An Efficient GUI-Based Clustering Software for Simulation and Bayesian Cluster Analysis of Single-Molecule Localization Microscopy Data

Saskia Kutz; Ando C Zehrer; Roman Svetlitckii; Gülce S Gülcüler Balta; Lucrezia Galli; Susanne Kleber; Jakob Rentsch; Ana Martin-Villalba; Helge Ewers

doi:10.3389/fbinf.2021.723915

An Efficient GUI-Based Clustering Software for Simulation and Bayesian Cluster Analysis of Single-Molecule Localization Microscopy Data

Front Bioinform. 2021 Oct 11:1:723915. doi: 10.3389/fbinf.2021.723915. eCollection 2021.

Authors

Saskia Kutz¹, Ando C Zehrer¹, Roman Svetlitckii¹, Gülce S Gülcüler Balta², Lucrezia Galli², Susanne Kleber², Jakob Rentsch¹, Ana Martin-Villalba², Helge Ewers¹

Affiliations

¹ Institut für Biochemie, Freie Universität Berlin, Berlin, Germany.
² Department of Molecular Neurobiology, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Abstract

Ligand binding of membrane proteins triggers many important cellular signaling events by the lateral aggregation of ligand-bound and other membrane proteins in the plane of the plasma membrane. This local clustering can lead to the co-enrichment of molecules that create an intracellular signal or bring sufficient amounts of activity together to shift an existing equilibrium towards the execution of a signaling event. In this way, clustering can serve as a cellular switch. The underlying uneven distribution and local enrichment of the signaling cluster's constituting membrane proteins can be used as a functional readout. This information is obtained by combining single-molecule fluorescence microscopy with cluster algorithms that can reliably and reproducibly distinguish clusters from fluctuations in the background noise to generate quantitative data on this complex process. Cluster analysis of single-molecule fluorescence microscopy data has emerged as a proliferative field, and several algorithms and software solutions have been put forward. However, in most cases, such cluster algorithms require multiple analysis parameters to be defined by the user, which may lead to biased results. Furthermore, most cluster algorithms neglect the individual localization precision connected to every localized molecule, leading to imprecise results. Bayesian cluster analysis has been put forward to overcome these problems, but so far, it has entailed high computational cost, increasing runtime drastically. Finally, most software is challenging to use as they require advanced technical knowledge to operate. Here we combined three advanced cluster algorithms with the Bayesian approach and parallelization in a user-friendly GUI and achieved up to an order of magnitude faster processing than for previous approaches. Our work will simplify access to a well-controlled analysis of clustering data generated by SMLM and significantly accelerate data processing. The inclusion of a simulation mode aids in the design of well-controlled experimental assays.

Keywords: GUI; SMLM; cell membrane proteins; cluster analyis; software.