Biology subjects

James, H. P.

Publications and source records attributed to James, H. P..

3 recordsLinked to original sources

bioRxiv2026

Multivalency in endocytic accessory proteins orchestrates membrane shape transitions

Fast endophilin-mediated endocytosis (FEME) relies on the coordinated activities of membrane-remodeling proteins and multivalent protein interaction networks. Endophilin is a potent membrane curvature generator through its N-BAR domain and forms multivalent interactions with the proline-rich motifs (PRMs) of lamellipodin via its SH3 domain. Although these interactions are known to promote biomolecular condensation, their functional consequences for membrane remodeling have remained unclear. Here, we investigate how SH3/PRM mediated phase separation regulates endophilin-driven membrane curvature generation. Using purified recombinant proteins, giant unilamellar vesicles, quantitative fluorescence microscopy, and in vitro phase separation assays, we show that multivalent interactions between endophilin and lamellipodin drive the formation of membrane-associated protein-rich phases that strongly suppress endophilin mediated membrane tubulation. Competitive disruption of SH3/PRM interactions with the synaptojanin-derived peptide PP19 (containing only two PRM motifs) rapidly dissolves condensates and restores membrane curvature generation without affecting the intrinsic curvature-generating activity of endophilin. We further demonstrate that this regulatory mechanism is conserved across endophilin A1, A2, and A3 isoforms and depends critically on interaction valency: a multivalent PRM7 construct recapitulates condensate formation and curvature suppression, whereas mono or divalent PRM constructs does not. Together, these findings identify multivalent SH3/PRM interactions as a reversible molecular switch that balances biomolecular condensation and membrane remodeling. Multiple mechanisms, including post-translational modifications, likely regulate the multivalency of these interactions. Our results thus establish a broad functional link between protein phase separation and membrane mechanics, providing a framework for understanding how multivalent interaction networks regulate membrane shaping in multiple endocytic pathways.

biophysics↗

bioRxiv2025

Genetic Code Expansion and Enzymatic Modifications as Accessible Methods for Studying Site-Specific Post-Translational Modifications of Alpha-Synuclein and Tau

Alpha-synuclein (S) and tau play important roles in the pathology of Parkinsons Disease and Alzheimers Disease, respectively, as well as numerous other neurodegenerative diseases. Both proteins are classified as intrinsically disordered proteins (IDPs), as they have no stable structure that underlies their function in healthy tissue, and both proteins are prone to aggregation in disease states. There is substantial interest in understanding the roles that post-translational modifications (PTMs) play in regulating the structural dynamics and function of S and tau monomers, as well as their propensity to aggregate. While there have been many valuable insights into site-specific effects of PTMs garnered through chemical synthesis and semi-synthesis, these techniques are often outside of the expertise of biochemistry and biophysics laboratories wishing to study S and tau. Therefore, we have assembled a primer on genetic code expansion and enzymatic modification approaches to installing PTMs into S and tau site-specifically, including isotopic labeling for NMR and fluorescent labeling for biophysics and microscopy experiments. These methods should be enabling for those wishing to study authentic PTMs in S or tau as well as the broader field of IDPs and aggregating proteins.

biochemistry↗

bioRxiv2024

VASP phase separation with priming proteins of fast endophilin mediated endocytosis modulates actin polymerization

Actin polymerization is essential in several clathrin-independent endocytic pathways including fast endophilin mediated endocytosis (FEME), however the actin machinery involved in FEME has been elusive. Here, we show that the actin polymerase VASP colocalizes and interacts directly with the FEME priming complex. We identify Endophilin as a VASP binding partner and establish novel non-canonical interactions between Endophilin and VASP. The major FEME regulators Endophilin and Lamellipodin interact multivalently with VASP to form liquid-like condensates both in solution and on lipid membranes that localize actin polymerization with the extent of actin polymerized regulated by multivalent Endophilin-Lamellipodin interactions. We identify a novel function for Endophilin condensates in bundling actin filaments and show that Endophilin directly binds filamentous actin. Our findings support a model that explains the connection between local actin polymerization and dynamic formation and dissolution of endocytic priming patches in FEME.

biochemistry↗