Regular Article Quantitative morphological analysis of arrestin2 clustering upon G protein-coupled receptor stimulation by super-resolution microscopy Zinnia Truan a,1 , Laura Tarancón Díez a,1 , Claudia Bönsch b , Sebastian Malkusch c , Ulrike Endesfelder c , Mihaela Munteanu b , Oliver Hartley b , Mike Heilemann c,⇑ , Alexandre Fürstenberg a,⇑ a Department of Human Protein Sciences, University of Geneva, CMU, Rue Michel-Servet 1, 1211 Genève 4, Switzerland b Department of Pathology and Immunology, University of Geneva, CMU, Rue Michel-Servet 1, 1211 Genève 4, Switzerland c Institute for Physical and Theoretical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 7, 60438 Frankfurt, Germany article info Article history: Received 1 April 2013 Received in revised form 20 September 2013 Accepted 24 September 2013 Available online 30 September 2013 Keywords: Super-resolution microscopy Clustering Arrestin G-protein coupled receptor Single-molecule fluorescence microscopy abstract Clustering of arrestins upon G protein-coupled receptor stimulation is a phenomenon that is well-known but difficult to describe quantitatively due to the size of the clusters close to the diffraction limit of visible light. We introduce a general method to quantitatively investigate the clustering of arrestin following stimulation of the C–C chemokine receptor 5 (CCR5) using single-molecule super-resolution imaging and coordinate and image-based cluster analysis. We investigated the effect of potent anti-HIV ligands of CCR5 with different pharmacological profiles on arrestin2 cluster formation and found that only the ligands capable of inducing CCR5 internalization induced arrestin2 recruitment and clustering. We fur- ther demonstrate that the fraction of arrestin2 molecules found in clusters larger than 100 nm correlates with the magnitude of ligand-induced CCR5 internalization, but not with G protein activation, indicating that recruitment of arrestin2 to CCR5 is independent of G protein activation. Pre-treatment of the cells with the drug cytochalasin D, which blocks actin polymerization, led to the formation of larger clusters, whereas the inhibitor of microtubule polymerization nocodazole had little effect on arrestin2 recruit- ment, suggesting an active role of actin in the organization and dynamics of these aggregates. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction G protein-coupled receptors (GPCRs) are proteins with seven transmembrane domains that have been extensively investigated owing to the fact that they are targeted by a third of currently mar- keted medicines (Rosenbaum et al., 2009). They mediate a wide variety of signals across the cell membrane as diverse as ions, small molecules, peptides, or hormones that regulate important physio- logical processes ranging from heart beat to smell to taste through the recruitment and activation of an array of adapter proteins in- side the cell. They are however not simple ‘on–off’ switches, since chemically very similar ligands that bind the same receptor often trigger distinct pharmacological outcomes. The detailed underlying activation mechanism is still poorly understood and likely related to the existence of various receptor conformations inducing differ- ential recruitment of cytosolic partners (Kenakin, 2009; Kobilka and Deupi, 2007). Upon agonist binding, GPCRs are stabilized in an ensemble of active conformations which catalyze the exchange of GDP for GTP on the a subunit of a cytoplasmic heterotrimeric G protein, promoting dissociation of Ga. Free Ga-GTP and Gbc sub- units subsequently mediate effector functions through second messengers that control the activity of key cellular enzymatic pro- cesses. In parallel, GPCR stimulation leads to the recruitment of arrestin proteins which block the interaction of receptors with their G protein, thereby arresting downstream second-messenger signaling (Lefkowitz and Shenoy, 2005). Independently of G pro- tein signaling, arrestins also induce clathrin-mediated endocytosis of the GPCRs, which are internalized and then either targeted to lysosomes for degradation or recycled to the cell surface after li- gand dissociation in endosomal compartments (Moore et al., 2007). Endocytosis of cell-surface receptors renders the cell less responsive to subsequent stimuli and is part of the desensitization process widespread among GPCRs. There are four known arrestins, two of which are localized pri- marily in visual sensory tissue. The two others, arrestin2 and arres- tin3, are ubiquitously expressed and interact with most GPCRs, but are also involved in the endocytosis of other proteins, suggesting a general role of arrestins in clathrin-mediated endocytosis (Luttrell and Gesty-Palmer, 2010). Upon stimulation, arrestins are recruited from cytosolic pools to the plasma membrane where they bind to clathrin and AP-2 components of the endocytic machinery and 1047-8477/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jsb.2013.09.019 ⇑ Corresponding authors. Fax: +49 6979829560 (M. Heilemann), +41 223795502 (A. Fürstenberg). E-mail addresses: heilemann@chemie.uni-frankfurt.de (M. Heilemann), alexan- dre.fuerstenberg@unige.ch (A. Fürstenberg). 1 These authors contributed equally to this work. Journal of Structural Biology 184 (2013) 329–334 Contents lists available at ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi