Fluorescence correlation spectroscopy investigation of a GFP mutant-enhanced cyan fluorescent protein and its tubulin fusion in living cells with two-photon excitation

This study investigates the feasibility of using the enhanced cyan mutant of green fluorescent protein (ECFP) as a probe for two-photon fluorescence correlation spectroscopy (FCS). Molecular dynamics and other properties of ECFP and an ECFP-tubulin fusion protein were investigated in living Potorous tridactylis (PTK2) cells. ECFP has high molecular brightness in the nucleus $(η=3.3 kcpsm)$ and in the cytoplasm (3.2 kcpsm) under our experimental conditions. The diffusion constants of ECFP were determined to be $20±7 μm2/s$ in the nucleus and $21±8 μm2/s$ in the cytoplasm. ECFP has stable molecular characteristics with negligible photobleaching and photodynamic effects in our measurements. At the highest concentration of monomer ECFP (425 nM) the amount of dimer ECFP was estimated to be negligible (∼1.8 nM), consistent with our data analysis using a single species model. ECFP-tubulin has a diffusion constant of 6 μm²/s in the living cells. In addition, we demonstrate that analysis of the molecular brightness can provide a new avenue for studying the polymerization state of tubulin. We suggest that the tubulin in the vicinity of the nucleus exists primarily as a heterodimer subunit while those in the area away from the nucleus $(d>5 μm)$ are mostly oligomers. We conclude that ECFP is a useful genetic fluorescent probe for FCS studies of various cellular processes when in fusion to other biomolecules of interest. © 2004 Society of Photo-Optical Instrumentation Engineers.