Triggered recruitment of ESCRT machinery promotes endolysosomal 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.