Novel Experimental Methods to Resolve Nanoscale Membrane Organizaion and Curvature

Abstract

The structures and functions of cell membranes are regulated by their diverse compositions, distinctive intermolecular interactions, and dynamic lateral organization. In particular, the combination of nanoscale membrane curvature and lateral organization of membrane components play a driving force in the initiation and regulation of intricate cellular processes such as endocytosis and exocytosis. However, many hypotheses concerning the nanoscale lipid redistribution in response to curvature are unanswered due to the diffraction limit of current optical imaging techniques. We aim to resolve nanoscale membrane curvature and the redistribution of proteins and lipids phase separated into liquid domains by inducing nanoscale curvature on giant unilamellar vesicles and novel microcopy methods. We achieve this through polarized total internal reflection fluorescence localization microscopy to yield sub-diffraction-limited resolution of membrane curvature and molecular organization. Polystyrene nanoparticles melted into agarose hydrogel substrates create a nanoengineered surface with small curved regions with controlled radii of curvature from 20 nm to 2 μm. These substrates work well with the evanescent field for total internal reflection microscopy because they are thin (<40 nm thick) and have a low index of refraction (<1.4). Micropipette aspiration techniques coupled with nanoengineered substrates enable dynamic control of membrane curvature and noticeable differences in lipid domain appearances. Preliminary data shows that when the vesicle is pressed against a surface, vesicles with coexisting liquid-­order and liquid-disorder phases demonstrate an increased propensity for the liquid-disordered phase to localize at the curved regions. Having the capability to resolve these previously irresolvable details allows us to better understand membrane dynamics and molecular sorting, in addition to exploring vital biological and disease related processes that are sensitive to membrane curvature.