The structure and some properties of the isolated mitotic apparatus

Abstract

The morphology of the isolated sea-urchin mitotic apparatus (MA) was examined by light and electron microscopy. With the polarization microscope and the Nomarski differential interference microscope, the isolated MAs appeared to be similar to in vivo MAs. Electron microscopy of the isolated MAs revealed the presence of microtubules, ribosome-like particles and vesicles. A close association between the ribosome-like particles and the MA microtubules resulted in the appearance of chains of particles running along the length of the microtubules. Isolated MAs washed two to three times in isolation medium showed fine-structural changes in the electron microscope, which were reflected by lower retardation values obtained with the polarization microscope. The addition of magnesium and calcium or sucrose to the washing medium prevented these structural changes. Varying the pH of the isolation medium also resulted in changes in birefringence and ultrastructure of unwashed MAs. Isolated MAs stored in the original isolation medium gradually became less birefringent and lost their microtubules. At pH 6·1 and pH 6·2 a residual birefringence was retained, even after several weeks of storage. Electron microscopy of these MAs revealed the presence of linear aggregates of ribosome-like particles oriented parallel to the long axis of the spindle. On the other hand, at pH 6·3 and pH 6·4, MAs lost their birefringence completely, and the ribosome-like particles became more randomly dispersed. 2M sucrose or 0·003 M Mg²⁺ greatly retarded the loss of birefringence in stored MAs. Glutaraldehyde-fixed MAs stained intensely with azure B bromide, demonstrating the presence of RNA. Treatment with RNase resulted in a loss of this staining. RNase-treated MAs examined with the electron microscope, revealed changes in the ribosome-like particles. The results are discussed in the light of recent biochemical analyses of the isolated MA, structural similarities to in situ MAs and the interpretation of the birefringence of the MA.