Live Imaging Approach for Quantitative Characterization of Transcription Factor Properties

Accurate spatiotemporal patterns of gene expression orchestrated by transcription factors (TFs) are fundamental to developmental progression. As such, the knowledge of biophysical properties of transcription factors (TFs) is crucial for understanding the mechanism of gene regulation. While fluorescence intensity, a commonly accessible parameter, gives an idea about relative bulk concentration, it provides an incomplete picture of eukaryotic transcriptional regulation owing to the involvement of a multitude of other factors in the process. The bulk concentration of TFs is composed of populations with distinct diffusivities, such as freely diffusing population with high diffusivity, DNA-bound population with low diffusivity, and population with intermediate diffusivity likely representing mobile clusters; each playing disparate but crucial roles in gene regulation. Biophysical parameters such as the diffusivity, concentrations of the distinct populations, as well as their spatiotemporal dynamics can shed light on mechanisms of TF mediated gene regulation. Here, we provide a detailed workflow spanning sample collection, sample preparation, image acquisition, and image analysis to quantify the aforementioned parameters in live Drosophila embryos using raster image correlation spectroscopy (RICS). The pipeline can be used to characterize the populations with distinct diffusivities and their variations over space and time for any protein of interest tagged with a fluorescent protein. In addition to the cross-correlation between a TF and DNA to characterize the TF-DNA binding, this pipeline can be extended to study protein-protein interactions by the cross-correlation between multiple proteins of interest by making use of spectrally distinct fluorescent tags. This quantitative approach is expected to enhance our mechanistic understanding of protein-based signaling networks.