Microtubule organization and the effects of GFP-tubulin expression in Dictyostelium discoideum

We have labeled microtubules in living Dictyostelium amoebae by incorporati on of a GFP-alpha-tubulin fusion protein. The GFP-alpha-tubulin incorporate s into microtubules and, as reported by others [Neujahr et al., 1998], the labeled microtubules are highly motile. Electron microscopy (EM) analysis o f the distribution and organization of microtubules in the amoebae shows th at some cytoplasmic microtubules form close associations. These association s could allow motor proteins attached to one microtubule to walk along an a djacent microtubule and thus generate some of the observed motility. Protei n blot analysis indicates that the GFP-alpha-tubulin incorporates into micr otubules at a lower efficiency than does the endogenous oc-tubulin. EM and immunofluorescence (IF) analyses suggest that the GFP-alpha-tubulin interfe res with microtubule nucleation. We have also observed an increased sensiti vity of the GFP-alpha-tubulin expressing cells to blue light, as compared t o wild-type cells. These results suggest that although GFP-alpha-tubulin ca n be used as a marker for microtubules in living cells, the use of this mar ker is not recommended for certain types of studies such as assembly dynami cs. (C) 2000 Wiley-Liss, Inc.