Diffusion coefficient of fluorescein-labeled tubulin in the cytoplasm of embryonic cells of a sea urchin: video image analysis of fluorescence redistribution after photobleaching.
The diffusion coefficient of tubulin has been measured in the cytoplasm of eggs and embryos of the sea urchin Lytechinus variegatus. We have used brain tubulin, conjugated to dichlorotriazinyl-aminofluorescein, to inject eggs and embryos. The resulting distributions of fluorescence were perturbed by bleaching with a microbeam of light from the 488-nm line of an argon ion laser. Fluorescence redistribution after photobleaching was monitored with a sensitive video camera and photography of the television-generated image. With standard photometric methods, we have calibrated this recording system and measured the rates of fluorescence redistribution for tubulin, conjugated to dichlorotriazinyl-aminofluorescein, not incorporated into the mitotic spindle. The diffusion coefficient (D) was calculated from these data using Fick's second law of diffusion and a digital method for analysis of the photometric curves. We have tested our method by determining D for bovine serum albumin (BSA) under conditions where the value is already known and by measuring D for fluorescein-labeled BSA in sea urchin eggs with a standard apparatus for monitoring fluorescence redistribution after photobleaching. The values agree to within experimental error. Dcytoplasmtubulin = 5.9 +/- 2.2 X 10(-8) cm2/s; DcytoplasmBSA = 8.6 +/- 2.0 X 10(-8) cm2/s. Because DH2OBSA = 68 X 10(-8) cm2/s, these data suggest that the viscosity of sea urchin cytoplasm for protein is about eight times that of water and that most of the tubulin of the sea urchin cytoplasm exists as a dimer or small oligomer, which is unbound to structures that would impede its diffusion. Values and limitations of our method are discussed, and we draw attention to both the variations in D for single proteins in different cells and the importance of D for the upper limit to the rates of polymerization reactions.
D000818:Animals; D002417:Cattle; D004058:Diffusion; D005260:Female; D005452:Fluoresceins / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D007700:Kinetics; D008027:Light; D008868:Microtubule Proteins / Q000378:metabolism; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D012617:Sea Urchins / Q000196:embryology*; D014404:Tubulin / Q000378:metabolism* / Q000528:radiation effects
D005452:Fluoresceins; D005456:Fluorescent Dyes; D008868:Microtubule Proteins; D014404:Tubulin; C039083:5-((4,6-dichloro-1,3,5-triazin-2-yl)amino)fluorescein