Twelve of these genes also satisfied the coexpression criterion (Segal 2004 )

Twelve of these genes also satisfied the coexpression criterion (Segal 2004 ). of centralspindlin, as knockdown of centralspindlin abolished the localization of Cep55 to the spindle midzone. Our study defines a cellular mechanism that links centralspindlin to Cep55, which, in turn, controls the midbody structure and membrane fusion at the terminal stage of cytokinesis. INTRODUCTION Cytokinesis, the division of a cell, involves the selection of the cleavage site, the assembly of the contractile ring, the ingression of the cleavage furrow, and finally the abscission of the cell (Glotzer, 2001 ). The initial events of cytokinesis are mediated by the concerted actions of the central spindle/spindle midzone and the contractile ring. At the molecular level, a key regulator is the centralspindlin complex, consisting of the mitotic kinesin MKLP1 and the GTPase-activating protein MgcRacGAP (Mishima 2002 ). At anaphase, centralspindlin, localized to the spindle midzone, interacts with and possibly activates the guanine nucleotide exchange factor, ECT2, which, in turn, activates the small GTPase RhoA to determine the positioning of the cleavage site and to induce the assembly and ingression of the contractile ring (Kamijo 2005 ; Yuce 2005 ; Zhao and Fang, 2005b ). Other regulatory and structural proteins also accumulate and function at the midzone; these include mitotic kinases Aurora B (Terada 1998 ) and Plk1 (Golsteyn 1995 ; Lane and Nigg, 1996 ), a mitotic kinesin MKLP2 (Hill 2000 ; Fontijn 2001 ), and a microtubule-associated protein PRC1 (Jiang 1998 ; Mollinari 2002 ). Phosphorylation of MKLP1 (Guse 2005 Rabbit polyclonal to ZNF564 ; Gruneberg 2006 ) and MgcRacGAP (Minoshima 2003 ) by Aurora B is necessary for the completion of cytokinesis, whereas phosphorylation of ICA MKLP1 (Liu 2004 ) and MKLP2 (Neef 2003 ) by Plk1 also contributes to the regulation of cytokinesis. On the other hand, localization of Aurora B and Plk1 to the spindle midzone requires MKLP2 (Neef 2003 ; Gruneberg 2004 ). The terminal stage of cytokinesis involves cell abscission to generate two separate daughter ICA cells (Glotzer, 2001 ). Although it has been established that both the midbody structure and membrane fusion are essential for cell abscission (Albertson 2005 ; Otegui 2005 ), the biochemical machinery and the cellular processes for abscission remain ill-defined. Several proteins have been shown to function at this terminal stage. PRC1, a microtubule-bundling protein (Mollinari 2002 , 2005 ), MKLP1, a subunit of the centralspindlin complex (Matuliene and Kuriyama, 2002 ), and annexin 11, a calcium-dependent phospholipid-binding protein (Tomas 2004 ), are all required for the integrity of the midbody structure and for the completion of cytokinesis. On the other hand, both t-SNARE syntaxin 2 and v-SNARE endobrevin localize to the midbody and play an essential role at the terminal stage of cytokinesis (Low 2003 ). Centriolin, a coiled-coil protein required for membrane fusion and cell abscission, anchors the membrane-vesicle-tethering exocyst complex at the midbody and controls the localization of syntaxin 2 and endobrevin during cytokinesis (Gromley 2003 , 2005 ). Thus, separation of two daughter cells requires membrane trafficking and fusion (Finger and White, 2002 ; Strickland and Burgess, 2004 ; Albertson 2005 ). To understand the molecular mechanism of cytokinesis, we initiated a functional genomic screen for novel regulators of cytokinesis and identified Cep55 as a protein that controls the terminal stage of cytokinesis. During the preparation of this article, Cep55 was also reported as a centrosomal protein required for cytokinesis, although the mechanism of its function was not clear (Fabbro 2005 ). We reported here the molecular and cellular pathway in which Cep55 acts. We found that Cep55 is required for the establishment and proper function of the midbody structure. In addition, Cep55 facilitates membrane fusion at the terminal stage of cytokinesis. Interestingly, Cep55 directly interacts with MKLP1 and its cellular localization is under the control of centralspindlin. ICA Our study provides a molecular and cellular mechanism that controls the terminal stage of cytokinesis. MATERIALS AND METHODS Gene Expression Analyses Whitfield (2002) performed microarray analysis on genome-wide gene.