Berlin 2008 – scientific programme

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BP: Fachverband Biologische Physik

BP 26: Posters II

BP 26.32: Poster

Thursday, February 28, 2008, 17:00–19:30, Poster A

Live imaging of signalling complexes using pulsed dual-colour microscopy — •Steffen Steinert¹, Felix Neugart¹, Andrea Zappe¹, Lutz Graeve², Carsten Tietz¹, and Jörg Wrachtrup¹ — ¹University Stuttgart — ²University Hohenheim

Communication between cells is mediated via messenger molecules which bind to a receptor that eventually induces oligomerisation with other molecules and transduce a certain signal. The CNTF receptor is a GPI-anchored protein involved in many signalling pathways which control apoptosis, differentiation and other key cellular functions. In order to gain information about the characteristics of the CNTF receptor at the membrane as well as its internal trafficking, colocalisation analysis with other distinct markers is required. For that purpose we set up a widefield and confocal microscope with pulsed dual-colour excitation which enables us to track two different proteins without crosstalk between the fluorophores. The high sensitivity and adjustable temporal resolution in ms-range of the widefield system facilitates a rapid image acquisition even on single-molecule level. Due to the simultaneous detection of the emission of both fluorophores, FRET signals are detected automatically. FCCS is used to study potential interactions at almost native protein concentrations. It was shown that gp130, an important glycoprotein which is known to associate with CNTF-receptor upon stimulation, and CNTF receptor have comparable diffusion constants in the range of 10-9cm*/s. However, FCCS revealed that both proteins are not pre-associated in the membrane, but both become part of the signalling complex after stimulation.