Investigating interactions mediated by the presynaptic protein bassoon in living cells by Foerster's resonance energy transfer and fluorescence lifetime imaging microscopy.

Neuronal synapses are highly specialized structures for communication between nerve cells. Knowledge about their molecular organization and dynamics is still incomplete. The large multidomain protein Bassoon plays a major role in scaffolding and organizing the cytomatrix at the active zone of neurotransmitter release in presynaptic boutons. Utilizing immunofluorescence techniques, we show that Bassoon is essential for corecruitment of its synaptic interaction partners, C-terminal binding protein 1/brefeldin A-dependent ADP-ribosylation substrate and CAZ-associated structural protein, into protein complexes upon heterologous expression in COS-7 cells. A combination of Foerster's resonance energy transfer and fluorescence lifetime imaging microscopy in the time domain was adopted to investigate the potential for the association of these proteins in the same complexes. A direct physical association between Bassoon and CtBP1 could also be observed at synapses of living hippocampal neurons. Simultaneous analysis of fluorescence decays of the donor and the acceptor probes along with their decay-associated spectra allowed a clear discrimination of energy transfer.

FRET-FLIM at nanometer spectral resolution from living cells.

We report the investigation of Foerster's Resonance Energy Transfer dynamics in GFP based tandem constructs in living T-cells using a combination of Fluorescence Lifetime Imaging Microscopy (FLIM) and Fluorescence Lifetime Micro-Spectroscopy (FLMS) at picosecond time resolution and nanometer spectral resolution. The involvement of multiple lifetimes of CFP in energy transfer was analyzed by plotting pre-exponential factors of individual lifetimes along the wavelength resulting in the Decay Associated Spectra (DAS). A change in the amplitude of pre-exponential factors from positive to negative at the acceptor emission maxima was used as a confirmation of FRET in the multiexponential lifetime analysis.

Photophysics of Clomeleon by FLIM: discriminating excited state reactions along neuronal development.

In this work, fluorescence lifetime imaging microscopy in the time domain was used to study the fluorescence dynamics of ECFP and of the ratiometric chloride sensor Clomeleon along neuronal development. The multiexponential analysis of fluorophores combined with the study of the contributions of the individual lifetimes (decay-associated spectra) was used to discriminate the presence of energy transfer from other excited state reactions. A characteristic change of sign of the pre-exponential factors of lifetimes from positive to negative near the acceptor emission maxima was observed in presence of energy transfer. By fluorescence lifetime imaging microscopy, we could show that the individual conformations of CFP display differential quenching properties depending on their microenvironment. Suitability of Clomeleon as an optical indicator to obtain a direct readout of the intracellular chloride concentrations in living cells was verified by steady-state and time-resolved spectroscopy. The simultaneous study of the photophysical properties of Clomeleon, the calcium indicator Cameleon, and ECFP with neuronal development provided a kinetic model for the mechanism when competitive quenching effects as well as energy transfer occur in the same molecule. Simultaneous analysis of donor and acceptor kinetics was necessary to discriminate Försters resonance energy transfer along neuronal development due to the different cellular effects involved.