Live-cell single-molecule microscopy
Despite being first introduced in biomedical research over a century ago, fluorescence microscopy is ageing extremely well. Thanks to genetically encoded fluorescent reporters such as GFP (Nobel Prize in Chemistry, 2008), it is possible to localize and follow biological events in real time in living cells. Further, the interaction between light and fluorescent molecules allows to circumvent physical barriers once thought unsurpassable and collect images of biological samples with details on the nanometer spatial scale, as demonstrated by the recent advances in super-resolution microscopy (Nobel Prize in Chemistry 2014). Fluorescence microscopy is also sensitive enough to detect and track an individual ("single") molecule as it moves and interacts with its partners, leading to quantitative analysis on a "molecule by molecule" base of protein dynamics, protein-protein interactions, gene expression and more.
Our R&D team focuses on developing and applying single molecule approaches to quantifying dynamic events in individual living cells. In particular, we have pioneered a single- molecule approach to quantify the interaction kinetics between nuclear proteins (transcription factors) and its binding sites on DNA, and we are currently applying it to dissect the causes that underlie activation and inactivation of those transcription factors that act as tumor suppressors or oncogenes in cancer settings.
Together with these activities, we support members of our scientific community interested in developing/applying assays in advanced fluorescence microscopy, ranging from protocol development, to data acquisition and data analysis for F-techniques ultrasensitive, single molecule and single-particle imaging and super-resolution microscopy.
Loffreda A, Jacchetti E, Antunes S, Rainone P, Morisaki T, Daniele T, Bianchi M.E, Tacchetti C and Mazza D. Live-cell p53 single molecule binding is modulated by C-terminal acetylation and correlates with transcriptional activity. Nature communications. 2017; 8, 313.
Rhodes J, Mazza D, Nasmyth K and Uphoff S. Scc2/Nipbl hops between chromosomal cohesin rings after loading. eLife. 2017; 6, e30000.
Caldieri G, Barbieri E, Nappo G, Raimondi A, Bonora M, Conte A, Verhoef LGGC, Confalonieri S, Malabarba MG, Bianchi F, Cuomo A, Bonaldi T, Martini E, Mazza D, Pinton P, Tacchetti C, Polo S, Di Fiore PP, Sigismund S. Reticulon3-dependent ER-PM contact sites control EGFR non-clathrin endocytosis. Science. 2017; 356, 617.
Swinstead EE, Miranda TB, Paakinaho V, Baek S, Goldstein I, Hawkins M, Karpova T, Ball D, Mazza D, Lavis LD, Morisaki T, Grøntved L, Presman DM and Hager GL. Steroid Receptors Reprogram FoxA1 Occupancy through Dynamic Chromatin Transitions. Cell,. 2016;165, 593.
Maltecca F, Baseggio E, Consolato F, Mazza D, Podini P, Youn SM, Drago I, Bahr BA, Puliti AM, Codazzi F, Quattrini A, Casari G. Purkinje neuron Ca2+ influx reduction rescues ataxia in SCA28 model. J. Clin. Invest. 2015; 125, 263.
Morisaki T, Mueller WG, Golob N, Mazza D* and McNally JG*. Single molecule analysis of transcription factor binding at transcription sites in live cells. Nature Communications. 2014; 5, 4456.
Mazza D, Mueller F, Stasevich TJ and McNally JG. Convergence of chromatin binding estimates in living cells. Nat. Meth. 2013; 10, 692.
Mazza D, Abernathy A, Golob N, Morisaki T and McNally JG. A benchmark for chromatin binding measurements in live cells. Nucl.. Ac. Res. 2012; 40, e119.
Michelman-Ribeiro A* , Mazza D*, Rosales T, Stasevich TJ, Boukari H, Rishi V, Vinson C, Knutson JR and McNally JG. Direct measurement of association and dissociation rates of DNA binding in live cells by fluorescence correlation spectroscopy. Biophys. J. 2009; 97, 337.
Mazza D, Braeckmans K, Cella F, Testa I, Vercauteren D, Demeester J, De Smedt SS and Diaspro A. A new FRAP/FRAPa method for 3D diffusion measurements based on multi- photon excitation microscopy. Biophys. J. 2008; 95, 3457.