Spindle birefringence of isolated mitotic apparatus: further evidence for two birefringent spindle components

Abstract

The authors studied sea urchin zygote mitotic apparatus (MA) isolated in hexylene glycol, transferred immediately to a glycerol dimethylsulphoxide medium and stored for 2 weeks at room temperature. Treatment with 0.5 M KCl caused loss of 45% of the birefringence, but microtubules remained intact (as seen electron microscopically in glutaraldehyde fixed MA), and tubulin was not extracted (as determined by polyacrylamide gel electrophoresis). These results suggest that a non tubulin component which is extracted by the KCl contributes 45% of the MA birefringence. Further evidence for this conclusion came from indirect immunofluorescence experiments. Non extracted (control) MA were fixed with formaldehyde and reacted with antibody against tubulin: there was intense staining of the spindle fibres and astral rays. Electron microscopically, however, microtubules were not present in formaldehyde fixed MA. Since formaldehyde fixation caused breakdown of microtubules but the tubulin remained in the MA (as judged by reaction with antibodies) the authors suggest that after microtubule breakdown the tubulin remains in the MA because it is bound to a peri microtubule spindle component (which we call 'substance γ'). When KCl extracted MA were fixed with formaldehyde and reacted with antibody against tubulin there was very little staining of spindle fibres and astral rays. Electron microscopically, formaldehyde caused microtubule breakdown, and since the tubulin is lost from formaldehyde fixed, KCl extracted MA (as judged by reaction with antibodies), we suggest that the tubulin binding component, substance γ, is extracted by the 0.5 M KCl. Pressure treatment caused the asters not to stain with antibody against tubulin, suggesting that the stability of substance γ is different in different regions of the mitotic apparatus.