In vitro nucleation of microtubules from microtubule-organizing center prepared from cellular slime mold.

Nucleus associated bodies (NABs) were isolated from Dictyostelium discoideum or Dictyostelium mucoroides and their ability to nucleate microtubules in vitro was examined. NABs were localized at the tapered ends of the nuclei and released from lysed cells in complex with the nuclei. Microtubules radiating from the NAB could also be isolated with the complex under microtubule stabilizing conditions. The ultrastructure of the isolated NAB showed it to be composed of a core structure surrounded by an amorphous matrix. The ability of isolated NABs to nucleate microtubules in vitro was demonstrated by incubation with exogenous brain microtubule protein. Microtubule assembly was easily visualized by dark-field or immunofluorescence microscopy. Polymerization of microtubules seemed to be initiated not from the core structure but from the surrounding matrix. The number of microtubules polymerized from the NAB was directly counted in whole-mount preparations by electron microscopy, which provided a quantitative assay for the NAB activity. The nucleating activity of NAB was quite unstable and its half-life was calculated as about 5 hours. The activity was sensitive to protease digestion and was also temperature sensitive but could be stabilized by addition of glycerol or storage at - 80 degrees C or in liquid nitrogen. These characteristics are analogous to those of the centrosomes in cultured mammalian cells and a possible explanation of their similarity is discussed.