Research Article

Triggered recruitment of ESCRT machinery promotes endolysosomal repair

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Science  06 Apr 2018:
Vol. 360, Issue 6384, eaar5078
DOI: 10.1126/science.aar5078

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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

Structured Abstract

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.

Abstract

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.

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