Masaru Toriyama

The Mitotic Apparatus with Unusually Many Microtubules from Sea Urchin Eggs Treated by Hexyleneglycol

The effect of hexyleneglycol on the structure of the mitotic apparatus was studied by light and electron microscopies. By treating sea urchin eggs at prometaphase and metaphase with hexyleneglycol, the mitotic apparatus was found to become remarkably decorated with unusually many astral microtubules which were conspicuously uniform in length. These microtubules appeared to be associated with the granular materials which are most likely microtubule initiating sites or microtubule‐organizing centers.

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

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.

Purification of a low molecular weight microtubule binding protein from sea urchin eggs

A low molecular weight microtubule binding protein(SU-MAP34) was purified from sea urchin eggs. This protein bound strongly to the microtubule formed from purified echinoderm tubulin but showed no cross-linking of microtubules. Monospecific antibody against SU-MAP34 was produced and an immunoblotting analysis showed that this protein was not a breakdown product of a protein of a higher molecular mass. Whole cell staining and confocal laser scanning microscope observation showed that SU-MAP34 localized on the filamentous structure of mitotic apparatus and this structure was identified as the microtubule with double staining using anti-SU-MAP34 and anti-tubulin. An immunoblotting experiment showed an enrichment of SU-MAP34 in a microtubule protein fraction prepared using taxol from a crude extract of the cell.

Calcium in Mitosis: Role of 51-kD Protein in the Centrosome of Sea Urchin Egg in Aster Formation

It has been proposed that cytoplasmic free calcium is a regulator of many cellular events during cell cycle. Signal transduction in the trigger of cell multiplication includes transient increase in free calcium through the inosital turnover (Berridge & Irvine, 1984; Rozengurt, 1986) similar to a burst of free calcium observed at fertilization of eggs (Whitaker & Steinhardt, 1985). This is probably followed by a variety of chains of reactions towards replication of genomes and construction of mitotic machineries. Since microtubules which are susceptible to calcium have been shown to be main skeletons of the mitotic apparatus, calcium has been proposed as a regulator in mitosis.