A Tethered Vesicle Assay for High-Throughput Quantification of Membrane Fission

Abstract

Membrane fission, which divides membrane surfaces into separate compartments, is essential to diverse cellular processes including membrane trafficking and cell division. Quantitative assays are needed to elucidate the physical mechanisms by which proteins drive membrane fission. Toward this goal, several experimental tools have been developed, including visualizing fission products using electron microscopy, measuring membrane shedding from a lipid reservoir, and observing fission of individual membrane tubes pulled from giant vesicles. However, no existing assay of membrane fission provides a quantitative, high-throughput measure of the distribution of vesicle curvatures generated by fission-driving proteins. Toward addressing this challenge, here we describe a novel approach that uses confocal fluorescence imaging to quantify the diameter distribution of membrane vesicles that have been tethered to a coverslip surface following exposure to fission-driving proteins. We employ this assay to measure the progressive appearance of high curvature fission products upon exposure of vesicles to increasing protein concentration. Results from this approach are in quantitative agreement with measurements from electron microscopy, but can be collected with considerably greater throughput, enabling examination of a broad range of experimental conditions. Using the tethered vesicle approach, we have recently found that membrane-bound intrinsically disordered proteins are surprisingly potent drivers of membrane fission. The capacity to drive fission arises from steric pressure generated when disordered domains with large hydrodynamic radii bind to membranes at high local densities. More broadly, the experimental tools described here have the potential to improve our mechanistic understanding of membrane fission in diverse biophysical contexts.

A Förster Resonance Energy Transfer-Based Sensor of Steric Pressure on Membrane Surfaces

“…Here, we used a quantitative fluorescence approach to measure the size distribution of vesicles following protein exposure, a technique that we have reported previously. 25 Briefly, vesicles were incubated for 1 h at room temperature with wtENTH or his-ENTH at concentrations ranging from 100 nM to 2 μM in solution. Following incubation, the distribution of vesicle sizes was determined using the quantitative fluorescence approach described above, producing the histograms shown in Figure 5A.…”

“…Finally, we examined the ability of wtENTH and his-ENTH to drive changes in vesicle diameter. Here, we used a quantitative fluorescence approach to measure the size distribution of vesicles following protein exposure, a technique that we have reported previously . Briefly, vesicles were incubated for 1 h at room temperature with wtENTH or his-ENTH at concentrations ranging from 100 nM to 2 μM in solution.…”

Molecular mechanisms of steric pressure generation and membrane remodeling by disordered proteins

“…Therefore, we estimated the coverage of the membrane by AP180CTD-FL as a function of the protein concentration in solution. For these measurements we used a tethered vesicle assay, an approach we have previously validated, Figure 1D (10,26). In brief, biotinylated vesicles were tethered to a biotin-NeutrAvidin functionalized surface.…”

“…Specifically, we compared membrane vesiculation by AP180CTD-FL and AP180CTD-1/3, Figure 5A. We used a quantitative fluorescence approach to measure the distribution of vesicle diameters following protein exposure, a technique that we have reported previously, Figure 5B (26,33). Briefly, vesicles were incubated with either AP180CTD-FL or AP180CTD-1/3 at concentrations varying from 20 nM to 4 µM for 1 hour at room temperature.…”

Characterization of Protein–Membrane Interactions in Yeast Autophagy

“…A major advantage of this technique is that it is used to directly visualize and quantify vesicle clustering and tubule width [ 57 , 58 , 59 , 120 ]. One limitation of using EM to monitor membranes is that it requires the manual analysis of membrane structures, thereby limiting the throughput and potentially introducing bias by the limited field of view [ 121 ].…”