Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

26-POS Board 14 COMPETITIVE INTERACTIONS AT THE ENDOCYTIC PIT: LIQUID-PHASE SEPARATION DURING ENDOCYTIC PROGRESSION Carl Helmer 1 ; Sigrid Milles 1 ; 1 Forschungsinstitut für Molekulare Pharmakologie, FMP, Berlin, Germany Clathrin-mediated endocytosis (CME) is an essential mechanism by which eukaryotic cells internalize cargo from the extracellular environment to enable nutrient uptake, signaling, and synaptic function. However, the molecular principles underlying endocytic progression remain poorly understood. CME is initiated by pioneering endocytic proteins, such as Eps15 and FCHo2, which contain large intrinsically disordered regions (IDRs) and lack stable three dimensional structures. These IDRs mediate dynamic, multivalent interactions that promote liquid-liquid phase separation (LLPS), a process proposed to facilitate the initiation of endocytosis. As endocytosis progresses, these pioneer proteins relocate to the periphery of the clathrin-coated pit and are excluded from the final matured vesicle. Their central position is instead occupied by a distinct set of proteins that also comprise extensive IDRs. In this project, we study the molecular basis for relocation of Eps15 and FCHo2, and how other endocytic disordered proteins are involved in this. We will combine high-resolution molecular characterization by nuclear magnetic resonance (NMR) spectroscopy and single-molecule Förster resonance energy transfer (smFRET), in vitro reconstitution of multicomponent protein systems and in cellulo validation assays. This integrated approach will identify the constructive and competitive interactions that regulate CME and drive endocytosis progression. Ultimately, this project will bridge in vitro structural studies with cellular readouts, providing a framework for investigating structural dynamics in the cellular context.

76