Early stage researcher 11 (ESR11) project – Ali Isibilir
Supervision: Prof MJ Lohse, Dr D Calebiro, Dr T Sungkaworn, Prof C Hoffmann
Host: UWUE (DE) – University of Würzburg, Institute of Pharmacology and Toxicology and Rudolf Virchow Center

I- Project proposal:

Aim:
1. Determine localization and trafficking of CXCR4 and CXCR7 in response to chemokines using high-resolution microscopy.
2. Determine the effect of CXCR4 and CXCR7 heteromerization on localization and trafficking
3. Evaluate the effect of phosphorylation barcoding and intracellular partners on CXCR4, CXCR7, and heteromer localization and trafficking.

Methodology:
We have pioneered single molecule microscopy in the analysis of GPCRs using fluorescently labeled receptors and total internal reflection fluorescence (TIRF) microscopy. With these methods, we can actually see receptors how they move at the cell surface and how they interact with each other and with other proteins. Analysis of receptor movements can then be done by single particle tracking. This allows the determination of mobility, subcellular localization as well as aggregation and transient interactions of cell surface proteins. In this project, we will apply these technologies to chemokine receptors in order to investigate their behaviour and interactions at the cell surface, and to study how regulatory mechanisms influence the receptors’ behavior.
Planned secondments: CNRS (FR), UNOTT (UK).

II- Requirement candidate:

Required diploma: MSc degree in life or natural sciences (e.g. biophysics, biochemistry, biology, pharmacy)
Required expertise: Molecular biology, cell culture, microscopy
Recommended expertise: Fluorescence microscopy, basic biophysics courses, interest in mathematics and basic programming

Key publications:
1. Lohse MJ, Nuber S, Hofmann C (2012) Fluorescence/bioluminescence resonance energy transfer techniques to study G-protein-coupled receptor activation and signaling. Pharmacol Rev. 64, 299–336.

2. Emami-Nemini A, Roux T, Leblay M, Bourrier E, Lamarque L, Trinquet E, Lohse MJ (2013) Time-resolved fluorescence ligand binding for G-protein-coupled receptors. Nature Prot. 8, 1307-1320.

3. Calebiro D, Rieken F, Wagner J, Sungkaworn T, Zabel U, Borzi A, Cocucci E, Zürn A, Lohse MJ (2013) Single-molecule analysis of fluorescently labeled GPCRs reveals receptor-specific complexes with distinct dynamics and organization. Proc. Natl. Acad. Sci. USA 110, 743–748.

4. Sungkaworn T, Rieken F, Lohse MJ, Calebiro D (2014) High-resolution spatiotemporal analysis of receptor dynamics by single-molecule fluorescence microscopy. J. Vis. Exp. 2014, 89.