Microtubule organization and the effects of GFP-tubulin expression in dictyostelium discoideum.
We have labeled microtubules in living Dictyostelium amoebae by incorporation of a GFP-alpha-tubulin fusion protein. The GFP-alpha-tubulin incorporates into microtubules and, as reported by others [Neujahr et al., 1998], the labeled microtubules are highly motile. Electron microscopy (EM) analysis of the distribution and organization of microtubules in the amoebae shows that some cytoplasmic microtubules form close associations. These associations could allow motor proteins attached to one microtubule to walk along an adjacent microtubule and thus generate some of the observed motility. Protein blot analysis indicates that the GFP-alpha-tubulin incorporates into microtubules at a lower efficiency than does the endogenous alpha-tubulin. EM and immunofluorescence (IF) analyses suggest that the GFP-alpha-tubulin interferes with microtubule nucleation. We have also observed an increased sensitivity of the GFP-alpha-tubulin expressing cells to blue light, as compared to wild-type cells. These results suggest that although GFP-alpha-tubulin can be used as a marker for microtubules in living cells, the use of this marker is not recommended for certain types of studies such as assembly dynamics.
Kimble
M
M
Department of Biology, University of South Florida, Tampa 33620-5150, USA. mkimble@chuma.cas.usf.edu
Kuzmiak
C
C
McGovern
K N
KN
de Hostos
E L
EL
D000818:Animals; D018929:Cell Culture Techniques; D002465:Cell Movement; D004023:Dictyostelium / Q000378:metabolism* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000494:pharmacology; D008027:Light; D008164:Luminescent Proteins / Q000235:genetics / Q000494:pharmacology*; D008854:Microscopy, Electron; D022101:Microtubule-Organizing Center / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D009068:Movement; D015800:Protozoan Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014404:Tubulin / Q000235:genetics / Q000378:metabolism*
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D049452:Green Fluorescent Proteins