Abstract
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.
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Acknowledgements
We are grateful to Jean Gruenberg, Stefania Vossio, and Elina Ikonen for thoughtful discussions about the role of cholesterol in vesicle trafficking. Andrea Picco is acknowledged for technical support in TIRF imaging, and Rafael Ferreira Caetano and Frédéric Humbert for protein purification. We thank Roux and Kaksonen labs for technical support and helpful discussions. We thank the Photonic Light Microscopy Facility and ACCESS Facility at the University of Geneva, the Microscopy Imaging Center (MIC) of the University of Bern, the Dubochet Center for Imaging (DCI) Bern, the Light Microscopy Unit (LMU) at the Institute of Biotechnology at the University of Helsinki, and IBPS electron microscopy platform at Sorbonne University in Paris for microscope access and support with data collection.
Funding
This work was supported by EMBO (ALTF 703-2020 for M.H., and 989-2022 for J.E.), Research Council of Finland (grant 369982 for M.H.) Fundación Alfonso Martín Escudero (Postdoctoral fellowship for C.B-S.), and Swiss National Science Foundation grant (310030_212288) for Ma.K. A.R. acknowledges funding from the Swiss National Fund for Research grant numbers #CRSII5_189996 and #310030_200793 and the European Research Council Synergy grant number #951324-R2-TENSION. Work in the group of W.K. was supported by the University of Bern and the SNSF project 201158. We would also like to acknowledge funding by the Agence Nationale pour la Recherche (ANR-20-CE13-0024-01, ANR-21-CE13-0018-01 to SV), Horizon Europe DREAMS project under grant agreement N°101080229, Sorbonne Université, INSERM, Association Institut de Myologie core funding to S.V. Mi.K. is supported by the Helmholtz Society and the Heisenberg Award from the DFG (KU3222/3-1). A.C. Acknowledges PID2022-140687NB-I00 funded by MCIN/AEI /10.13039/501100011033/ and European Union Unión Europea Next GenerationEU/ PRTR, CNS2024-154624 founded by MICIU/AEI/10.13039/501100011033 CNS2024-154624, MICIU/AEI/10.13039/501100011033 and the Basque Resource for Advanced Light Microscopy (BRALM) located at Instituto Biofisika (CSIC, EHU) for their support and assistance in this work.
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Hakala, M., Moparthi, S.B., Ganeva, I. et al. Two-dimensional HRS condensates drive the assembly of flat clathrin lattices on endosomes. Nat Commun (2026). https://doi.org/10.1038/s41467-026-73132-x
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DOI: https://doi.org/10.1038/s41467-026-73132-x
