imaging techniques, we demonstrate here for the first time the nuclear accumulation of the autophagic marker MAP1LC3/LC3 (microtubule associated protein1 light chain 3)-II, endogenous and exogenously added (AdGFP-LC3, AdtfLC3), in response to cyclic mechanical stress (CMS). Wheat germ agglutinin (WGA) and leptomycin B treatment suggest LC3 to enter the nucleus by passive diffusion, but to exit in an XPO1/CRM1 (exportin 1)-dependent manner in human TM (hTM) cells. While blockage of nuclear export leads to accumulation of LC3 with promyelocytic leukemia (PML) bodies, nuclear LC3 localizes in the nucleolus in cells under CMS. Moreover, nuclear LC3 co-immunoprecipitated with NUFIP1, a ribosome receptor for starvation-induced ribophagy. More interestingly, we further demonstrate that NUFIP1 translocates from the nucleus to LAMP2 (lysosomal associated membrane protein 2)-positive organelles in the stretched cells without triggering ribophagy, suggesting a more general role of NUFIP1 as a selective autophagy receptor for another yet-to-be-identified target in CMS and a surveillance role of nuclear LC3 against stretch-induced damage. AdGFP: adenovirus encoding GFP; ATG: autophagy-related; BSA: bovine serum albumin; CMS: cyclic mechanical stretch; Co-IP: coimmunoprecipitation; DAPI: 4′,6-diamidino-2-phenylindole; DFCs: dense fibrillar components; EM: electron microscopy; FCs: fibrillar centers; GCs: granular components; GFP: green fluorescent protein; hTM: human trabecular meshwork; HBSS: Hanks balanced salt solution; IOP: intraocular pressure; LAMP1/2: lysosomal associated membrane protein 1/2; LepB: leptomycin B; MTOR: mechanistic target of rapamacyin kinase; NES: nuclear export signals; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NLS: nuclear localization signal; NPCs: nuclear pore complexes; NUFIP1: nuclear FMR1 interacting protein 1; NS: non-stretched; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; pfu: plaque-forming units; PML: promyelocytic leukemia; RFP: red fluorescent protein; RPS15A: ribosomal protein S15a; RPL26: ribosomal protein L26; rRNA: ribosomal RNA; SIRT1: sirtuin 1; SQSTM1/p62: sequestosome 1; tfLC3: mRFP-GFP tandem fluorescent-tagged LC3; TM: trabecular meshwork; WB: western blot; WDR36: WD repeat domain 36; WGA: wheat germ agglutinin; XPO1/CRM1: exportin 1.

小梁网（trabecular meshwork, TM）是眼前房内眼压（intraocular pressure, IOP）的关键调控组织。TM功能障碍会导致房水流出阻力增加，进而引发眼压升高——这是青光眼性神经变性的主要危险因素。由于眼压存在波动，TM细胞持续暴露于机械形变之中。我们此前曾报道，机械牵张刺激后的TM细胞会激活巨自噬/自噬（macroautophagy/autophagy），这是其生理应答之一。本研究通过生化分级分离分析与成像技术，首次证实了自噬标志物微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3, MAP1LC3/LC3）-II，以及内源性和外源性添加的（AdGFP-LC3、AdtfLC3），在周期性机械牵张（cyclic mechanical stretch, CMS）刺激下发生核聚集。
麦胚凝集素（wheat germ agglutinin, WGA）与亮霉素B（leptomycin B, LepB）处理实验表明，LC3可通过被动扩散进入细胞核，但在人TM（human trabecular meshwork, hTM）细胞中需依赖输出蛋白1（exportin 1, XPO1/CRM1）完成核输出。核输出阻断会导致LC3与早幼粒细胞白血病（promyelocytic leukemia, PML）小体共聚集，而在CMS刺激下的细胞中，核内LC3则定位于核仁。此外，核LC3可与核FMR1相互作用蛋白1（nuclear FMR1 interacting protein 1, NUFIP1）发生免疫共沉淀，后者是饥饿诱导的核糖体自噬的核糖体受体。
更有趣的是，我们进一步证实，在牵张刺激的细胞中，NUFIP1会从细胞核转位至溶酶体相关膜蛋白2（lysosomal associated membrane protein 2, LAMP2）阳性细胞器，且这一过程不会触发核糖体自噬。这提示NUFIP1可能作为一种选择性自噬受体，在CMS刺激下靶向尚未被鉴定的其他底物，同时也表明核LC3在应对牵张诱导的损伤中发挥监测功能。

附：相关缩写及术语释义
AdGFP：编码GFP的腺病毒；ATG：自噬相关（autophagy-related）；BSA：牛血清白蛋白；CMS：周期性机械牵张；Co-IP：免疫共沉淀；DAPI：4′,6-二脒基-2-苯基吲哚；DFCs：致密纤维组分；EM：电子显微镜；FCs：纤维中心；GCs：颗粒组分；GFP：绿色荧光蛋白；hTM：人小梁网；HBSS：汉克斯平衡盐溶液；IOP：眼压；LAMP1/2：溶酶体相关膜蛋白1/2；LepB：亮霉素B；MTOR：雷帕霉素机制性靶激酶；NES：核输出信号；MAP1LC3/LC3：微管相关蛋白1轻链3；NLS：核定位信号；NPCs：核孔复合物；NUFIP1：核FMR1相互作用蛋白1；NS：非牵张组；PBS：磷酸盐缓冲液；PE：磷脂酰乙醇胺；pfu：噬斑形成单位；PML：早幼粒细胞白血病；RFP：红色荧光蛋白；RPS15A：核糖体蛋白S15a；RPL26：核糖体蛋白L26；rRNA：核糖体RNA；SIRT1：沉默信息调节因子1；SQSTM1/p62：自噬结合蛋白p62/Sequestosome 1；tfLC3：mRFP-GFP串联荧光标记LC3；TM：小梁网；WB：蛋白质印迹；WDR36：WD重复结构域36；WGA：麦胚凝集素；XPO1/CRM1：输出蛋白1。