A quick fix for leaky endosomes Cells internalize diverse material through various forms of endocytosis into an extensive endolysosomal network. Protecting the integrity of endolysosomal membranes in both physiological and pathophysiological contexts is critical to cell health. Skowyra et al. describe a role for the ESCRT (endosomal sorting complex required for transport) machinery on endolysosomal organelles during membrane repair (see the Perspective by Gutierrez and Carlton). The ESCRTs act as first responders to repair limited membrane damage and thereby restore compartmental integrity and function. This ESCRT activity is distinct from organelle disposal pathways. These findings will be important in understanding cellular responses to invading pathogens and potentially disruptive proinflammatory particulates. Science, this issue p. eaar5078; see also p. 33 Damaged endosomes and lysosomes rapidly recruit ESCRT machinery to promote membrane repair. INTRODUCTION Lysosomes are degradative organelles that break down diverse materials delivered from inside and outside the cell by specialized vesicles called endosomes. Collectively, these membrane-enclosed compartments constitute the endolysosomal network. Endolysosomes can be ruptured or otherwise damaged by materials that they transport or accumulate. Damage can occur intentionally as in the case of incoming pathogens that seek to access the cytoplasm. Alternatively, damage can arise incidentally by membrane destabilizing molecules or by particulates such as crystals and protein aggregates that can puncture the lipid bilayer. To guard against these toxic or harmful substances and preserve pathway function, cells must be able to maintain and restore the integrity of their endolysosomal membranes. The mechanisms responsible for this vital function remain unclear. RATIONALE Extensively damaged compartments can be sequestered and degraded by a form of selective autophagy called lysophagy, which is facilitated by cytosolic damage sensors such as galectins that bind to luminal glycans exposed on injured organelles. More limited damage is likely to require alternative responses for efficient resolution and repair. The endosomal sorting complex required for transport (ESCRT) machinery comprises a collection of proteins that form polymeric filaments to promote budding and fission of membranes in numerous contexts, notably during the formation of multivesicular endosomes. Recent studies highlight an additional role for ESCRT proteins in resolving small wounds on the plasma membrane and tears in the nuclear envelope. We investigated whether ESCRT machinery might also be recruited to damaged endolysosomes to promote their repair. RESULTS Using common peptide reagents that accumulate within acidic endolysosomes and selectively trigger their disruption, we demonstrate that ESCRT machinery rapidly and coherently assembles on the limiting membrane of injured endolysosomal organelles. This response was observed in multiple types of cells, including phagocytes, and was especially prominent on endolysosomes damaged by internalized silica crystals. Notably, damage-triggered ESCRT recruitment required calcium as well as known ESCRT-nucleating factors including TSG101 and ALIX, and was distinct from lysophagy. To investigate the role played by ESCRT machinery on damaged endolysosomes, we used live-cell imaging of fluorescently tagged ESCRT proteins together with probes to dynamically monitor compartmental integrity. These experiments established that ESCRT recruitment correlates with the onset of small perforations permeable to protons, but not with larger ruptures that allow exchange of high molecular weight material, including internalized dextrans and cytoplasmic glycan-sensing galectins. Imaging ESCRT dynamics during a pulse of transient membrane disruption further revealed that ESCRT recruitment precedes recovery of compartmental function, monitored with a fluorogenic indicator of lysosomal protease activity. Accordingly, depleting cells of relevant ESCRT recruitment factors impaired both the reacidification and functional recovery of transiently injured organelles. CONCLUSION Our kinetic and functional data reveal a role for ESCRTs in the repair of small perforations in endolysosomes. This activity enables a restorative response to limited membrane damage that is likely to be protective in pathological contexts involving endolysosomal leakage and may help counter damage-induced inflammation. Transiently pacifying this response could additionally benefit efforts aimed at maximizing targeted drug and nanoparticle delivery through endocytosis. ESCRT participation in endolysosomal repair shares similarities with previously described roles for this machinery in mending nanometer-sized wounds at the plasma membrane and resealing the nuclear envelope, suggesting that ESCRT-promoted membrane repair may constitute a generic cellular response to limited membrane disruption. Based on the geometric constraints of endolysosomes and the type of damage they incur, we speculate that repair could proceed through closure of the membrane wound by ESCRT-containing filaments. Alternative fates of damaged endolysosomes. Materials that are transported by or accumulate in endolysosomes can disrupt their membranes. Small disruptions trigger Ca2+-dependent recruitment of ESCRT machinery to promote repair of the injured organelle. More extensively damaged compartments instead accumulate galectins and are degraded by lysophagy. Endolysosomes can be damaged by diverse materials. Terminally damaged compartments are degraded by lysophagy, but pathways that repair salvageable organelles are poorly understood. Here we found that the endosomal sorting complex required for transport (ESCRT) machinery, known to mediate budding and fission on endolysosomes, also plays an essential role in their repair. ESCRTs were rapidly recruited to acutely injured endolysosomes through a pathway requiring calcium and ESCRT-activating factors that was independent of lysophagy. We used live-cell imaging to demonstrate that ESCRTs responded to small perforations in endolysosomal membranes and enabled compartments to recover from limited damage. Silica crystals that disrupted endolysosomes also triggered ESCRT recruitment. ESCRTs thus provide a defense against endolysosomal damage likely to be relevant in physiological and pathological contexts.

中文翻译：

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ESCRT 机制的触发招募促进内溶酶体修复

快速修复泄漏的内体细胞通过各种形式的内吞作用将不同的物质内化为广泛的内溶酶体网络。在生理和病理生理环境中保护内溶酶体膜的完整性对细胞健康至关重要。Skowyra 等人。描述了 ESCRT（运输所需的内体分选复合体）机器在膜修复过程中对内溶酶体细胞器的作用（参见 Gutierrez 和 Carlton 的观点）。ESCRT 作为第一响应者修复有限的膜损伤，从而恢复隔室完整性和功能。这种 ESCRT 活动不同于细胞器处理途径。这些发现对于理解细胞对入侵病原体和潜在破坏性促炎颗粒的反应非常重要。科学，这个问题 p。eaar5078; 另见第。33 受损的内体和溶酶体迅速募集 ESCRT 机制以促进膜修复。介绍 溶酶体是一种降解细胞器，可分解通过称为内体的特化囊泡从细胞内外传递的各种物质。总的来说，这些膜封闭的隔室构成了内溶酶体网络。内溶酶体可能会被它们运输或积累的材料破裂或以其他方式损坏。损害可能是有意发生的，就像试图进入细胞质的传入病原体一样。或者，膜不稳定分子或颗粒（如晶体和蛋白质聚集体）可能会刺破脂质双层，从而偶然引起损伤。防范这些有毒或有害物质，保护通路功能，细胞必须能够维持和恢复其内溶酶体膜的完整性。负责这一重要功能的机制仍不清楚。基本原理 广泛受损的隔室可以通过一种称为溶菌的选择性自噬形式进行隔离和降解，细胞溶质损伤传感器（例如与暴露在受损细胞器上的腔内聚糖结合的半乳糖凝集素）促进了这种形式。更有限的损坏可能需要替代响应以有效解决和修复。运输所需的内体分选复合物 (ESCRT) 机器包括一组蛋白质，这些蛋白质形成聚合物细丝，以在许多情况下促进膜的出芽和裂变，特别是在多泡内体形成期间。最近的研究强调了 ESCRT 蛋白在解决质膜上的小伤口和核膜撕裂方面的额外作用。我们研究了 ESCRT 机器是否也可能被招募到受损的内溶酶体以促进它们的修复。结果 使用在酸性内溶酶体中积累并选择性触发其破坏的常见肽试剂，我们证明了 ESCRT 机器在受损的内溶酶体细胞器的限制膜上快速且连贯地组装。在包括吞噬细胞在内的多种类型的细胞中观察到这种反应，并且在被内化二氧化硅晶体损坏的内溶酶体上尤为突出。值得注意的是，损伤触发的 ESCRT 募集需要钙以及已知的 ESCRT 成核因子，包括 TSG101 和 ALIX，并且与溶食症不同。为了研究 ESCRT 机制对受损内溶酶体的作用，我们使用荧光标记的 ESCRT 蛋白的活细胞成像以及探针来动态监测隔室完整性。这些实验证实，ESCRT 募集与质子可渗透的小穿孔的发生相关，但与允许交换高分子量材料（包括内化葡聚糖和细胞质聚糖感应半乳糖凝集素）的较大破裂无关。在瞬时膜破裂脉冲期间对 ESCRT 动力学进行成像进一步揭示，ESCRT 募集先于室功能恢复，并通过溶酶体蛋白酶活性的荧光指示剂进行监测。因此，消耗相关 ESCRT 募集因子的细胞会损害暂时性受损细胞器的再酸化和功能恢复。结论我们的动力学和功能数据揭示了 ESCRT 在修复内溶酶体小穿孔中的作用。这种活动能够对有限的膜损伤产生恢复性反应，这可能在涉及内溶酶体渗漏的病理环境中具有保护作用，并且可能有助于对抗损伤引起的炎症。暂时平息这种反应可以另外有益于旨在通过内吞作用最大化靶向药物和纳米颗粒递送的努力。ESCRT 参与内溶酶体修复与之前描述的这种机制在修复质膜上的纳米级伤口和重新密封核膜方面的作用有相似之处，这表明 ESCRT 促进的膜修复可能构成对有限膜破坏的一般细胞反应。基于内溶酶体的几何限制和它们引起的损伤类型，我们推测修复可以通过含有 ESCRT 的细丝闭合膜伤口来进行。受损内溶酶体的替代命运。由内溶酶体运输或积累的物质会破坏它们的膜。小的破坏会触发 Ca2+ 依赖的 ESCRT 机制的募集，以促进受损细胞器的修复。更广泛受损的隔室反而会积聚半乳糖凝集素并被溶食降解。内溶酶体可被多种材料破坏。最终受损的隔室会被溶食降解，但对修复可挽救细胞器的途径知之甚少。在这里，我们发现运输 (ESCRT) 机械所需的内体分选复合物，已知介导内溶酶体的出芽和裂变，也在其修复中发挥重要作用。通过需要钙和 ESCRT 激活因子的途径，ESCRT 被迅速招募到急性损伤的内溶酶体，该途径与溶食无关。我们使用活细胞成像来证明 ESCRT 对内溶酶体膜中的小穿孔有反应，并使隔室能够从有限的损伤中恢复。扰乱内溶酶体的二氧化硅晶体也引发了 ESCRT 招募。因此，ESCRT 可以防御可能与生理和病理背景相关的内溶酶体损伤。已知介导内溶酶体的出芽和裂变，也在其修复中发挥重要作用。通过需要钙和 ESCRT 激活因子的途径，ESCRT 被迅速招募到急性损伤的内溶酶体，该途径与溶食无关。我们使用活细胞成像来证明 ESCRT 对内溶酶体膜中的小穿孔有反应，并使隔室能够从有限的损伤中恢复。扰乱内溶酶体的二氧化硅晶体也引发了 ESCRT 招募。因此，ESCRT 可以防御可能与生理和病理背景相关的内溶酶体损伤。已知介导内溶酶体的出芽和裂变，也在其修复中发挥重要作用。通过需要钙和 ESCRT 激活因子的途径，ESCRT 被迅速招募到急性损伤的内溶酶体，该途径与溶食无关。我们使用活细胞成像来证明 ESCRT 对内溶酶体膜中的小穿孔有反应，并使隔室能够从有限的损伤中恢复。扰乱内溶酶体的二氧化硅晶体也引发了 ESCRT 招募。因此，ESCRT 可以防御可能与生理和病理背景相关的内溶酶体损伤。我们使用活细胞成像来证明 ESCRT 对内溶酶体膜中的小穿孔有反应，并使隔室能够从有限的损伤中恢复。扰乱内溶酶体的二氧化硅晶体也引发了 ESCRT 招募。因此，ESCRT 可以防御可能与生理和病理背景相关的内溶酶体损伤。我们使用活细胞成像来证明 ESCRT 对内溶酶体膜中的小穿孔有反应，并使隔室能够从有限的损伤中恢复。扰乱内溶酶体的二氧化硅晶体也引发了 ESCRT 招募。因此，ESCRT 可以防御可能与生理和病理背景相关的内溶酶体损伤。