Cell‐cycle‐dependent changes in labelling of specific phosphoproteins by the monoclonal antibody MPM‐2 in plant cells

Abstract

The MPM‐2 antibody, which recognizes a mitosis‐specific phosphorylated epitope, has been used to study cell‐cycle‐related proteins in partially synchronized cell suspension cultures and root meristem cells. Immunofluorescence revealed that the epitope recognized by MPM‐2 is located in the nucleus in interphase cells. In mitotic cells, MPM‐2 labels the prophase nucleus, the spindle and some cytoplasmic components. The relative amount of the epitope changes significantly during the cell cycle. Labelling is lowest in G1 and S‐phase cells and increases 2–3‐fold during G2. Prophase and metaphase show four to five times the labelling of G1 cells. Labelling decreases rapidly after metaphase and is at a very low level by telophase. One‐ (1‐D) and two‐dimensional (2‐D) immunoblots showed that MPM‐2 labels a family of phosphorylated proteins. The labelling shows significant cell cycle dependence. Subfractionation shows at least one of these proteins is a component of the detergent‐insoluble cytoskeleton cell fraction. This component is resolved on 2‐D immunoblots to two to three spots of slightly different isoelectric point, possibly charge isomers, at a relative molecular mass of approximately 65 kDa. The same spots are labelled by IFA, an antibody against intermediate filament proteins. Another three of the spots at lower relative molecular mass are labelled on 2‐D immunoblots of the nuclear matrix fraction.