resumo
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.
palavras-chave
VESICLE FORMATION; BINDING; RECOGNITION; EXPLANATION; RECRUITMENT
categoria
Life Sciences & Biomedicine - Other Topics; Science & Technology - Other Topics
autores
Maestro, A; Zaccai, NR; Gonzalez-Martinez, JF; Sanchez-Puga, P; Tajuelo, J; Rubio, MA; Santamaria, A; Carrascosa-Tejedor, J; Pereira, D; Marín-Montesinos, I; Gutfreund, P; Campbell, R; Kotar, J; Kelly, BT; Cicuta, P; Owen, DJ
nossos autores
Daniel António Cunha Pereira
Estudante de doutoramento
Ildefonso Marin Montesinos
Investigador Auxiliaragradecimentos
The authors thank the Institut Laue-Langevin for the allocation of beamtime (https://doi.org/10.5291/ILL-DATA.8-02-780, https://doi.org/10.5291/ILL-DATA.8-02-795, and https://doi.org/10.5291/ILL-DATA.8-02-804) and the Partnership for Soft Condensed Matter (PSCM) for the lab support. A.M. acknowledges the financial support from The Wellcome Trust Institutional Strategic Support Fund (ISSF) for a Junior Interdisciplinary Fellowship; from MICIU under grant PID2021-129054NA-I00, the Department of Education of the Basque Government under grant PIBA-2023-1-0054 and from the IKUR Strategy under the collaboration agreement between Ikerbasque Foundation and Materials Physics Center. N.R.Z and D.J.O were supported by Wellcome Trust grant WT 207455/Z/17/Z awarded to D.J.O.