Fig. 6.
Analyses of the nucleoplasmic mobility of wild-type, phosphomimetic and phosphorylation-deficient lamin A by fluorescence correlation spectroscopy. The mobilities of wild-type (WT) GFP–lamin A and the double mutants S22D S392D, S22D S392A, S22A S392A and S22A S392D in the nucleoplasm of HeLa cells were determined by fluorescence correlation spectroscopy analyses. (A) A representative fluorescence image of WT GFP-lamin A during fluorescence correlation spectroscopy measurements. The white crosses indicated the points where fluorescence correlation spectroscopy measurements were obtained. Scale bar: 10 µm. (B) The mobility of each lamin A protein compared with that of the WT, measured by using fluorescence correlation spectroscopy. The fluctuating fluorescence signals for all of these proteins were plotted. (C) Autocorrelation curves were calculated from the fluorescence intensities and then plotted (see Materials and Methods). Normalized G(τ) means that for comparison of the mobility all curves were normalized to the amplitude G = 2 at 0 µs. Pack: all of which was normalized to the same amplitude of G (0) = 2 for a comparison of the mobility. (D) Diffusion coefficients in slow (Dslow) and fast (Dfast) fractions were obtained from the fluorescence correlation spectroscopy data (see Materials and Methods). The mobilities of GFP–lamin A S22D S392D, S22D S392A and S22A S392D were significantly faster than that of WT GFP–lamin A. Error bars represent the s.e.m. *P<0.005 compared with that of WT GFP–lamin A.