Lysosomal damage sensing and lysophagy initiation by SPG20-ITCH.

Cells respond to lysosomal membrane permeabilization by membrane repair or selective macroautophagy of damaged lysosomes, termed lysophagy, but it is not fully understood how this decision is made. Here, we uncover a pathway in human cells that detects lipid bilayer perturbations in the limiting membrane of compromised lysosomes, which fail to be repaired, and then initiates ubiquitin-triggered lysophagy. We find that SPG20 binds the repair factor IST1 on damaged lysosomes and, importantly, integrates that with the detection of damage-associated lipid-packing defects of the lysosomal membrane. Detection occurs via sensory amphipathic helices in SPG20 before rupture of the membrane. If lipid-packing defects are extensive, such as during lipid peroxidation, SPG20 recruits and activates ITCH, which marks the damaged lysosome with lysine-63-linked ubiquitin chains to initiate lysophagy and thus triages the lysosome for destruction. With SPG20 being linked to neurodegeneration, these findings highlight the relevance of a coordinated lysosomal damage response for cellular homeostasis.

Applying a homogeneous pressure distribution to the upper vertebral endplate: Validation of a new loading system, pilot application to human vertebral bodies, and finite element predictions of DIC measured displacements and strains.

Image-based personalized Finite Element Models (pFEM) could detect alterations in physiological deformation of human vertebral bodies, but their accuracy has been seldom reported. Meaningful validation experiments should allow vertebral endplate deformability and ensure well-controlled boundary conditions. This study aimed to (i) validate a new loading system to apply a homogeneous pressure on the vertebral endplate during vertebral body compression regardless of endplate deformation; (ii) perform a pilot study on human vertebral bodies measuring surface displacements and strains with Digital Image Correlation (DIC); (iii) determine the accuracy of pFEM of the vertebral bodies. Homogeneous pressure application was achieved by pressurizing a fluid silicone encased in a rubber silicone film acting on the cranial endplate. The loading system was validated by comparing DIC-measured longitudinal strains and lower-end contact pressures, measured on three homogeneous pseudovertebrae of constant transversal section at 2.0 kN, against theoretically calculated values. Longitudinal strains and contact pressures were rather homogeneous, and their mean values close to theoretical calculations (5% underestimation). DIC measurements of surface longitudinal and circumferential displacements and strains were obtained on three human vertebral bodies at 2.0 kN. Complete displacement and strain maps were achieved for anterolateral aspects with random errors ≤0.2 µm and ≤30 µstrain, respectively. Venous plexus and double curvatures limited the completeness and accuracy of DIC data in posterior aspects. pFEM of vertebral bodies, including cortical bone mapping, were built from computed tomography images. In anterolateral aspects, pFEM accuracy of the three vertebrae was: (i) comparable to literature in terms of longitudinal displacements (R2>0.8); (ii) extended to circumferential displacements (pooled data: R2>0.9) and longitudinal strains (zero median error, 95% error: <27%). Circumferential strains were overestimated (median error: 39%). The new methods presented may permit to study how physiological and pathologic conditions influence the ability of vertebral endplates/bodies to sustain loads.

The ubiquitin-conjugating enzyme UBE2QL1 coordinates lysophagy in response to endolysosomal damage.

The autophagic clearance of damaged lysosomes by lysophagy involves extensive modification of the organelle with ubiquitin, but the underlying ubiquitination machinery is still poorly characterized. Here, we use an siRNA screening approach and identify human UBE2QL1 as a major regulator of lysosomal ubiquitination, lysophagy, and cell survival after lysosomal damage. UBE2QL1 translocates to permeabilized lysosomes where it associates with damage sensors, ubiquitination targets, and lysophagy effectors. UBE2QL1 knockdown reduces ubiquitination and accumulation of the critical autophagy receptor p62 and abrogates recruitment of the AAA-ATPase VCP/p97, which is essential for efficient lysophagy. Crucially, it affects association of LC3B with damaged lysosomes indicating that autophagosome formation was impaired. Already in unchallenged cells, depletion of UBE2QL1 leads to increased lysosomal damage, mTOR dissociation from lysosomes, and TFEB activation pointing to a role in lysosomal homeostasis. In line with this, mutation of the homologue ubc-25 in Caenorhabditis elegans exacerbates lysosome permeability in worms lacking the lysosome stabilizing protein SCAV-3/LIMP2. Thus, UBE2QL1 coordinates critical steps in the acute endolysosomal damage response and is essential for maintenance of lysosomal integrity.