Double immunofluorescence labeling of calmodulin and tubulin in dividing plant cells

Using rabbit antibodies to purified spinach calmodulin and monoclonal anti-tubulin, we have investigated via immunofluorescence microscopy the distribution of calmodulin relative to that of tubulin at all stages of the cell cycle in onion and pea root meristematic cells. Calmodulin is associated with the mitotic spindle and with the phragmoplast at cytokinesis, but patterns of calmodulin that coincide with those of interphase microtubule arrays have not been demonstrated with any of the several fixation and processing regimes tested. Calmodulin is not likely to be involved in regulating the formation of the preprophase band of microtubules or its disappearance during spindle formation because a localized cortical band of calmodulin is only very rarely seen in cells at these stages. Prolonged incubation with the calmodulin antagonist calmidazolium has variable effects on calmodulin localization patterns, sometimes reducing immunofluorescence associated with spindles, but not altering phragmoplast patterns. Compound 48/80, another calmodulin inhibitor, appears to inhibit division, and when mitotic spindles and phragmoplasts are found again after a 1-2 day continuous exposure to the drug, some are labeled with anti-calmodulin only very weakly or not at, all. Material treated briefly with 48/80 both before and during fixation likewise shows some reduction in the numbers of dividing cells displaying localization of calmodulin and in the intensity of localized staining.