Structural organization and dynamics of FCHo2 docking on membranes
Fatima El Alaoui; Ignacio Casuso; David Sanchez-Fuentes; Charlotte Arpin-Andre; Raissa Rathar; Volker Baecker; Anna Castro; Thierry Lorca; Julien Viaud; Stéphane Vassilopoulos; Adrian Carretero-Genevrier; Laura Picas.


Clathrin-mediated endocytosis (CME) is a central trafficking pathway in eukaryotic cells regulated by phosphoinositides. The plasma membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an instrumental role in driving CME initiation. The F-BAR domain-only protein 1 and 2 complex (FCHo1/2) is among the early proteins that reach the plasma membrane, but the exact mechanisms triggering its recruitment remain elusive. Here, we show the molecular dynamics of FCHo2 self-assembly on membranes by combining minimal reconstituted in vitro and cellular systems. Our results indicate that PI(4,5)P2 domains assist FCHo2 docking at specific membrane regions, where it self-assembles into ring-like-shaped protein patches. We show that the binding of FCHo2 on cellular membranes promotes PI(4,5)P2 clustering at the boundary of cargo receptors and that this accumulation enhances clathrin assembly. Thus, our results provide a mechanistic framework that could explain the recruitment of early PI(4,5)P2-interacting proteins at endocytic sites

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Silica thin-film nanostructuration

SiO2 vertical nanostructures were prepared on conventional borosilicate coverslips with precision thickness no. 1.5 (0.170 ± 0.005 mm), as previously reported (Sansen et al., 2020; Zhang et al., 2020). Briefly, Si masters were elaborated using LIL lithography as detailed in Zhang et al., 2020 and Zhang et al., 2019. Polydimethylsiloxane (PDMS) reactants (90 w% RTV141A; 10 w% RTV141B from Bluesil) were transferred onto the master and dried at 70°C for 1 hr before unmolding.

Silica precursor solution was prepared by adding 4.22 g tetraethyl orthosilicate (TEOS) into 23.26 g absolute ethanol, then 1.5 g HCl (37%), and stirring the solution for 18 hr. The final molar composition was TEOS:HCl:EtOH = 1:0.7:25. All the chemicals were from Sigma. Gel films were obtained by dipcoating the coverslips with a ND-DC300 dip-coater (Nadetech Innovations) equipped with an EBC10 Miniclima device to control the surrounding temperature and relative humidity to 20°C and 45–50%, respectively. The thickness of the film was controlled by the withdrawal rate at 300 mm/min. After dipcoating, gel films were consolidated at 430°C for 5 min. Then, a new layer of the same solution was deposited under the same conditions for printing with the PDMS mold. After imprinting, the samples were transferred to a 70°C oven for 1 min and then to a 140°C oven for 2 min to consolidate the xerogel films before peeling off the PDMS mold. Finally, the sol–gel replicas were annealed at 430°C for 10 min for consolidation.