THE ANTI-CENTROSOME MONOCLONAL-ANTIBODY 6C6 REACTS WITH A PLASMA MEMBRANE-ASSOCIATED POLYPEPTIDE OF 77 KDA FROM NICOTIANA-TABACUM POLLEN TUBES

In the pollen and pollen tube of higher plants, the distribution of th e microtubular cytoskeleton has been extensively studied. Even though the pattern of microtubules is known, one of the most remarkable defic iencies is the absence of data on the localization of microtubule-nucl eation sites in the pollen tubes. In order to get insights about the l ocalization of centrosome-like structures in the pollen tube of Nicoti ana tabacam L., we have used the monoclonal antibody 6C6 to search for pericentriolar antigen(s). The antibody was initially raised against a component of animal centrosomes and has been already employed to loc ate centrosomal structures in other plant cell types. By immunoblottin g analysis, a polypeptide of M(r) 77,000 was identified specifically i n the membrane-associated protein fraction of the pollen tube, and is absent from the soluble protein pool. Immunofluorescence observations have shown the polypeptide to be located in the apical part of the pol len tube (about 40-50 mu m from the tip) in association with the corti cal area. A purified plasma membrane fraction from the growing pollen tubes has been obtained, using H+-ATPase activity as an organelle mark er. The plasma membrane fraction was shown to be enriched in the M(r) 77,000 polypeptide, which can be extracted from membranes by treatment with the detergent CHAPS at a concentration of 0.5%. These data open new research perspectives on the localization and analysis of putative cortical microtubule nucleation sites in the pollen tube.