The curvature of biological membranes at the nanometer scale is critically important for vesicle trafficking, organelle morphology, and disease propagation. The initial membrane bending events that initiate these complex processes occur at a length scale that is irresolvable by most super-resolution optical microscopy methods. This manuscript reports the development of polarized localization microscopy (PLM), a pointillist optical imaging technique for the detection of nanoscale membrane curvature in correlation with single-molecule dynamics and molecular sorting. PLM combines polarized total internal reflection fluorescence microscopy (TIRFM) and single-molecule localization microscopy to reveal membrane orientation with a sub-diffractionlimited resolution without reducing localization precision by point spread function (PSF) manipulation. Membrane curvature detection with PLM requires fewer localization events to detect curvature than 3D single-molecule localization microscopy (e.g., PALM or STORM), which enables curvature detection 10x faster via PLM. With rotationally confined lipophilic fluorophores and the polarized incident fluorescence excitation, membrane-bending events are revealed with super-resolution. Engineered hemispherical membrane curvature with a radii ≥ 24 nm was detected with PLM and individual fluorophore localization precision was 13 ± 5 nm.Further, deciphering molecular mobility as a function of membrane topology was enabled. The diffusion coefficient of individual DiI molecules was 7.6x higher in planar supported lipid bilayers than within nanoscale membrane curvature. Through the theoretical foundation and experimental demonstration provided here, PLM is poised to become a powerful technique for revealing the underlying biophysical mechanisms of membrane bending at physiological length scales.

AB5bacterial toxins and polyomaviruses induce membrane curvature as a mechanism to facilitate their entry into host cells. How membrane bending is accomplished is not yet fully understood but has been linked to the simultaneous binding of the pentameric B subunit to multiple copies of glycosphingolipid receptors. Here, we probe the toxin membrane binding and internalization mechanisms by using a combination of superresolution and polarized localization microscopy. We show that cholera toxin subunit B (CTxB) can induce membrane curvature only when bound to multiple copies of its glycosphingolipid receptor, GM1, and the ceramide structure of GM1 is likely not a determinant of this activity as assessed in model membranes. A mutant CTxB capable of binding only a single GM1 fails to generate curvature either in model membranes or in cells, and clustering the mutant CTxB−single-GM1 complexes by antibody cross-linking does not rescue the membrane curvature phenotype. We conclude that both the multiplicity and specific geometry of GM1 binding sites are necessary for the induction of membrane curvature. We expect this to be a general rule of membrane behavior for all AB5toxins and polyomaviruses that bind glycosphingolipids to invade host cells.

For endocytosis and exocytosis, membranes transition among planar, budding, and vesicular topographies through nanoscale reorganization of lipids, proteins, and carbohydrates. However, prior attempts to understand the initial stages of nanoscale bending have been limited by experimental resolution. Through the implementation of polarized localization microscopy, this article reports the inherent membrane bending capability of cholera toxin subunit B (CTxB) in quasi-one-component-supported lipid bilayers. Membrane buds were first detected with <50 nm radius, grew to >200 nm radius, and extended into longer tubules with dependence on the membrane tension and CTxB concentration. Compared to the concentration of the planar-supported lipid bilayers, CTxB was (12 ± 4)× more concentrated on the positive curvature top and (26 ± 11)× more concentrated on the negative Gaussian curvature neck of the nanoscale membrane buds. CTxB is frequently used as a marker for liquid-ordered lipid phases; however, the coupling between CTxB and membrane bending provides an alternate understanding of CTxB-induced membrane reorganization. These findings allow for the reinterpretation of prior observations by correlating CTxB clustering and diffusion to CTxB-induced membrane bending. Single-particle tracking was performed on single lipids and CTxB to reveal the correlations among single-molecule diffusion, CTxB accumulation, and membrane topography. Slowed lipid and CTxB diffusion was observed at the nanoscale bud locations, suggesting a local increase in the effective membrane viscosity or molecular crowding upon membrane bending. These results suggest inherent CTxB-induced membrane bending as a mechanism for initiating CTxB internalization in cells that could be independent of clathrin, caveolin, actin, and lipid phase separation.

Recent advances in nanoengineering and super-resolution microscopy have enabled new capabilities for creating and observing membrane curvature. However, the effects of curvature on single-lipid diffusion have yet to be revealed. The simulations presented here describe the capabilities of varying experimental methods for revealing the effects of nanoscale curvature on single-molecule mobility. Traditionally, lipid mobility is revealed through fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single particle tracking (SPT). However, these techniques vary greatly in their ability to detect the effects of nanoscale curvature on lipid behavior. Traditionally, FRAP and FCS depend on diffraction-limited illumination and detection. A simulation of FRAP shows minimal effects on lipids diffusion due to a 50 nm radius membrane bud. Throughout the stages of the budding process, FRAP detected minimal changes in lipid recovery time due to the curvature versus flat membrane. Simulated FCS demonstrated small effects due to a 50 nm radius membrane bud that was more apparent with curvature-dependent lipid mobility changes. However, SPT achieves a sub-diffraction-limited resolution of membrane budding and lipid mobility through the identification of the single-lipid positions with ≤15 nm spatial and ≤20 ms temporal resolution. By mapping the single-lipid step lengths to locations on the membrane, the effects of membrane topography and curvature could be correlated to the effective membrane viscosity. Single-fluorophore localization techniques, such SPT, can detect membrane curvature and its effects on lipid behavior. These simulations and discussion provide a guideline for optimizing the experimental procedures in revealing the effects of curvature on lipid mobility and effective local membrane viscosity.

AB5 bacterial toxins and polyomaviruses induce membrane curvature as a mechanism to facilitate their entry into host cells. How membrane bending is accomplished is not yet fully understood but has been linked to the simultaneous binding of the pentameric B-subunit to multiple copies of their glycosphingolipid receptors. Here, we probe the toxin membrane binding and internalization mechanisms by using a combination of super-resolution and polarized localization microscopy. We show that cholera toxin subunit B (CTxB) can induce membrane curvature only when bound to multiple copies of its glycosphingolipid receptor, GM1, and the ceramide structure of GM1 is likely not a determinant of this activity as assessed in model membranes. A mutant CTxB capable of binding only a single GM1 fails to generate curvature either in model membranes or in cells and clustering the mutant CTxB-single-GM1 complexes by antibody cross-linking does not rescue the membrane curvature phenotype. We conclude that both the multiplicity and specific geometry of GM1 binding sites are necessary for the induction of membrane curvature. We expect this to be a general rule of membrane behavior for all AB5 toxins and polyomaviruses that bind glycosphingolipids to invade host cells.SIGNIFICANCE STATEMENTMembrane binding toxins demonstrate both a public health challenge and a bioengineering opportunity due to their efficient internalization into cells. These toxins multivalently bind to naturally occurring lipid receptors at the plasma membrane and initiate endocytosis. This manuscript reports the importance of structured lipid-receptor clustering for the induction of membrane bending. We also observed that the magnitude of membrane curvature was correlated to the stoichiometry of toxin-bound receptors. By identifying how these bacterial proteins initiate membrane curvature, these findings provide mechanistic insights into the early steps of pathogenic endocytosis.