Tubulin and calmodulin. Effects of microtubule and microfilament inhibitors on localization in the mitotic apparatus.

Abstract

Indirect immunofluorescence was used to determine the distribution of calmodulin in the mitotic apparatus of rat kangaroo PtK2 and Chinese hamster ovary (CHO) cells. The distribution of calmodulin in PtK2 cells was compared to the distribution of tubulin, also as revealed by indirect immunofluorescence. During mitosis, calmodulin was found to be a dynamic component of the mitotic apparatus. Calmodulin first appeared in association with the forming mitotic apparatus during midprophase. In metaphase and anaphase, calmodulin was found between the spindle poles and the chromosomes. While tubulin was found in the interzonal region throughout anaphase, calmodulin appeared in the interzone region only at late anaphase. The interzonal calmodulin of late anaphase condensed during telophase into two small regions, one on each side of the midbody. Calmodulin was not detected in the cleavage furrow. In view of the differences in the localization of calmodulin, tubulin, and actin in the mitotic apparatus, experiments were designed to determine the effects of various antimitotic drugs on calmodulin localization. Cytochalasin B, an inhibitor of actin microfilaments, had no apparent effect on calmodulin or tubulin localization in the mitotic apparatus of CHO cells. Microtubule inhibitors, such as colcemid and N2O, altered the appearance of tubulin- and calmodulin-specific fluorescence in mitotic CHO cells. Cold temperature (0 degrees C) altered tubulin-specific fluorescence of metaphase PtK2 cells but did not alter calmodulin-specific fluorescence. From these studies, it is concluded that calmodulin is more closely associated with the kinetichore-to-pole microtubules than other components of the mitotic apparatus.