Supporting material for "Cathepsin-dependent amyloid formation drives mechanical rupture of lysosomal membranes"

Supporting material and source data for: Title: Cathepsin-dependent amyloid formation drives mechanical rupture of lysosomal membranes Authors: Delong Li1,2†, Wenxin Zhang1,2†, Michaela Medina3, Jan F. M. Stuke4, Andre Schwarz5, Jonas Brill6, Johann Brenner1,7, Felix Kraus2,8, Simon Ohlerich1, Javier Lizarrondo1,2, Jeremy Pflaum1, Julia H. Grass1, Lena-Marie Soltow1, Dietmar Hammerschmid9,10, Natalie Weber9,10, Sonja Welsch11, Julian D. Langer9,10, Maike Windbergs6, J. Wade Harper2,8, Erin Schuman5, Gerhard Hummer2,4,12, Danielle A. Grotjahn3, Florian Wilfling1,2* Affiliations: 1Mechanism of Cellular Quality Control, Max Planck Institute of Biophysics; 60438 Frankfurt am Main, Germany. 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network; Chevy Chase, MD 20815, USA. 3Department of Integrative Structural and Computational Biology, The Scripps Research Institute; La Jolla, CA 92037, USA. 4Department of Theoretical Biophysics, Max Planck Institute of Biophysics; 60438 Frankfurt am Main, Germany. 5Department of Synaptic Plasticity, Max Planck Institute for Brain Research; 60438 Frankfurt am Main, Germany. 6Institute of Pharmaceutical Technology, Goethe University Frankfurt; 60438 Frankfurt am Main, Germany. 7Research Group CryoEM Technology, Max Planck Institute of Biochemistry; 82152 Martinsried, Germany. 8Department of Cell Biology, Blavatnik Institute, Harvard Medical School; Boston, MA 02115, USA. 9Proteomics and Mass Spectrometry, Max Planck Institute of Biophysics; 60438 Frankfurt am Main, Germany. 10Mass Spectrometry, Max Planck Institute for Brain Research; 60438 Frankfurt am Main, Germany. 11Central Electron Microscopy Facility, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany. 12Institute of Biophysics, Goethe University Frankfurt; 60438 Frankfurt am Main, Germany. †These authors contributed equally to this work. *Corresponding author. Email: [e-mail] Abstract: Lysosomal membrane integrity is essential for cellular homeostasis, and its failure drives lysosomal storage disorders (LSD) and neurodegeneration. The dipeptide L-leucyl-L-leucine methyl ester (LLOMe) is widely used to model lysosomal damage, yet its mechanism remains poorly understood. The prevailing view holds that LLOMe polymerizes into membrane-permeabilizing peptide chains within the lysosomal lumen. Using cryo-electron tomography in cultured cells and primary neurons, we visualized the structural basis of LLOMe-induced lysosomal damage. We reveal that LLOMe forms amyloid structures within lysosomes that directly interact with and rupture the limiting membrane through mechanical stress. In vitro reconstitution confirms this amyloid-mediated mechanism. These findings establish a structural paradigm for lysosomal membrane disruption and provide insights into how disease-relevant protein aggregates, implicated in neurodegeneration and LSD, may compromise lysosomal integrity.