Nanoscale Membrane Buds Induced by CTxB-GM1 in One Component Bilayer Detected by Polarized Localization Microscopy (PLM)

Abstract

Membrane biomolecules binding, organization, and clustering play a vital role in cell function. For instance, Cholera toxin subunit B (CTxB) binds to the ganglioside (GM1) and preferentially partitions on highly curved membranes as an initial stage for its internalization into the cell. Further, CTxB-GM1 demonstrates confined diffusion at nanoscale sites in synthetic bilayers and cells. In our attempt to understand the mechanism of the CTxB-GM1 complex, we introduced CTxB on a quasi-one component bilayer of 99.4% POPC + 1:1 GM1/DiI while imaging membrane topology and protein dynamics by Polarized Localization Microscopy (PLM). PLM, a super-resolution technique developed in our lab, combines super-resolution fluorescence localization microscopy (FLM) with polarized total internal reflection fluorescence microscopy (pTIRFM) to reveal nanoscale membrane orientation. In particular, PLM enabled the detection of nanoscale membrane budding events that initiated upon adding CTxB. Single particle tracking (SPT) was then performed on lipid (DiI) and CTxB trajectories revealing correlated diffusion between CTxB and the membrane structure. Confined CTxB diffusion was observed at the nanoscale buds locations. In this study, we obtained super-resolution, time-lapse images of CTxB locations and the corresponding membrane topology. CTxB was recruited to the negative Gaussian curved membrane; single-event and correlation analysis demonstrated increased clustering of lipids as function of CTxB incubation time with the membrane. Finally, we explored the effect of changing CTxB concentration on the bud formation process and bud sizes. The presented study is the first known report of bud formation induced by CTxB-GM1 in one component bilayer and in the absence of lipid phases. Our work demonstrates a possible mechanism for CTxB internalization and confinement in cells. PLM and this discovery may open up new avenues in the investigation of the processes governing cell signaling triggered by bacterial toxins.