Studies of a microtubule-associated protein using a monoclonal antibody elicited against mammalian mitotic spindles.

Abstract

A procedure was devised for the identification of individual molecules which are associated with the mitotic spindle apparatus and cytoskeleton in mammalian cells. Monoclonal antibody-producing hybridomas were prepared by immunizing mice with mitotic spindles isolated from cultured HeLa cells. Among several antibody-producing clones obtained, 1 hybridoma (22MA2) produced an antibody that recognizes a putative microtubule-associated protein which exhibits unusual distribution characteristics in cultured cells. Immunofluorescence studies showed that during mitosis the 22MA2 antigen is distributed in parallel with the spindle fibers of the mitotic apparatus, and that during interphase the antigen is always associated to a limited extent with cytoplasmic microtubules. Also, the co-distribution of the antigen with microtubules was colcemid sensitive. The 22MA2 antibody immunofluorescently stained the nuclei of cells in the exponential growth phase, but did not stain the nuclei of cells that had grown to confluence. This nuclear fluorescence appears to be directly related to cell density rather than nutritional (serum) factors in the growth medium. The results suggest that the antigen undergoes some change in structure or distribution in response to changes in the proliferative capacity of the cell. Biochemical analyses of cytoplasmic, nuclear and mitotic spindle subcellular fractions show that the antigen exhibits a polypeptide MW of 240,000, is found in various mammalian cells ranging from marsupial to human, and is particularly susceptible to proteolysis.