The Coding Region Determinant-Binding Protein (CRDBP)
is a carcinoembryonic
protein, and it is overexpressed in various cancer cells in the form
of granules. We speculated the formation of CRDBP granules possibly
through liquid–liquid phase separation (LLPS) processes due
to the existence of intrinsically disordered regions (IDRs) in CRDBP.
So far, we did not know whether or how phase separation processes
of CRDBP occur in single living cells due to the lack of invivo methods for studying intracellular protein
phase separation. Therefore, to develop an in situ method for studying protein phase separation in living cells is
a very urgent task. In this work, we proposed an efficient method
for studying phase separation behavior of CRDBP in a single living
cell by combining in situ fluorescence correlation
spectroscopy (FCS) and fluorescence cross-correlation spectroscopy
(FCCS) with a fluorescence protein fusion technique. We first predicted
and confirmed that CRDBP has phase separation in solution by conventional
fluorescence imaging and FCS methods. And then, we in situ studied the phase separation behaviors of CRDBP in living cells
and observed three states of CRDBP phase separation such as monomer
state, cluster state, and granule state. We studied the effects of
CRDBP truncated forms and its inhibitor on the CRDBP phase separation.
Furthermore, we discovered the recruitment of CRDBP to β-catenin
protein in living cells and investigated the effects of CRDBP structures
and inhibitor on CRDBP recruitment behavior. This finding may help
us to further understand the mechanism of CRDBP protein for regulating
Wnt signaling pathway. Additionally, our results documented that FCS/FCCS
is an efficient and alternative method for studying protein phase
separation in situ in living cells.