Figure 7. Graphical presentation of the data collected during FRAP experiments with farnesylation incompetent lamin Dm mutants in HeLa cells.

Lamin Dm and all pseudophosphorylated mutants except lamin Dm T⁴³⁵E show the same low diffusion rate. Lack of recovery after photobleaching was observed for control human lamins (A, C and B1) (data not shown) as well as D. melanogaster wild type lamin Dm and the majority of lamin Dm mutants (S²⁵E, S45E, S⁵⁹⁵E), excluding lamin Dm T⁴³⁵E. They showed no measurable recovery after several minutes. In contrast, lamin Dm mutant with threonine 435 substituted by glutamic acid to mimic stable, permanent phosphorylation displayed increased dynamics (D = 2.7 µm²/s; Mf = 20%). The presented fluorescence recovery curve shows that Drosophila lamins show similar mobility as human lamins in HeLa cells. Only specific mutation (T⁴³⁵E) can increase protein mobility, indicating lower polymerization ability in vivo. For control experiments we used HeLa cells expressing free EGFP. EGFP expression was seen to be evenly distributed within the nucleus and cytoplasm. Cytoplasmic fraction of EGFP shows a slower recovery (t1/2 = 2.05 seconds) versus that observed in the nucleus (t1/2 = 1.2 seconds).