Studying the mechanism of splicing factors using in-vivo dynamic methods

FCS (Fluorescence Correlation Spectroscopy) is a novel technique, which allows exploring the dynamic properties of proteins inside a living cell. FCS is based on the measurements of the minute spontaneous fluctuations in fluorescence emission of the molecules. These fluctuations result from the random motion of the molecules, while entering and exiting the focal volume that is illuminated through the microscope. We use FCS technique in order to study the dynamic properties of splicing factors in living cells. We found that splicing factors demonstrate existence of two types of diffusion: fast diffusion attributed to free diffusion molecules and slow diffusion attributed to transiently binding molecules. We compare the influence of the splicing factors location in the nucleus (nucleoplasm, active transcription site and speckles – compartments where the splicing factors accumulate) on their ability to diffuse. We found that fast diffusion which indicates the diffusion of free molecule is identical in all nuclear compartments. However, slow diffusion, which indicates the transiently binding molecule, is significantly slower in the speckles compared to nucleoplasm.

Left: Random motion of the molecules via the focal volume that is illuminated through the microscope.
Center: Spontaneous fluctuations in fluorescence emission of the molecules, due to the random motion at the focal volume.
Right: Quantification of this random signal by using the autocorrelation function (ACF- blue points) which could be fitted to an appropriate analytical model (red line) to the ACF curve.

This research is done in collaborate with Yaron Shav-Tal group.