A Structurally Robust Phospholipid Microtube Constructed by Membrane Phase Separation as a Scaffold for <i>On-Tube</i> Characterization of Membrane-Bound Proteins

Artificial phospholipid assemblies, such as liposomes, have become indispensable scaffolds for the characterization of membrane proteins. Phospholipid microtubes (PMTs) are universal biological architectures, as seen in the endoplasmic reticulum and neurites, that are constructed by curvature-sensing membrane-bound proteins such as Bin/Amphiphysin/Rvs (BAR) proteins. Inspired by the biological PMTs, artificial PMTs have been constructed by physically pulling the membranes using optical tweezers or kinesin motors. However, the inherent low stability of artificial PMTs, which collapse after the removal of the energy source, has critically limited their applications as scaffolds. Here, we report the construction of structurally robust PMTs as practically useful scaffolds for <i>on-tube</i> characterization of membrane-bound proteins. We focused on a membrane deformation driven by phase separation between saturated and unsaturated phospholipids. We developed a polycationic peptide lipid (PCaL) that dissociates the phase separation. Interestingly, complexation of PCaL with an anionic ligand prompted the spontaneous formation of phospholipid microtubes (PMT^PCaL). Importantly, PMT^PCaL exhibited high robustness against harsh physical stresses, including increased temperatures, increased salt concentrations, osmotic stress, physical pulling using optical tweezers, and molecular crowding. Taking advantage of the high structural stability, PMT^PCaL was utilized as a scaffold for <i>on-tube</i> characterization of a curvature-sensing membrane-bound protein. We revealed that sorting nexin-1 enhances its binding property with a tubular membrane under highly crowded cell-mimicking conditions relative to noncrowding conditions.