FluoSim: simulator of single molecule dynamics for fluorescence live-cell and super-resolution imaging of membrane proteins

Abstract

Fluorescence live-cell and super-resolution microscopy methods have considerably advanced our understanding of the dynamics and mesoscale organization of macro-molecular complexes that drive cellular functions. However, different imaging techniques can provide quite disparate information about protein motion and organization, owing to their respective experimental ranges and limitations. To address these issues, we present here a robust computer program, called FluoSim, which is an interactive simulator of membrane protein dynamics for live-cell imaging methods including SPT, FRAP, PAF, and FCS, and super-resolution imaging techniques such as PALM, dSTORM, and uPAINT. FluoSim integrates diffusion coefficients, binding rates, and fluorophore photo-physics to calculate in real time the localization and intensity of thousands of independent molecules in 2D cellular geometries, providing simulated data directly comparable to actual experiments. FluoSim was thoroughly validated against experimental data obtained on the canonical neurexin-neuroligin adhesion complex at cell–cell contacts. This unified software allows one to model and predict membrane protein dynamics and localization at the ensemble and single molecule level, so as to reconcile imaging paradigms and quantitatively characterize protein behavior in complex cellular environments.

MDGAs are fast-diffusing molecules that delay excitatory synapse development by altering neuroligin behavior

“…Experiments were performed at DIV 8, 10, or 14, a time window of active excitatory synapse differentiation ( Chanda et al, 2017 ). As a control, we electroporated neurons with GFP-GPI and labeled them with an anti-GFP nanobody conjugated to Atto 647 N, as described ( Lagardère et al, 2020 ). At DIV 8, recombinant AP-MDGA1 and AP-MDGA2 diffused very fast in the dendritic membrane, showing a single peak of diffusion coefficient around 0.30 µm²/s, very similar to GFP-GPI ( Figure 3A and B ).…”

“…dSTORM stacks were analyzed using the PALM-Tracer program, allowing the reconstruction of a unique super-resolved image of 32 nm pixel size (zoom 5 compared to the original images) by summing the intensities of all localized single molecules (1 detection per frame is coded by an intensity value of 1). The localization precision of our imaging system in dSTORM conditions is around 60 nm (FWHM) ( Lagardère et al, 2020 ). To analyze protein enrichment at post-synapses, the average number of detections within Homer1c puncta was divided by the the average number of extra-synaptic detections, both normalized per unit area.…”

High-Resolution Fluorescence Imaging Combined With Computer Simulations to Quantitate Surface Dynamics and Nanoscale Organization of Neuroligin-1 at Synapses

“…We previously applied such a modeling approach to evaluate the mechanisms controlling AMPA receptor trafficking at synapses ( Czöndör et al, 2012 ). In this study, we took advantage of our recently released simulator of membrane protein dynamics, FluoSim, that was thoroughly validated against live-cell and super-resolution imaging experiments performed on lamellipodial contacts mediated by NRXN-NLGN adhesions in heterologous cells ( Lagardère et al, 2020 ). We extended this analysis to model NLGN1 dynamics and organization in the neuronal membrane, with a systematic comparison to single molecule tracking and localization studies, as well as long-term FRAP experiments performed in primary hippocampal neurons.…”

An efficient GUI-based clustering software for simulation and Bayesian cluster analysis of single-molecule localization microscopy data

“…The execution of thought experiments is essential in developing reliable experimental strategies and is especially important for data processing-intensive assays. To allow for the freehand design of ground-truth data while simulating realistic experimental output, we included a simulation module similar to FluoSim (Lagardère et al, 2020). This module allows the generation of user-defined clusters of molecules combined with a selected level of randomly placed background molecules.…”