{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,26]],"date-time":"2026-01-26T23:10:15Z","timestamp":1769469015470,"version":"3.49.0"},"reference-count":52,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,1,26]],"date-time":"2026-01-26T00:00:00Z","timestamp":1769385600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100004412","name":"Human Frontier Science Program","doi-asserted-by":"publisher","award":["RGP0019\/20"],"award-info":[{"award-number":["RGP0019\/20"]}],"id":[{"id":"10.13039\/100004412","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Membranes"],"abstract":"<jats:p>The combination of supported lipid bilayers (SLBs) with the Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) has been proven to be a powerful tool to simultaneously monitor mass and viscoelastic changes related to membrane binding-events. In this work, the above methodology is employed for the study of the interaction of the Early Endosomal Antigen 1 (EEA1) to a model lipid bilayer that mimics the early endosome (EE) membrane, focusing on the membrane composition. Starting with the formation of a lipid bilayer through the vesicles fusion technique, we investigated the formation of SLBs that incorporate phosphatidylinositol 3-phosphate (PI(3)P), a key component for EEA1 binding, in combination with other lipids, e.g., (1,2-dioleoyl-sn-glycero-3)-phosphocholine (DOPC), -phosphoserine (DOPS), -phosphoethanolamine (DOPE), and cholesterol (Chol). The interaction of the full-length coiled-coil EEA1 to the formed SLBs was further studied in real time with the QCM-D and characterized with respect to the lipid composition and pH. Our findings confirm that PI(3)P is essential for the EEA1\u2013membrane interaction, while it was shown that Chol and phosphatidylserine greatly influence the binding event. In fact, including 30% Chol in a PI(3)P (3%):PS (6%) SLB resulted in almost double EEA1 binding than in the absence of Chol. Moreover, we employed the QCM-viscoelastic model available to analyze the QCM-D data with emphasis on the study of the protein conformation. Our results showed that, in our in vitro system, EEA1 is not fully extended and\/or highly packed, but is mainly in a bent, distorted conformation with an average size close to 100 nm. This study complements previous works employing in vitro assays, also demonstrating the ability to reconstitute more complex biomimetic EE membranes containing inositol phospholipids on a QCM surface for the study of EEA1 binding.<\/jats:p>","DOI":"10.3390\/membranes16020049","type":"journal-article","created":{"date-parts":[[2026,1,26]],"date-time":"2026-01-26T12:41:16Z","timestamp":1769431276000},"page":"49","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A QCM-D Study of the Interaction of Early Endosomal Antigen 1 (EEA1) Protein with Supported Lipid Bilayers Mimicking the Early Endosomal Lipid Composition"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2196-1049","authenticated-orcid":false,"given":"Fotini","family":"Papagavriil","sequence":"first","affiliation":[{"name":"Department of Biology, University of Crete, Vassilika Vouton, 70013 Heraklion, Greece"},{"name":"Institute of Molecular Biology and Biotechnology (IMBB), Foundation for Research and Technology-Hellas (FORTH), 100 N. Plastira Str., 70013 Heraklion, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pablo","family":"Mateos-Gil","sequence":"additional","affiliation":[{"name":"Dto. Qu\u00edmica F\u00edsica, Universidad Complutense de Madrid, 28040 Madrid, Spain"},{"name":"Instituto de Investigaci\u00f3n Hospital Doce de Octubre (i+12), 28041 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Janelle","family":"Lauer","sequence":"additional","affiliation":[{"name":"Arthrex, Naples, FL 34108, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marino","family":"Zerial","sequence":"additional","affiliation":[{"name":"Human Technopole, 20157 Milan, Italy"},{"name":"Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, Center for Systems Biology (CSBD), 01307 Dresden, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3598-5984","authenticated-orcid":false,"given":"Electra","family":"Gizeli","sequence":"additional","affiliation":[{"name":"Department of Biology, University of Crete, Vassilika Vouton, 70013 Heraklion, Greece"},{"name":"Institute of Molecular Biology and Biotechnology (IMBB), Foundation for Research and Technology-Hellas (FORTH), 100 N. Plastira Str., 70013 Heraklion, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"3497","DOI":"10.1021\/la052687c","article-title":"Formation of solid-supported lipid bilayers: An integrated view","volume":"22","author":"Richter","year":"2006","journal-title":"Langmuir"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2345","DOI":"10.1021\/acs.jpcb.1c10534","article-title":"Selective Recognition of Phosphatidylinositol Phosphate Receptors by C-Terminal Tail of Mitotic Kinesin-like Protein 2 (MKlp2)","volume":"126","author":"Tae","year":"2022","journal-title":"J. Phys. Chem. B"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Bonet, N.F., Cava, D.G., and V\u00e9lez, M. (2022). Quartz crystal microbalance and atomic force microscopy to characterize mimetic systems based on supported lipids bilayer. Front. Mol. Biosci., 9.","DOI":"10.3389\/fmolb.2022.935376"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"113341","DOI":"10.1016\/j.colsurfb.2023.113341","article-title":"Engineering phosphatidylinositol-4,5-bisphosphate model membranes enriched in endocytic cargo: A neutron reflectometry, AFM and QCM-D structural study","volume":"227","author":"Pereira","year":"2023","journal-title":"Colloids Surf. B Biointerfaces"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"6267","DOI":"10.1021\/ac2009178","article-title":"Pleckstrin homology-phospholipase C-\u03b4 1 interaction with phosphatidylinositol 4,5-bisphosphate containing supported lipid bilayers monitored in Situ with dual polarization interferometry","volume":"83","author":"Baumann","year":"2011","journal-title":"Anal. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"852","DOI":"10.1002\/prot.22392","article-title":"Membrane insertion of the FYVE domain is modulated by pH","volume":"76","author":"He","year":"2009","journal-title":"Proteins"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1006\/jcis.2001.8054","article-title":"A Silicate Gel for Promoting Deposition of Lipid Bilayers","volume":"246","author":"Melzak","year":"2002","journal-title":"J. Colloid. Interface Sci."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1681","DOI":"10.1021\/la0263920","article-title":"Intact vesicle adsorption and supported biomembrane formation from vesicles in solution: Influence of surface chemistry, vesicle size, temperature, and osmotic pressure","volume":"19","author":"Reimhult","year":"2003","journal-title":"Langmuir"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Yousefi, N., and Tufenkji, N. (2016). Probing the Interaction between Nanoparticles and Lipid Membranes by Quartz Crystal Microbalance with Dissipation Monitoring. Front. Chem., 4.","DOI":"10.3389\/fchem.2016.00046"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Yamamoto, Y., Ito, D., Akatsuka, H., Noguchi, H., Matsushita, A., Kinekawa, H., Nagano, H., Yoshino, A., Taga, K., and Shervani, Z. (2024). The Interaction between Anesthetic Isoflurane and Model-Biomembrane Monolayer Using Simultaneous Quartz Crystal Microbalance (QCM) and Quartz Crystal Impedance (QCI) Methods. Membranes, 14.","DOI":"10.3390\/membranes14030062"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.bpc.2014.09.003","article-title":"Differentiating antimicrobial peptides interacting with lipid bilayer: Molecular signatures derived from quartz crystal microbalance with dissipation monitoring","volume":"196","author":"Wang","year":"2014","journal-title":"Biophys. Chem."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"3699","DOI":"10.1074\/jbc.275.5.3699","article-title":"The FYVE domain of early endosome antigen 1 is required for both phosphatidylinositol 3-phosphate and Rab5 binding. Critical role of this dual interaction for endosomal localization","volume":"275","author":"Lawe","year":"2000","journal-title":"J. Biol. Chem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"24048","DOI":"10.1074\/jbc.271.39.24048","article-title":"Endosomal localization of the autoantigen EEA1 is mediated by a zinc-binding FYVE finger","volume":"271","author":"Stenmark","year":"1996","journal-title":"J. Biol. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"13052","DOI":"10.1073\/pnas.0503900102","article-title":"Targeting of the FYVE domain to endosomal membranes is regulated by a histidine switch Stephanie","volume":"102","author":"Lee","year":"2005","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e54434","DOI":"10.7554\/eLife.54434","article-title":"A non-linear system patterns Rab5 GTPase on the membrane","volume":"9","author":"Cezanne","year":"2020","journal-title":"eLife"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1053","DOI":"10.1002\/cbic.201700046","article-title":"The Lipids of the Early Endosomes: Making Multimodality Work","volume":"18","author":"Arumugam","year":"2017","journal-title":"ChemBioChem"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1016\/j.semcdb.2014.04.010","article-title":"Lipid compartmentalization in the endosome system","volume":"31","author":"Taguchi","year":"2014","journal-title":"Semin. Cell Dev. Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1016\/S0092-8674(00)81966-2","article-title":"Oligomeric Complexes Link Rab5 Effectors with NSF and Drive Membrane Fusion via Interactions between EEA1 and Syntaxin 13","volume":"98","author":"McBride","year":"1999","journal-title":"Cell"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1038\/nature19326","article-title":"An endosomal tether undergoes an entropic collapse to bring vesicles together","volume":"537","author":"Murray","year":"2016","journal-title":"Nature"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2657","DOI":"10.1091\/mbc.11.8.2657","article-title":"EEA1, a tethering protein of the early sorting endosome, shows a polarized distribution in hippocampal neurons, epithelial cells, and fibroblasts","volume":"11","author":"Wilson","year":"2000","journal-title":"Mol. Biol. Cell"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"4652","DOI":"10.1038\/s41467-023-40428-1","article-title":"Deterministic early endosomal maturations emerge from a stochastic trigger-and-convert mechanism","volume":"14","author":"York","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"206","DOI":"10.1007\/BF01337937","article-title":"Verwendung von Schwingquarzen zur W\u00e4gung d\u00fcnner Schichten und zur Mikrow\u00e4gung","volume":"55","author":"Sauerbrey","year":"1959","journal-title":"Z. Fiir Physik"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"7092","DOI":"10.1021\/la025911w","article-title":"Sauerbrey equation of quartz crystal microbalance in liquid medium","volume":"18","author":"Kankare","year":"2002","journal-title":"Langmuir"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1038\/s43586-024-00340-4","article-title":"Quartz crystal microbalance with dissipation monitoring for studying soft matter at interfaces","volume":"4","author":"Johannsmann","year":"2024","journal-title":"Nat. Rev. Methods Primers"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"9275","DOI":"10.1021\/ac102495q","article-title":"Label-Free Detection of Clustering of Membrane-Bound Proteins","volume":"82","author":"Carton","year":"2010","journal-title":"Anal. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"5306","DOI":"10.1021\/ja068768s","article-title":"Membrane-Grafted Hyaluronan Films: A Well-Defined Model System of Glycoconjugate Cell Coats","volume":"129","author":"Richter","year":"2007","journal-title":"J. Am. Chem. Soc."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"8838","DOI":"10.1021\/ac201778h","article-title":"Hearing What You Cannot See and Visualizing What You Hear: Interpreting Quartz Crystal Microbalance Data from Solvated Interfaces","volume":"83","author":"Reviakine","year":"2011","journal-title":"Anal. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"010801","DOI":"10.1116\/6.0003312","article-title":"Quartz crystal microbalance in soft and biological interfaces","volume":"19","author":"Reviakine","year":"2024","journal-title":"Biointerphases"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"824","DOI":"10.1002\/bip.21684","article-title":"The intrinsic viscosity of linear DNA","volume":"95","author":"Tsortos","year":"2011","journal-title":"Biopolymers"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"srep02033","DOI":"10.1038\/srep02033","article-title":"Acoustic detection of DNA conformation in genetic assays combined with PCR","volume":"3","author":"Papadakis","year":"2013","journal-title":"Sci. Rep."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1854","DOI":"10.1021\/ac202515p","article-title":"Direct Detection of DNA Conformation in Hybridization Processes","volume":"84","author":"Papadakis","year":"2012","journal-title":"Anal. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"6541","DOI":"10.1039\/C6CC02127A","article-title":"Monitoring structural changes in intrinsically disordered proteins using QCM-D: Application to the bacterial cell division protein ZipA","volume":"52","author":"Tsortos","year":"2016","journal-title":"Chem. Commun."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"8186","DOI":"10.1021\/acs.analchem.0c00366","article-title":"Acoustic Methodology for Selecting Highly Dissipative Probes for Ultrasensitive DNA Detection","volume":"92","author":"Milioni","year":"2020","journal-title":"Anal. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1397","DOI":"10.1016\/S0006-3495(98)74057-3","article-title":"Surface Specific Kinetics of Lipid Vesicle Adsorption Measured with a Quartz Crystal Microbalance","volume":"75","author":"Keller","year":"1998","journal-title":"Biophys. J."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Swana, K.W., Camesano, T.A., and Nagarajan, R. (2022). Formation of a Fully Anionic Supported Lipid Bilayer to Model Bacterial Inner Membrane for QCM-D Studies. Membranes, 12.","DOI":"10.3390\/membranes12060558"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"46","DOI":"10.3389\/fmats.2018.00046","article-title":"Quartz crystal microbalance with dissipation monitoring: A versatile tool to monitor phase transitions in biomimetic membranes","volume":"5","author":"Neupane","year":"2018","journal-title":"Front Mater."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"202","DOI":"10.1016\/j.colsurfb.2018.09.031","article-title":"Towards biomimics of cell membranes: Structural effect of phosphatidylinositol triphosphate (PIP3) on a lipid bilayer","volume":"173","author":"Luchini","year":"2019","journal-title":"Colloids Surf. B Biointerfaces"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"14877","DOI":"10.1021\/la503203a","article-title":"Formation and characterization of supported lipid bilayers containing phosphatidylinositol-4,5-bisphosphate and cholesterol as functional surfaces","volume":"30","author":"Grill","year":"2014","journal-title":"Langmuir"},{"key":"ref_39","unstructured":"Perini, E.D.E.D. (2012). In Vitro Reconstitution of the Molecular Mechanisms of Vesicle Tethering and Membrane Fusion. [Ph.D. Thesis, Technische Universit\u00e4t Dresden]. Available online: https:\/\/tud.qucosa.de\/en\/landing-page\/https%3A%2F%2Ftud.qucosa.de%2Fapi%2Fqucosa%253A26774%2Fmets\/."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Johannsmann, D. (2014). The Quartz Crystal Microbalance in Soft Matter Research: Fundamentals and Modeling, Springer. Available online: http:\/\/books.google.com\/books?id=IkAqBAAAQBAJ&pgis=1.","DOI":"10.1007\/978-3-319-07836-6_1"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1016\/0014-5793(85)80541-X","article-title":"The effect of bilayer order and fluidity on detergent-induced liposome fusion","volume":"179","author":"Saez","year":"1985","journal-title":"FEBS Lett."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Lind, T.K., Wacklin, H., Schiller, J., Moulin, M., Haertlein, M., Pomorski, T.G., and C\u00e1rdenas, M. (2015). Formation and Characterization of Supported Lipid Bilayers Composed of Hydrogenated and Deuterated Escherichia coli Lipids. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0144671"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"612","DOI":"10.1038\/nature04399","article-title":"Role of cholesterol and lipid organization in disease","volume":"438","author":"Maxfield","year":"2005","journal-title":"Nature"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1016\/j.chemphyslip.2014.02.003","article-title":"Cholesterol stabilizes fluid phosphoinositide domains","volume":"182","author":"Jiang","year":"2014","journal-title":"Chem. Phys. Lipids"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"868","DOI":"10.1016\/j.bbalip.2006.03.011","article-title":"Phosphatidylinositol 3-phosphate recognition and membrane docking by the FYVE domain","volume":"1761","author":"Kutateladze","year":"2006","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"472","DOI":"10.1016\/j.colsurfb.2014.01.036","article-title":"Antimicrobial peptide alamethicin insertion into lipid bilayer: A QCM-D exploration","volume":"116","author":"Wang","year":"2014","journal-title":"Colloids Surf. B Biointerfaces"},{"key":"ref_47","first-page":"4752","article-title":"Alpha-Helical Peptide-Induced Vesicle Rupture Revealing New Insight into the Vesicle Fusion Process As Monitored in Situ by Quartz Crystal Microbalance-Dissipation and Reflectometry","volume":"95","author":"Cho","year":"1998","journal-title":"Bioorganic Med. Chem. Lett."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"494","DOI":"10.1038\/28879","article-title":"EEA1 links PI(3)K function to Rab5 regulation of endosome fusion","volume":"394","author":"Simonsen","year":"1998","journal-title":"Nature"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"835","DOI":"10.1021\/ac0204911","article-title":"Comparative study of IgG binding to proteins G and A: Nonequilibrium kinetic and binding constant determination with the acoustic waveguide device","volume":"75","author":"Saha","year":"2003","journal-title":"Anal. Chem."},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Johannsmann, D., Langhoff, A., Leppin, C., Reviakine, I., and Maan, A.M.C. (2023). Effect of Noise on Determining Ultrathin-Film Parameters from QCM-D Data with the Viscoelastic Model. Sensors, 23.","DOI":"10.3390\/s23031348"},{"key":"ref_51","first-page":"1","article-title":"Lipid bilayers: Phase behavior and nanomechanics","volume":"86","author":"Giannotti","year":"2020","journal-title":"Curr. Top. Membr."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"947","DOI":"10.1016\/S1097-2765(01)00385-9","article-title":"Multivalent Endosome Targeting by Homodimeric EEA1","volume":"8","author":"Dumas","year":"2001","journal-title":"Mol. Cell"}],"container-title":["Membranes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2077-0375\/16\/2\/49\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,26]],"date-time":"2026-01-26T13:01:49Z","timestamp":1769432509000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2077-0375\/16\/2\/49"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,26]]},"references-count":52,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["membranes16020049"],"URL":"https:\/\/doi.org\/10.3390\/membranes16020049","relation":{},"ISSN":["2077-0375"],"issn-type":[{"value":"2077-0375","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,26]]}}}