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.

FBAR syndapin 1 recognizes and stabilizes highly curved tubular membranes in a concentration dependent manner.

Syndapin 1 FBAR, a member of the Bin-amphiphysin-Rvs (BAR) domain protein family, is known to induce membrane curvature and is an essential component in biological processes like endocytosis and formation and growth of neurites. We quantify the curvature sensing of FBAR on reconstituted porcine brain lipid vesicles and show that it senses membrane curvature at low density whereas it induces and reinforces tube stiffness at higher density. FBAR strongly up-concentrates on the high curvature tubes pulled out of Giant Unilamellar lipid Vesicles (GUVs), this sorting behavior is strongly amplified at low protein densities. Interestingly, FBAR from syndapin 1 has a large affinity for tubular membranes with curvatures larger than its own intrinsic concave curvature. Finally, we studied the effect of FBAR on membrane relaxation kinetics with high temporal resolution and found that the protein increases relaxation time of the tube holding force in a density-dependent fashion.