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

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

Early activation of CD95 is limited and localized to the cytotoxic synapse

The cytotoxic synapse formed between cytotoxic T lymphocytes or natural killer cells expressing CD95L and target cells with CD95 on their surface is a key pathway for apoptosis induction by the immune system. Despite similarities with the immune synapse in antigen presenting cells, little is known about the role of the spatiotemporal organization of agonistic proteins/receptor interactions for CD95 signaling. Here, we have developed an artificial cytotoxic synapse to examine how mobility and geometry of an anti-CD95 agonistic antibody affect receptor aggregation and mobility, ie the first step of receptor activation. By measuring the distribution, diffusion coefficient, and fraction of immobile CD95 receptor in living cells, we show that at short times, the initial activation of CD95 occurs locally and is limited to the contact region of the cytotoxic synapse. This anisotropic activation of apoptotic signaling supports a role for confined interactions on the efficiency of signal transduction that may have implications for biomedical applications of extrinsic apoptosis induction.Fil: Sánchez, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Murad, Fabronia. Universität Tübingen; AlemaniaFil: Gülcüler Balta, Gülce S.. German Cancer Research Center; Alemania. Heidelberg University; AlemaniaFil: Martin Villalba, Ana. German Cancer Research Center; AlemaniaFil: García Sáez, Ana J.. Universität Tübingen; AlemaniaFil: Carrer, Dolores Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentin

3D Cellular Architecture Modulates Tyrosine Kinase Activity, Thereby Switching CD95-Mediated Apoptosis to Survival

The death receptor CD95 is expressed in every cancer cell, thus providing a promising tool to target cancer. Activation of CD95 can, however, lead to apoptosis or proliferation. Yet the molecular determinants of CD95’s mode of action remain unclear. Here, we identify an optimal distance between CD95Ligand molecules that enables specific clustering of receptor-ligand pairs, leading to efficient CD95 activation. Surprisingly, efficient CD95 activation leads to apoptosis in cancer cells in vitro and increased tumor growth in vivo. We show that allowing a 3D aggregation of cancer cells in vitro switches the apoptotic response to proliferation. Indeed, we demonstrate that the absence or presence of cell-cell contacts dictates the cell response to CD95. Cell contacts increase global levels of phosphorylated tyrosines, including CD95’s tyrosine. A tyrosine-to-alanine CD95 mutant blocks proliferation in cells in contact. Our study sheds light into the regulatory mechanism of CD95 activation that can be further explored for anti-cancer therapies