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Masanori Mishima

Publications and source records attributed to Masanori Mishima.

6 recordsLinked to original sources
PubMed2006

Structural analysis of the ZEN-4/CeMKLP1 motor domain and its interaction with microtubules.

The centralspindlin complex is required for the assembly and maintenance of the central spindle during late anaphase and the completion of cytokinesis. It is composed of two copies each of the kinesin-like protein ZEN-4, a Caenorhabditis elegans MKLP-1 (Kinesin-6 family), and the RhoGAP CYK-4. By using cryo-electron microscopy and helical 3D reconstruction, we are investigating the structural features of the interactions between monomeric and dimeric motor domain constructs of ZEN-4 and microtubules. We have calculated helically averaged 3D maps of microtubules decorated with ZEN-4 motor domain in the presence of AMP-PNP, ADP, ADP-AlF(4)(-), and nucleotide-free conditions. We used statistical difference mapping to compare these maps among each other and to related maps obtained from microtubules decorated with a well-characterized Kinesin-1 motor domain from Neurospora crassa. Thereby, we found distinct structural features in microtubule-ZEN-4 complexes that may directly relate to the functional properties of ZEN-4 and centralspindlin. Furthermore, we investigated the location, structure, and function of a highly conserved extension of approximately 50 residues unique to the Kinesin-6 subfamily, located in the motor core loop6/beta4 region.

Adenosine Diphosphate↗
PubMed2005

Phosphorylation of ZEN-4/MKLP1 by aurora B regulates completion of cytokinesis.

The central spindle regulates the formation and positioning of the contractile ring and is essential for completion of cytokinesis [1]. Central spindle assembly begins in early anaphase with the bundling of overlapping, antiparallel, nonkinetochore microtubules [2, 3], and these bundles become compacted and mature into the midbody. Prominent components of the central spindle include aurora B kinase and centralspindlin, a complex containing a Kinesin-6 protein (ZEN-4/MKLP1) and a Rho family GAP (CYK-4/MgcRacGAP) that is essential for central spindle assembly [4]. Centralspindlin localization depends on aurora B kinase [5]. Aurora B concentrates in the midbody and persists between daughter cells. Here, we show that in C. elegans embryos and in cultured human cells, respectively, ZEN-4 and MKLP1 are phosphorylated by aurora B in vitro and in vivo on conserved C-terminal serine residues. In C. elegans embryos, a nonphosphorylatable mutant of ZEN-4 localizes properly but does not efficiently support completion of cytokinesis. In mammalian cells, an inhibitor of aurora kinase acutely attenuates phosphorylation of MKLP1. Inhibition of aurora B in late anaphase causes cytokinesis defects without disrupting the central spindle. These data indicate a conserved role for aurora-B-mediated phosphorylation of ZEN-4/MKLP1 in the completion of cytokinesis.

Animals↗
PubMed2004

Cell cycle regulation of central spindle assembly.

The bipolar mitotic spindle is responsible for segregating sister chromatids at anaphase. Microtubule motor proteins generate spindle bipolarity and enable the spindle to perform mechanical work. A major change in spindle architecture occurs at anaphase onset when central spindle assembly begins. This structure regulates the initiation of cytokinesis and is essential for its completion. Central spindle assembly requires the centralspindlin complex composed of the Caenorhabditis elegans ZEN-4 (mammalian orthologue MKLP1) kinesin-like protein and the Rho family GAP CYK-4 (MgcRacGAP). Here we describe a regulatory mechanism that controls the timing of central spindle assembly. The mitotic kinase Cdk1/cyclin B phosphorylates the motor domain of ZEN-4 on a conserved site within a basic amino-terminal extension characteristic of the MKLP1 subfamily. Phosphorylation by Cdk1 diminishes the motor activity of ZEN-4 by reducing its affinity for microtubules. Preventing Cdk1 phosphorylation of ZEN-4/MKLP1 causes enhanced metaphase spindle localization and defects in chromosome segregation. Thus, phosphoregulation of the motor domain of MKLP1 kinesin ensures that central spindle assembly occurs at the appropriate time in the cell cycle and maintains genomic stability.

Adenosine Triphosphatases↗
PubMed2003

Cytokinesis: a logical GAP.

Two complexes localize to the central spindle and regulate completion of cytokinesis: one, centralspindlin, contains a kinesin-like protein and a Rho-family GAP; the second contains Aurora B kinase. Aurora B kinase is known to regulate localization of centralspindlin and may regulate the activity of the RhoGAP component of centralspindlin.

Amino Acid Sequence↗
PubMed2002

Central spindle assembly and cytokinesis require a kinesin-like protein/RhoGAP complex with microtubule bundling activity.

A late step in cytokinesis requires the central spindle, which forms during anaphase by the bundling of antiparallel nonkinetochore microtubules. Microtubule bundling and completion of cytokinesis require ZEN-4/CeMKLP-1, a kinesin-like protein, and CYK-4, which contains a RhoGAP domain. We show that CYK-4 and ZEN-4 exist in a complex in vivo that can be reconstituted in vitro. The N terminus of CYK-4 binds the central region of ZEN-4, including the neck linker. Genetic suppression data prove the functional significance of this interaction. An analogous complex, containing equimolar amounts of a CYK-4 ortholog and MKLP-1, was purified from mammalian cells. Biochemical studies indicate that this complex, named centralspindlin, is a heterotetramer. Centralspindlin, but not its individual components, strongly promotes microtubule bundling in vitro.

Amino Acid Sequence↗