Fluorescent quantification of size and lamellarity of membrane nanotubes

Membrane nanotubes, ubiquitous in cellular systems, adopt a spectrum of curvatures and shapes that are dictated by their intrinsic physical characteristics as well as their interactions with the local cellular environment. A high bending flexibility is needed in the crowded cytoplasm where tubes often need to bend significantly in the axial direction at sub-micron length scales. We find the stiffness of spontaneously formed membrane nanotubes by measuring the persistence length of reconstituted membrane nanotubes freely suspended in solution and imaged by fluorescence microscopy. By quantifying the tube diameter we demonstrate for the first time that the persistence length scales linearly with radius. Although most tubes are uni-lamellar, the predicted linear scaling between tube radius and persistence length allows us to identify tubes that spontaneously form as multilamellar structures upon hydration. We provide the first experimental evidence that illumination of lipid fluorophores can have a profound effect on the lipid bilayer which we sensitively detect as a continuous change in the tube persistence length with time. The novel assay and methodology here presented has potential for quantification of the structural reinforcement of membrane tubes by scaffolding proteins.