NATIONAL INSITUTE FOR BASIC BIOLOGY

National Institute for Basic Biology

DIVISION OF CELL FUSION

(Adjunct)

Professor:
Issei Mabuchi
Associate Professor:
Hiroshi Abe
Research Associate:
Hirotaka Fujimoto


Cytokinesis in animal and some primitive eukaryotic cells is achieved by the progressive contraction of the cleavage furrow. The cleavage furrow contains a contractile apparatus, called the contractile ring, which is composed of a bundle of actin filaments that lies in the furrow cortex beneath the plasma membrane. It has been established that the contractile ring contracts as the result of interaction between actin filaments and myosin. However, little is known about process of its formation, mechanism that controls its formation, protein constituents, and its ultrastructure. The goal of our research is to solve these problems and thereby clarify the molecular mechanism of cytokinesis. For this purpose, we use three kinds of cells, namely, sea urchin egg, Xenopus egg, and the fission yeast Schizosaccharomyces pombe.

The isolation of cleavage furrows from dividing cells is one of the most important methods for elucidating the molecular mechanism underlying cytokinesis. We hand-isolated furrows from newt eggs and found some unique proteins in the furrow preparation (Mabuchi, Tsukita, Tsukita, and Sawai, Proc. Natl. Acad. Sci. USA 85: 5966-5970, 1988). However, this method has the disadvantage that it is not easy to obtain a sufficient amount of the furrows for analysis of the protein constituents. We have also reported a mass isolation method for cleavage furrows from eggs of the sand dollar, Clypeaster japonicus (Yonemura, Mabuchi, and Tsukita, J. Cell Sci. 100: 73-84, 1991). However, this method has not been applicable to the regular sea urchins.

We have recently developed an isolation method for cleavage furrows by which we were able to isolate furrows from various sea urchin eggs. The contractile ring was included in the isolated cleavage furrows, as seen on rhodamine-phalloidin staining of actin filaments. When the furrows were isolated with the isolation medium containing both NaF and b-glycerophosphate, which are potent protein phosphatase inhibitors, the isolated furrows were found to be accompanied by the mitotic apparatus. When the isolation was carried out without both of them, cleavage furrows without the mitotic apparatus were obtained (Fig. 1).

Fig. 1
Cleavage furrows of Hemicentrotus pulcherrimus eggs isolated with the isolation medium that does not contain protein phosphatase inhibitors. Fluorescence (A) and phase-contrast (B) micrographs of cleavage furrows stained with rhodamine-phalloidin are presented. Bar, 50 µm.


The development of a method of isolation of cleavage furrows from regular sea urchin eggs enabled us to compare protein constituents among furrows from different sea urchin and sand dollar species. We found that 32, 36, 51 kDa proteins were concentrated in common in the cleavage furrows isolated from eggs of the sand dollars, C. japonicus and Scaphechinus mirabilis, and sea urchins, Hemicentrotus pulcherrimus and Strongylocentrotus nudus, on two-dimensional gel electrophoreses. We are currently investigating the nature of these proteins.

Next we investigated the role of myosin in cytokinesis of S. pombe. Recently, it has been shown by Kitayama et al. (J. Cell Biol. 137: 1309-1319, 1997) that Myo2, a type II myosin heavy chain plays a role in the F-actin ring formation in this organism. We found another myosin II called Myo3. Myo3 is the same protein as Myp2 reported independently by Bezanilla et al. (Mol. Biol. Cell, 8: 2693-2705, 1997). Overexpression of Myo3 in the cell leads to formation of aberrant F-actin ring, F-actin cable, and septum. We knocked out the myosin genes in S. pombe. Since Myo3 is not essential, but Myo2 is essential for its growth, we made a myo2myo3 null strain containing pREP81-myo2 to control the expression of myo2. After shut off the myo2, no F-actin ring formation occurs during mitosis. Therefore, type-II myosin is necessary in the formation of the contractile ring in S. pombe.


Publication List:
Fujimoto, H. and Mabuchi, I. (1997) Isolation of cleavage furrows from eggs of regular sea urchins and identification of furrow-specific proteins. J. Biochem. 122, 518-524.

webmaster@nibb.ac.jp
Last Modified: 12:00, May 28, 1998