{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T10:29:35Z","timestamp":1785407375389,"version":"3.56.0"},"posted":{"date-parts":[[2026,5,26]]},"group-title":"Biophysics","reference-count":53,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,5,26]],"date-time":"2026-05-26T00:00:00Z","timestamp":1779753600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["R35GM147333"],"award-info":[{"award-number":["R35GM147333"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,5,26]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\u03b1-Synuclein (\u03b1Syn) is an intrinsically disordered protein that preferentially binds anionic membranes with lipid packing defects. Cholesterol is an abundant membrane component that regulates packing and organization within membranes, yet its effect on \u03b1Syn binding remains unclear as prior studies report both cholesterol-mediated enhancement and suppression. Here, we investigated whether these conflicting effects reflect differences in the intrinsic packing state of the phospholipid bilayer. Using a quantitative fluorescence microscopy-based binding assay, we measured \u03b1Syn binding preferences among reconstituted phosphatidylcholine\/phosphatidylserine membranes with varied cholesterol content, lipid tail chemistry, and vesicle curvature. We found that cholesterol\u2019s effect depended on the underlying packing regime of the membrane. In defect-rich membranes, cholesterol reduced \u03b1Syn binding, consistent with cholesterol tightening lipid packing and reducing \u03b1Syn-accessible defects. In membranes with intermediate defect content, cholesterol enhanced binding, whereas tightly packed membranes remained largely insensitive to cholesterol except when high cholesterol content was combined with high membrane curvature. Curvature further shaped these responses, with high curvature compressing cholesterol-dependent differences between membrane compositions. These results show that cholesterol does not universally promote or inhibit \u03b1Syn binding. Instead, cholesterol regulates \u03b1Syn-membrane interactions through a packing-regime-dependent mechanism shaped by both lipid tail chemistry and membrane curvature. This framework helps reconcile opposing reports in the literature and highlights membrane physical state as a key determinant of how cholesterol modulates \u03b1Syn binding.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance<\/jats:title>\n                  <jats:p>\u03b1Syn is a membrane-binding protein associated with Parkinson\u2019s disease, but the role of cholesterol in regulating its membrane interactions has remained unclear. Some studies report that cholesterol enhances \u03b1Syn association with membranes, whereas others show that cholesterol suppresses it. This work helps explain why both outcomes can occur. We show that cholesterol\u2019s effect depends on the membrane\u2019s underlying packing state: cholesterol can reduce, enhance, or have little effect on \u03b1Syn binding depending on the membrane environment. These findings shift the question from whether cholesterol is generally pro- or anti-binding to how cholesterol reshapes the membrane physical states that control \u03b1Syn association.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.05.21.726870","type":"posted-content","created":{"date-parts":[[2026,5,26]],"date-time":"2026-05-26T16:00:22Z","timestamp":1779811222000},"source":"Crossref","is-referenced-by-count":0,"title":["Cholesterol differentially regulates \u03b1-synuclein binding across membrane packing regimes"],"prefix":"10.64898","author":[{"given":"Orianna H.","family":"Kou","sequence":"first","affiliation":[{"name":"Department of Physics and Astronomy, University of Southern California, Los Angeles, California, 90089, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brian H.","family":"Kim","sequence":"additional","affiliation":[{"name":"Mork Family Department of Chemical Engineering and Materials Science, University of Southern 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S. ; Dema, A. ; Ch\u00e9rot, H. ; Dumesnil, C. ; Cohen, S. ; Shalom, H. S. ; Rimon, N. ; Sibony-Nevo, O. ; Beck, G. ; Ainbinder, E. ;, et al. \u03b1-Synuclein acts as a cholesteryl-ester sensor on lipid droplets regulating organelle size and abundance. openRxiv: 2024.","DOI":"10.1101\/2024.06.19.599670"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.05.21.726870","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T09:50:24Z","timestamp":1780048224000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.05.21.726870"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,26]]},"references-count":53,"URL":"https:\/\/doi.org\/10.64898\/2026.05.21.726870","relation":{},"subject":[],"published":{"date-parts":[[2026,5,26]]},"subtype":"preprint"}}