Combined thermodynamic and time-resolved structural analysis of interactions between AP2 and biomimetic plasma membranes provides insights into clathrin-mediated endocytosis
Clathrin-mediated endocytosis (CME), the main mechanism for swift, selective protein uptake in eukaryotic cells, initiates with adaptor protein AP2 recruitment to the plasma membrane (PM). AP2 recognizes PM-associated PtdIns(4,5)P2 and protein cargo for internalization. Nonetheless, many aspects of this process remain unclear due to their in vivo complexity. Here, a thermodynamic and time-resolved structural analysis of AP2 binding to different biomimetic PM was undertaken under physiological conditions using a combination of neutron reflectometry, interfacial tensiometry and rheology, and atomic force microscopy. The resultant in vitro data replicated previous in vivo observations, as well as yielded biophysical insights into normal and aborted CME. The presence of cargo may not be pivotal for the "activating" conformational change of AP2. However, the presence of cargo extends AP2's residence time on the membrane surface, due to slower on- and off-rates, thereby tentatively giving sufficient time for CME to proceed fully. Moreover, upon interaction with AP2, phospholipid lateral diffusion decreases markedly, inducing a gel phase attributed to creating a percolated network involving AP2 on the membrane, which could potentially serve as a mechanism for modulating subsequent clathrin binding. The subsequent clathrin polymerization at the membrane is dependent on the AP2's clathrin binding sequence.
Authors
- Richard A. Campbell (ORCID: https://orcid.org/0000-0002-6296-314X)
- David J. Owen (ORCID: https://orcid.org/0000-0002-8351-6322)
- Juan F. González-Martínez (ORCID: https://orcid.org/0000-0001-5518-4095)
- Javier Tajuelo (ORCID: https://orcid.org/0000-0002-4085-7610)
- Daniel Pereira (ORCID: https://orcid.org/0000-0001-8496-3063)
- Jurij Kotar (ORCID: https://orcid.org/0000-0001-6246-5178)
- Pietro Cicuta (ORCID: https://orcid.org/0000-0002-9193-8496)
- Nathan R. Zaccai (ORCID: https://orcid.org/0000-0002-1476-2044)
- Philipp Gutfreund (ORCID: https://orcid.org/0000-0002-7412-8571)
- Andreas Santamaría (ORCID: https://orcid.org/0000-0002-6151-4406)
- Pablo Sánchez-Puga (ORCID: https://orcid.org/0000-0002-1000-7527)
- Armando Maestro (ORCID: https://orcid.org/0000-0002-7791-8130)
- Javier Carrascosa-Tejedor (ORCID: https://orcid.org/0000-0003-0385-2383)
- Ildefonso Marín-Montesinos (ORCID: https://orcid.org/0000-0002-4206-2643)
- Miguel A. Rubio (ORCID: https://orcid.org/0000-0002-4210-0443)
- B.T. Kelly (ORCID: https://orcid.org/0000-0003-2666-6268)
Institutions
- Ikerbasque (ES)
- University of the Basque Country (ES)
- Universidad Nacional de Educación a Distancia (ES)
- University of Cambridge (GB)
- University of Manchester (GB)
- Universidad Politécnica de Cartagena (ES)
- Bridge University (SS)
- Institut Laue-Langevin (FR)
- Material Physics Center (ES)
- Donostia International Physics Center (ES)
- University of Aveiro (PT)
Publication Details
- Journal
- Communications Biology
- Published
- 2025-08-12
- DOI
- https://doi.org/10.1038/s42003-025-08597-5
- Primary Topic
- Cellular transport and secretion
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Wellcome
- Wellcome Trust
- International Seafood Sustainability Foundation
- Eusko Jaurlaritza
- Ikerbasque, Basque Foundation for Science