Characterization of Monoclonal Antibodies Against the Mitotic Apparatus Associated 51-kD Protein and the Effect of the Microinjection on Cell Division in Sand Dollar Eggs

Abstract

We prepared monoclonal antibodies against the protein fraction capable of reassembling into microtubule organizing granules (MTOGs) from eggs of sea urchin, Hemicentrotus pulcherrimus. Immunoblot analysis presented evidence that the antigen to the monoclonal antibodies corresponded to the mitotic apparatus associated 51-kD protein which has been shown to be involved in the aster forming activity in vitro. Indirect immunofluorescence staining of mitotic eggs showed that the monoclonal antibodies stained the centrosomal region, basal regions of asters and half spindles in the same fluorescence pattern stained by a polyclonal antibody to the 51-kD protein. Some of the monoclonal antibodies cross-reacted with mitotic apparatus (MA) proteins of several other kinds of sea urchins. The immunoreactive proteins had molecular weights close to that of the 51-kD protein of Hemicentrotus pulcherrimus. Localization of the immunoreactive 52-kD antigen in Clypeaster japonicus MAs was generally the same as that of the 51-kD protein in Hemicentrotus pulcherrimus MAs. When one of monoclonal antibodies was injected into Clypeaster japonicus eggs before prophase, the formation of the MA was not observed, thereby no cleavage occurred. When injected at prometaphase, the spindle formed was shorter than the control, causing failure of nuclear as well as cell division. These results suggested that the 52-kD antigen in Clypeaster japonicus eggs is prerequisite for the formation of a functional spindle.