Calmodulin is uniformly distributed during cell division in living stamen hair cells ofTradescantia virginiana

Summary Because the activity of calmodulin (CaM) may be dependent upon its structural distribution, we have examined its spatial localization in living cells. We have focused on cell division and cell plate formation, where conventional immunofluorescence studies report that CaM is specifically associated with microtubules (MTs) of the spindle and the phragmoplast. In dividing stamen hair cells ofTradescantia virginiana that were injected with fluorescently labeled CaM and examined by confocal laser scanning microscopy (CLSM), we found that the labeled protein is uniformly distributed throughout the cell and is not localized with the phragmoplast MTs or any other obvious structure. To explore why these images from live cells differ from those prepared by immunolabeling, we investigated the fate of CaM during fixation and compared it with the localization of fixable dextran and tubulin. The results show that fixation causes severe changes in cell morphology and in the distribution of CaM and dextran in three quarters of the cells. Conversely, injected rhodamine-tubulin did not show redistribution after fixation. We conclude that in the live cell, CaM is largely uniformly distributed throughout the cytoplasm, and secondly that conventional chemical fixation does not preserve CaM, and probably many other soluble proteins, in its in vivo distribution. The role postulated for CaM in mitosis, solely based on indirect immunofluorescence microscopy, has to be re-evaluated.Abbreviations BSA bovine serum albumin - CaM calmodulin - CLSM confocal laser scanning microscopy - Cy3 indocarbocyanine - EDTA ethylenediamine-tetraacetic acid - EGTA ethylene glycol bis (-aminoethyl ether)-N,N,NN-tetraacetic acid - FITC fluoresceinisothiocyanate - IAF 5-iodoacetamido-fluorescein - MT microtubule - PBS phosphate-buffered saline - TBS Tris-buffered saline