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In cells, phase separation can occur in a bulk solution or on a lipid membrane. To establish life-like features in artificial cells, much research has been carried out to reproduce multiphase separation behavior. However, this has been mostly restricted to bulk properties, whereas membrane interactions are much less investigated. Here, we introduce a membrane-based strategy in which interfacial electrostatic heterogeneity governs multiphase separation in amylose-based coacervates, enabling the formation of subcompartments localized near the membrane interface. By integrating bowl-shaped polymer vesicles (stomatocytes) decorated with negatively charged gold nanoparticles into a terpolymer membrane, we construct a patchy membrane architecture that introduces localized, highly charged interfacial domains, thereby stabilizing amylose-based coacervates. Upon the addition of succinylated bovine serum albumin as a second phase-forming component, the embedded stomatocytes allow precise control over multiphase separation. Modulating the stomatocytes\u2019 coverage at the coacervate interface enables systematic tuning of the size, spatial localization, and number of protein-enriched subdroplets. Complete stomatocyte coverage further enhances multiphase stability against variations in ionic strength and pH, while improving long-term storage stability. Moreover, the interfacial multiphase organization can be dynamically regulated by ionic strength and temperature. Furthermore, the enlarged interfacial area promotes enhanced cargo uptake, while the programmable positioning of enzyme-containing subcompartments enables spatial regulation of interprotocellular chemical communication. Together, these findings establish interfacial electrostatic heterogeneity as a versatile strategy for engineering membrane-associated multiphase protocells in which spatial organization governs molecular transport, catalysis, and communication.<\/jats:p>","DOI":"10.1021\/jacs.6c11826","type":"journal-article","created":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T13:36:31Z","timestamp":1785159391000},"page":"33945-33958","source":"Crossref","is-referenced-by-count":0,"title":["Patchy\nMembrane-Directed Multiphase Complex Coacervation"],"prefix":"10.1021","volume":"148","author":[{"given":"Siwen","family":"Sun","sequence":"first","affiliation":[{"name":"Eindhoven University of Technology , , ,","place":["Eindhoven, The Netherlands, 5600 MB"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nadia","family":"Erkamp","sequence":"additional","affiliation":[{"name":"Eindhoven University of Technology , , ,","place":["Eindhoven, The Netherlands, 5600 MB"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sander G. A. M.","family":"Huisman","sequence":"additional","affiliation":[{"name":"Eindhoven University of Technology , , ,","place":["Eindhoven, The Netherlands, 5600 MB"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5005-1039","authenticated-orcid":true,"given":"Alexander B.","family":"Cook","sequence":"additional","affiliation":[{"name":"Eindhoven University of Technology , , ,","place":["Eindhoven, The Netherlands, 5600 MB"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arjan","family":"Hazegh Nikroo","sequence":"additional","affiliation":[{"name":"Eindhoven University of Technology , , ,","place":["Eindhoven, The Netherlands, 5600 MB"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhuping","family":"Yin","sequence":"additional","affiliation":[{"name":"Eindhoven University of Technology , , ,","place":["Eindhoven, The Netherlands, 5600 MB"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianhong","family":"Wang","sequence":"additional","affiliation":[{"name":"Eindhoven University of Technology , , ,","place":["Eindhoven, The Netherlands, 5600 MB"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4445-937X","authenticated-orcid":true,"given":"Madelief A. M.","family":"Verwiel","sequence":"additional","affiliation":[{"name":"Eindhoven University of Technology , , ,","place":["Eindhoven, The Netherlands, 5600 MB"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7973-2404","authenticated-orcid":true,"given":"Jan C. M.","family":"van Hest","sequence":"additional","affiliation":[{"name":"Eindhoven University of Technology , , ,","place":["Eindhoven, The Netherlands, 5600 MB"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,7,27]]},"reference":[{"issue":"1","key":"2026081210075134000_cit1","doi-asserted-by":"publisher","DOI":"10.1038\/s41392-021-00678-1","article-title":"Liquid\u2013Liquid\nPhase Separation\nin Human Health and Diseases","volume":"6","author":"Wang","year":"2021","journal-title":"Signal Transduction\nTargeted Therapy"},{"issue":"41","key":"2026081210075134000_cit2","doi-asserted-by":"publisher","first-page":"37337","DOI":"10.1021\/jacs.5c10731","article-title":"Coacervate\nDroplets Drive Organocatalyzed Aqueous C\u2013C Bond Formation via\nInterfacial Activation","volume":"147","author":"Peyraud-Vicre\u0301","year":"2025","journal-title":"J. Am. Chem. 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