Two-dimensional HRS condensates drive the assembly of flat clathrin lattices on endosomes

Article Article scientifique Article Two-dimensional HRS condensates drive the assembly of flat clathrin lattices on endosomes eng 2026-05-12 author Hakala Markku 0000-0002-3485-6080 Department of Biochemistry, University of Geneva, Geneva, Switzerland.,Helsinki Institute of Life Sciences and the Institute of Biotechnology, University of Helsinki, Helsinki, Finland. 941496 author Moparthi Satish Babu 0000-0001-7493-3848 Sorbonne Université, INSERM, Institute of Myology, Centre of Research in Myology, Paris, France. author Ganeva Iva Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland. author Gül Mehmet In situ Structural Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany.,Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany. author Bernat Cesar Department of Biochemistry, University of Geneva, Geneva, Switzerland. 979062 Biochimie groupe 5 (CHIB5) author Marcuello Carlos 0000-0003-3459-8605 Biofisika Institute (CSIC, UPV/EHU) and Department of Biochemistry and Molecular Biology, University of the Basque Country, Leioa, Spain. author Espadas Javier 0000-0002-4559-5410 Department of Biochemistry, University of Geneva, Geneva, Switzerland. 960240 Biochimie groupe 5 (CHIB5) author Colom Diego Adai 0000-0003-4869-8981 Biofisika Institute (CSIC, UPV/EHU) and Department of Biochemistry and Molecular Biology, University of the Basque Country, Leioa, Spain. 815331 author Kudryashev Mikhail 0000-0003-3550-6274 In situ Structural Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany.,Institute of Medical Physics and Biophysics, Charité-Universitätsmedizin Berlin, Berlin, Germany. author Kukulski Wanda 0000-0002-2778-3936 Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland. author Vassilopoulos Stéphane 0000-0003-0172-330X Sorbonne Université, INSERM, Institute of Myology, Centre of Research in Myology, Paris, France. author Kaksonen Marko 0000-0003-3645-7689 Department of Biochemistry, University of Geneva, Geneva, Switzerland. Marko.Kaksonen@unige.ch. 830048 Département de biochimie (CHIBI) author Roux Aurélien 0000-0002-6088-0711 Department of Biochemistry, University of Geneva, Geneva, Switzerland. Aurelien.Roux@unige.ch. 716946 Département de biochimie (CHIBI) 10.1038/s41467-026-73132-x 42120412 2041-1723 https://www.nature.com/articles/s41467-026-73132-x In cells, the curved clathrin structures in vesicle budding are well characterized, while the flat ones remain poorly understood. Here, we reconstitute the flat assembly of ESCRT-0 protein HRS and clathrin onto lipid membranes in vitro. HRS forms gel-like protein condensates at micromolar concentrations in solutions. These condensates spread as a two-dimensional layer on negatively charged membranes and, together with clathrin, form multilayered coats. Importantly, the two-dimensional condensates spontaneously form only on membranes at HRS concentrations below 50 nM, its cytoplasmic concentration. Correlative cryo-electron tomography of HRS-labelled endosomes in cells reveals a multilayered structure containing a flat clathrin layer 16 nm away from the membrane, consistent with our in vitro findings. Cholesterol enhances HRS recruitment to the membrane both in cells and in supported bilayers. Furthermore, cholesterol promotes the phase separation of HRS onto membranes, which in turn concentrates cholesterol underneath. This positive feedback promotes the formation of HRS-clathrin microdomains that sorts reconstituted ubiquitinated cargoes. Altogether, our results show that the distinct architecture of ESCRT-0 is assembled by the two-dimensional phase-separation of HRS which drives the assembly of flat clathrin coats. Dewey 500 Swiss National Science Foundation Molecular mechanisms of clathrin-mediated endocytosis 212288 CH Swiss National Science Foundation METEORIC: MEmbrane TEnsion and tOR In Condensates 189996 CH Swiss National Science Foundation MEETICS: MEmbrane Extension Transduces Information between Cellular Scales 200793 CH European Commission In-situ spatiotemporal imaging of membrane hydration, electrostatics, tension and curvature to understand cell response to osmotic shocks and cell migration. 951324 R2-TENSION EU H2020 Swiss National Science Foundation Lipid transfer at organelle contact sites 201158 CH European Commission Drug REpurposing with Artificial intelligence for Muscular disorderS 101080229 DREAMS EU HE 2 Faculté des sciences 1 false 769 Groupe Roux 1397 Groupe Kaksonen Nature communications 82c14947-24f4-4e82-9666-bd8672866123 Article (Accepted version) Public s41467-026-73132-x_reference.pdf 12508787 application/pdf CC BY-NC-ND-4.0 a322406bca969080f79a8a395bc063e254ea16df256a2d98d4f90d5c68b89185 openAccess Faculté des sciences

Title	Published in	Year	Views	Downloads

Two-dimensional HRS condensates drive the assembly of flat clathrin lattices on endosomes	Nature communications	2026	12	15
Vps60 initiates alternative ESCRT-III filaments	The Journal of cell biology	2023	151	104

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