Removal of hypersignaling endosomes by simaphagy

Activated transmembrane receptors continue to signal following endocytosis and are only silenced upon ESCRT-mediated internalization of the receptors into intralumenal vesicles (ILVs) of the endosomes. Accordingly, endosomes with dysfunctional receptor internalization into ILVs can cause sustained receptor signaling which has been implicated in cancer progression. Here, we describe a surveillance mechanism that allows cells to detect and clear physically intact endosomes with aberrant receptor accumulation and elevated signaling. Proximity biotinylation and proteomics analyses of ESCRT-0 defective endosomes revealed a strong enrichment of the ubiquitin-binding macroautophagy/autophagy receptors SQSTM1 and NBR1, a phenotype that was confirmed in cell culture and fly tissue. Live cell microscopy demonstrated that loss of the ESCRT-0 subunit HGS/HRS or the ESCRT-I subunit VPS37 led to high levels of ubiquitinated and phosphorylated receptors on endosomes. This was accompanied by dynamic recruitment of NBR1 and SQSTM1 as well as proteins involved in autophagy initiation and autophagosome biogenesis. Light microscopy and electron tomography revealed that endosomes with intact limiting membrane, but aberrant receptor downregulation were engulfed by phagophores. Inhibition of autophagy caused increased intra- and intercellular signaling and directed cell migration. We conclude that dysfunctional endosomes are surveyed and cleared by an autophagic process, simaphagy, which serves as a failsafe mechanism in signal termination. <b>Abbreviations:</b> AKT: AKT serine/threonine kinase; APEX2: apurinic/apyrimidinic endodoexyribonuclease 2; ctrl: control; EEA1: early endosome antigen 1; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; ESCRT: endosomal sorting complex required for transport; GFP: green fluorescent protein; HGS/HRS: hepatocyte growth factor-regulated tyrosine kinase substrate; IF: immunofluorescence; ILV: intralumenal vesicle; KO: knockout; LIR: LC3-interacting region; LLOMe: L-leucyl-L-leucine methyl ester (hydrochloride); MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; NBR1: NBR1 autophagy cargo receptor; PAG10: Protein A-conjugated 10-nm gold; RB1CC1/FIP200: RB1 inducible coiled-coil 1; siRNA: small interfering RNA; SQSTM1: sequestosome 1; TUB: Tubulin; UBA: ubiquitin-associated; ULK1: unc-51 like autophagy activating kinase 1; VCL: Vinculin; VPS37: VPS37 subunit of ESCRT-I; WB: western blot; WT: wild-type.

激活的跨膜受体在胞吞作用后仍可持续传递信号，仅当转运必需内体分选复合物（ESCRT）介导受体向内体的管腔内囊泡（ILVs）内化时，信号才会被沉默。因此，受体向内体管腔内囊泡内化功能异常的内体，可引发受体信号持续激活，这一现象与癌症进展密切相关。本研究报道了一种细胞监视机制，可使细胞识别并清除受体异常聚集、信号通路异常激活且膜结构完整的内体。对转运必需内体分选复合物0亚基（ESCRT-0）缺陷型内体进行邻近生物素标记和蛋白质组学分析后发现，泛素结合型巨自噬/自噬受体SQSTM1与NBR1呈现显著富集，该表型在细胞培养体系与果蝇组织中均得到验证。活细胞显微镜成像结果显示，敲除ESCRT-0亚基HGS/HRS或ESCRT-I亚基VPS37后，内体表面泛素化与磷酸化受体水平显著升高。与此同时，NBR1、SQSTM1以及参与自噬起始与自噬体生物发生的相关蛋白会被动态招募至异常内体处。光学显微镜与电子断层扫描结果显示，尽管限制膜结构完整但受体下调异常的内体，会被吞噬泡所包裹。抑制自噬会导致细胞内与细胞间信号通路活性增强，并诱导细胞定向迁移。综上，本研究证实功能异常的内体可通过一种名为“simaphagy”的自噬过程被监视并清除，该过程作为信号终止的后备保障机制发挥作用。<b>缩写说明：</b> AKT：AKT丝氨酸/苏氨酸激酶；APEX2：无嘌呤/无嘧啶内切脱氧核糖核酸酶2；ctrl：对照；EEA1：早期内体抗原1；EGF：表皮生长因子；EGFR：表皮生长因子受体；ESCRT：转运必需内体分选复合物；GFP：绿色荧光蛋白；HGS/HRS：肝细胞生长因子调节的酪氨酸激酶底物；IF：免疫荧光；ILV：管腔内囊泡；KO：基因敲除；LIR：LC3互作结构域；LLOMe：L-亮氨酰-L-亮氨酸甲酯（盐酸盐）；MAP1LC3/LC3：微管相关蛋白1轻链3；MAPK1/ERK2：丝裂原活化蛋白激酶1；MAPK3/ERK1：丝裂原活化蛋白激酶3；NBR1：NBR1自噬 cargo受体；PAG10：蛋白A标记的10nm金颗粒；RB1CC1/FIP200：RB1诱导型卷曲螺旋1；siRNA：小干扰RNA；SQSTM1：Sequestosome 1；TUB：微管蛋白；UBA：泛素相关结构域；ULK1：Unc-51样自噬激活激酶1；VCL：黏着斑蛋白；VPS37：ESCRT-I复合物的VPS37亚基；WB：蛋白质免疫印迹；WT：野生型。