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Understanding spatiotemporal stoichiometry of proteins in living cells / A global view on HIV-1 fusion
Par Sergi Padilla / Luis Alvarez (Oxford)
Le 14 Novembre 2018 à 14h00 - Salle de séminaires 5ème étage, Tour 32-33


Number and Brightness : from theory to practice in understanding spatiotemporal stoichiometry of proteins in living cells - Luis Alvarez

The study of protein spatiotemporal time resolved dynamics in live cells is key to understanding the molecular mechanisms that underpin signal transduction events.

Protein oligomerisation has been historically difficult to explore by non-specialists, as there is high learning curve for fluorescence anisotropy and FCS for biologists. In 2008 Enrico Gratton’s group published a new way to probe oligomeric states, namely “Number and Brightness”(N&B Digman et al. 2008). This imaging based technique showed significant promise to be accessible and become quite powerful in addressing protein oligomerisation and also either direct protein-protein interactions or interactions as part of protein complexes with the use of cross correlation N&B (ccN&B). Nevertheless this techniques has not yet shown widespread adoption by biologists. We present here our own efforts to make N&B and ccN&B routine advance imaging modalities(Nolan, Iliopoulou, et al. 2017, Nolan et al. 2018) to study protein dynamics and interactions. Specifically how the use of a novel detrending algorithm is key to probe the biological time resolved stoichiometry of proteins in living cells (Nolan, Alvarez, et al. 2017).

A global view on HIV-1 fusion - Sergi Padilla

HIV-1 infection represents one of the current health challenges worldwide, particularly in developing countries where anti-retroviral therapy is not as readily available as in the western world. A key aspect to develop new strategies against HIV-1 is to understand the intermolecular dynamics that drive the fusion of the HIV-1 infectious particles to the target cell membranes (Jakobsdottir et al. 2017). We reveal though advance quantitative imaging (Number and Brightness, ccN&B and quantitative interaction analysis through dSTORM data) the time resolved dynamics of the prefusion complex [HIV-1 envelope (Env) with the HIV-1 receptor (CD4) and co- receptor(either CCR5 or CXCR4)].

We also show how monoclonal HIV1-neutralising antibodies affect this prefusion complexes (Iliopoulou et al. 2018). We additional explore the links between cellular metabolism and HIV-1 infection.


Digman, M. A., R. Dalal, A. F. Horwitz and E. Gratton (2008). "Mapping the number of molecules and brightness in the laser scanning microscope." Biophys J 94(6): 2320-2332.

Nolan, R., L. A. Alvarez, S. C. Griffiths, J. Elegheert, C. Siebold and S. Padilla-Parra (2018). "Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software." J Vis Exp(137).

Nolan, R., L. A. J. Alvarez, J. Elegheert, M. Iliopoulou, G. M. Jakobsdottir, M. Rodriguez-Munoz, A. R. Aricescu and S. Padilla-Parra (2017). "nandb-number and brightness in R with a novel automatic detrending algorithm." Bioinformatics 33(21): 3508-3510.

Nolan, R., M. Iliopoulou, L. Alvarez and S. Padilla-Parra (2017). "Detecting protein aggregation and interaction in live cells: A guide to number and brightness." Methods.

Iliopoulou, M., R. Nolan, L. Alvarez, Y. Watanabe, C. A. Coomer, G. M. Jakobsdottir, T. A. Bowden and S. Padilla-Parra (2018). "A dynamic three-step mechanism drives the HIV-1 pre-fusion reaction." Nat Struct Mol Biol 25(9): 814-822.

Jakobsdottir, G. M., M. Iliopoulou, R. Nolan, L. Alvarez, A. A. Compton and S. Padilla-Parra (2017). "On the Whereabouts of HIV-1 Cellular Entry and Its Fusion Ports." Trends Mol Med 23(10): 932-944.