Spatial Control of Epsin-induced Clathrin Assembly by Membrane Curvature

Abstract

Background: The clathrin adaptor epsin is indispensable for clathrin-mediated endocytosis, but the mechanism by which it regulates clathrin assembly remains unclear.Results: Epsin shows a preference to localize to regions of high membrane curvature.Conclusion: Epsin's membrane curvature sensing directs clathrin assembly to highly curved membranes.Significance: Membrane curvature could determine the hierarchy of molecular interactions during clathrin-mediated membrane budding.

Automated Multi-Peak Tracking Kymography (AMTraK): A Tool to Quantify Sub-Cellular Dynamics with Sub-Pixel Accuracy

“…While the parameters a and b are scaling factors, c determines the characteristic clathrin polymerization time, τ = 1/c. In our analysis the clathrin assembly time in presence of wild-type epsin is <τ> = 71.49±44.09 s while with mutant epsin <τ> = 70.16±29.89 s. In our estimate of the mutant assembly time is indistinguishable from wild-type, consistent with the previous report, which used manual quantification of the kymograph [24]. We proceed to examine if our tool, which appears to work successfully on in vitro data with low background noise, can also be used for the quantification of in vivo dynamics inside the crowded environment of an intact cell.…”

“…We proceed to quantify the assembly kinetics of clathrin on membranes from an in vitro reconstitution assay of clathrin assembly on vesicle precursors reported previously by Holkar et al [24]. This process has been analyzed using kymography due to its effectively 1D spatial extent and the multiple simultaneous events of assembly.…”

“…This process has been analyzed using kymography due to its effectively 1D spatial extent and the multiple simultaneous events of assembly. The published time-series of fluorescently labeled clathrin assembly kinetics in the presence of wild-type epsin (supplementary movie 3 in [24]) and L6W mutant epsin (supplementary movie 5 in [24]) in the form of 16 bit TIF images were provided by the authors (Sachin Holkar, personal communication). AMTraK was used to analyze this data without any pre-processing, resulting in tracked kymographs of assembly kinetics with wild-type (Fig 7A) and mutant epsin (Fig 7B).…”

“…The software outputs a text-file of grey-value intensities normalized by the bit-depth (maximum normalized, between 0–1) (S4 Data), which when multiplied by the bit-depth of the input images, produced intensity profiles of clathrin assembly in grey-values with time in the presence of wild-type (Fig 7C, S3A Fig) and mutant epsin (Fig 7D, S3B Fig). These intensity profiles were fit to a single phase exponential function y = a+(b-a)*(1-e -c*t ), where y is the intensity which increases with time t, and depends on three fit parameters, a, b and c, the same function as used by Holkar et al [24]. A large proportion of the assembly events were successfully tracked and most showed saturation kinetics that were fit by curves with R 2 >0.7 (S3 Fig).…”

“…Microscopy of in vitro reconstituted membrane bilayers has become a powerful tool to study the dynamics of protein assembly during vesicle formation [21,22]. Proteins such as epsin, which were reported to accelerate clathrin ‘recruitment’ [23] have been examined using kymography of the fluorescently labelled clathrin and the effect of mutant epsins on the process [24]. While such an approach lends itself to high-content screening, the analysis of the kymograph has been manual.…”