Kinesin-like Protein CHO1 Is Required for the Formation of Midbody Matrix and the Completion of Cytokinesis in Mammalian Cells

Abstract

CHO1 is a mammalian kinesin-like motor protein of the MKLP1 subfamily. It associates with the spindle midzone during anaphase and concentrates to a midbody matrix during cytokinesis. CHO1 was originally implicated in karyokinesis, but the invertebrate homologues of CHO1 were shown to function in the midzone formation and cytokinesis. To analyze the role of the protein in mammalian cells, we mutated the ATP-binding site of CHO1 and expressed it in CHO cells. Mutant protein (CHO1F') was able to interact with microtubules via ATP-independent microtubule-binding site(s) but failed to accumulate at the midline of the central spindle and affected the localization of endogenous CHO1. Although the segregation of chromosomes, the bundling of midzone microtubules, and the initiation of cytokinesis proceeded normally in CHO1F'-expressing cells, the completion of cytokinesis was inhibited. Daughter cells were frequently entering interphase while connected by a microtubule-containing cytoplasmic bridge from which the dense midbody matrix was missing. Depletion of endogenous CHO1 via RNA-mediated interference also affected the formation of midbody matrix in dividing cells, caused the disorganization of midzone microtubules, and resulted in abortive cytokinesis. Thus, CHO1 may not be required for karyokinesis, but it is essential for the proper midzone/midbody formation and cytokinesis in mammalian cells.

Cytokinesis microtubule organisers at a glance

“…In mitotic cells, the majority of MKLP1 and CYK4 are in the centralspindlin complex and there is no clear evidence for the presence of free components. The motor domain of MKLP1 possesses the microtubule plus-enddirected motor activity (Hizlan et al, 2006;Mishima et al, 2004;Nislow et al, 1992), which is essential for proper microtubule bundle formation and deposition of the midbody matrix (Matuliene and Kuriyama, 2002). The neck region of MKLP1, which links the motor domain to a relatively short coiledcoil stalk domain, is unusually long and contains the binding site for CYK4 (Mishima et al, 2002;PavicicKaltenbrunner et al, 2007;Somers and Saint, 2003).…”

Role of the Midbody Matrix in Cytokinesis: RNAi and Genetic Rescue Analysis of the Mammalian Motor Protein CHO1

“…The homologues of MKLP1/CHO1 studied thus far function in cytokinesis in diverse organisms (Adams et al, 1998;Powers et al, 1998;Raich et al, 1998;Chen et al, 2002;Matuliene and Kuriyama, 2002). However, there is some controversy regarding the stage of cytokinesis in which these proteins are involved.…”

“…The mechanochemical motor domain covering amino acids 1-423 might also be important for formation of the midbody matrix by bundling midzonal microtubules. This could be facilitated by the ATP-independent microtubule-binding site located at the C-terminal region of the CHO1 motor domain at the N terminus (amino acids 277-421; Matuliene and Kuriyama, 2002). Because the stalk domain facilitates dimerization of CHO1, the motorstalk construct can have multiple microtubule-binding sites, allowing the protein to bundle microtubules.…”

“…The midbody matrix has been shown to be a stable structure (Mullins and McIntosh, 1982) important in gluing microtubules together and stabilizing them at the midbody (Salmon et al, 1976). As disorganization of the midbody matrix closely correlates with regression of cleavage furrows (King and Akai, 1971;Mullins and Biesele, 1977;Matuliene and Kuriyama, 2002), the midbody matrix has been implicated in completion of cytokinesis. However, the molecular structure and function of the midbody matrix have not yet been elucidated.…”

“…However, CHO1 represents a unique isotype, in which an actin-binding sequence encoded by exon 18 is included in the middle of the tail domain . Members of the MKLP1 subfamily (Miki et al, 2001) have been shown to function in cytokinesis in different species (Adams et al, 1998;Powers et al, 1998;Raich et al, 1998;Chen et al, 2002;Matuliene and Kuriyama, 2002). In humans and C. elegans, the motor proteins interact with a GAP for Rho family GTPases, which facilitates microtubule bundling and formation of the spindle midzone and midbody (Mishima et al, 2002).…”