Mobility & Interaction Measurements
Fluorescence microscopy based techniques are essential tools for measuring the dynamics of molecules in living cells to approach a variety of biological questions. The microscopy techniques applied in our lab can be subdivided into Fluorescence Correlation Spectroscopy, Fluorescence Bleaching Techniques and Single Particle Experiments. A combination of these complementary methods allows for a comprehensive analysis on multiple length and time scales.
The concentration, mobility and interactions of molecules can be measured even in living cells by analyzing intensity fluctuations in the recorded fluorescence signal arising from fluorescent molecules entering and leaving a diffraction-limited focus volume (Figure 1).
Figure 1: Fluorescent molecules diffusing into and out of a diffraction-limited focus volume result in a fluctuating fluorescence signal according to the fluctuating particle number. These fluctuations can be analyzed by calculating so-called correlation curves. The molecules' diffusion coefficient is inversely proportional to the width of the correlation curve and the concentration inversely proportional to the amplitude.
Several modifications of this main principle are applied in our lab to cope with different biological questions. (i) We apply point FCS to measure the mobility of heterochromatin protein HP1 and associated factors at distinct spots in open and dense chromatin regions by conventional Confocal Laser Scanning Microscopes (CLSM) equipped with highly sensitive fluorescence detectors . (ii) The complex formation of differently labeled molecules can be determined by cross-correlating signal fluctuations from spectrally separated detection cannels (two-color FCCS). Two-color FCCS is applied in our lab for measuring the dimerization of different HP1 isoforms. (iii) For efficient mapping of molecule mobility and interactions in the cell  we developed a line-illuminating and -detecting confocal microscope , the Spatial & Temporal Fluctuation Microscope (STFM). Cross-correlation of fluorescence signals from spatially separated detection volumes mapped on a high-sensitive EM-CCD camera allows for measuring translocation rates between different nuclear compartments and observation of diffusion barriers (spatial FCCS).
A fundamentally different approach is bleaching of fluorescent molecules in a region of interest (ROI) and analyzing the fluorescence signal redistribution due to diffusion of unbleached molecules into this area.
Figure 2: For Fluorescence Recovery After Photobleaching (FRAP) experiments the fluorescence is bleached in a region of interest by fast scanning with high illumination intensity. Afterwards, the recovery of the fluorescence signal is recorded and analyzed for extracting mobility, binding rates and immobile molecule fractions.
According to the bleach process and subsequent data evaluation, different approaches can be distinguished. (i) If the fluorescent molecules are bleached at a single spot by continuous moderate illumination, the slowly decreasing fluorescence signal can be analyzed by a technique called Continuous Fluorescence Photo-bleaching (CP). In our lab, CP is applied for measuring slow binding processes of proteins related to heterochromatin formation with a good spatial resolution (1). (ii) The fluorescence in a ROI can be also be bleached by rapidly scanning the area with high illumination intensity and afterwards recording and analyzing the fluorescence signal recovery (Fluorescence Recovery After Photo-bleaching, FRAP) (Figure 2). FRAP is complementary to the correlation techniques, because in addition to freely mobile molecules, strongly bound immobilized fractions can be measured. We use FRAP for measuring slow molecule mobility, binding rates and immobile fractions of heterochromatin protein HP1 or chromatin remodelers , . (iii) By accounting for the pixel-wise bleaching of a ROI the resulting bleach profiles can be evaluated quantitatively for determining the mobility of fast proteins and their binding reactions within a remarkably short measurement time (Pixel-wise Photobleaching Profile Evolution Analysis, 3PEA). We apply 3PEA for measuring the mobility and interactions of chromatin remodelers .
In addition to the ensemble methods described above, we apply Single Particle techniques in our lab to study mobility and interactions on the molecular level. (i) Conventional CLSMs and the STFM can be used for fast tracking the trajectory of single fluorescent particles (Single Particle Tracking, SPT) and analyzing their movement by calculating the mean squared displacement (MSD). We use SPT for studying the mobility of distinct nuclear compartments, like PML bodies, heterochromatin spots and telomeres . (ii) The proximity of appropriately labeled molecules can be measured due to a distance sensitive dipole-dipole interaction between a fluorescence acceptor-donor pair by a technique called Förster Resonance Energy Transfer (FRET). In our lab, FRET is used for measuring the interaction between SUMO and PML proteins in the cell nucleus.
Müller KP, Erdel F, Caudron-Herger M, Marth C, Fodor BD, Richter M, Scaranaro M, Beaudouin J, Wachsmuth M, Rippe K. (2009).
Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy.
Biophys J. 97(11):2876-85.
Erdel F, Müller-Ott K, Baum M, Wachsmuth M, Rippe K. (2011).
Dissecting chromatin interactions in living cells from protein mobility maps.
Chromosome Res. 19(1):99-115.
Heuvelman G, Erdel F, Wachsmuth M, Rippe K. (2009).
Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy.
Eur Biophys J. 38(6):813-28.
Erdel F, Schubert T, Marth C, Längst G, Rippe K. (2010).
Human ISWI chromatin-remodeling complexes sample nucleosomes via transient binding reactions and become immobilized at active sites.
Erdel F, Rippe K. (2012).
Quantifying transient binding of ISWI chromatin remodelers in living cells by pixel-wise photobleaching profile evolution analysis.
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