5 C). recruitment. Thus, Plk1 activity negatively regulates Cep55 to ensure orderly abscission factor recruitment and ensures that this occurs only once cell contraction has completed. Introduction Polo-like kinase 1 (Plk1) is a conserved protein kinase controlling many of the key events of mitosis and cytokinesis (Barr et al., 2004). In prometaphase and metaphase, it is found on centrosomes USL311 and kinetochores where it promotes formation of a bipolar mitotic spindle (Lane and Nigg, 1996; Liu and Erikson, 2002; USL311 Sumara et al., 2004). Subsequently, it relocates to the central spindle in anaphase and activates Rho-GTPase regulators, initiating cleavage furrow formation and cell contractility (Golsteyn et al., 1995; Brennan et al., UCHL2 2007; Burkard et al., 2007, 2009; Petronczki et al., 2007; Santamaria et al., 2007; Wolfe et al., 2009). A conserved phosphopeptide-binding domain, the Polo-box domain, is responsible for this USL311 complex pattern of spatial and temporal control (Cheng et al., 2003; Elia et al., 2003a,b). Through this domain, Polo kinases bind to phosphorylated partners containing a conserved motif (Elia et al., 2003a). This leads to concentration at particular sites and increased levels of kinase activity. According to the prevailing model, Cdk1Ccyclin B creates these docking sites in prophase and metaphase (Cheng et al., 2003; Elia et al., 2003a,b), whereas in anaphase, self-priming by Plk1 itself predominates (Neef et al., 2007; Burkard et al., 2009). In anaphase, Plk1 self-primes a docking site at the C terminus of the anaphase spindle and microtubule-associated protein PRC1 on T602 and is then recruited to the central spindle (Neef et al., 2007). Consistent with these mitosis-specific functions, Plk1 is slowly degraded as cells exit mitosis and is essentially absent by the time cells undergo cytokinesis (Golsteyn et al., 1994, 1995; Lindon and Pines, 2004). The anaphase-promoting complex/cyclosomeCCdh1 ubiquitin ligase is required for Plk1 degradation (Lindon and Pines, 2004). Anaphase-promoting complex/cyclosomeCCdh1 recognizes Plk1 as a target for ubiquitylation through a conserved destruction box (D-box) motif and, thus, earmarks it for proteasomal degradation (Lindon and Pines, 2004). However, although degradation of Plk1 as animal cells exit mitosis is clearly important in the control of mitotic exit and cytokinesis (Lindon and Pines, 2004), the mechanistic detail of its function at this time remains unclear. Cytokinesis is terminated by a membrane-remodeling and fission event termed abscission that is mediated by the ESCRT-III membraneCremodeling proteins (Carlton and Martin-Serrano, 2007), which is an ancestral part of the cytokinesis machinery shared with Archaea (Samson et al., 2008). In dividing mammalian cells, ESCRT-III is specifically nucleated on the surface of the preassembled midbody by the Cep55 adaptor protein. Cep55 directly interacts with the central MKlp1 component of the midbody and the ESCRT proteins ALIX and TSG101 (Fabbro USL311 et al., 2005; Martinez-Garay et al., 2006; Zhao et al., 2006; Carlton and Martin-Serrano, 2007; Morita et al., 2007; Carlton et al., 2008; Lee et al., 2008). This series of events, whereby abscission components are recruited only after midbody formation when the plasma membrane is pulled down to a narrow 0.5-m-diameter tube of membrane connecting the two daughter cells, may be caused by the limitations of the membrane-remodeling properties of ESCRT-III (Wollert et al., 2009). At earlier times, when the plasma membrane is less closely apposed and has a larger radius of curvature, ESCRT-III cannot promote the membrane-remodeling event leading to abscission. In addition, other membrane delivery and microtubule-remodeling events have to occur before abscission can be triggered (Kouranti et al., 2006; Barr and Gruneberg, 2007; Simon et al., 2008). Thus, timing is critical for efficient abscission, yet it remains unclear how this is achieved. Results and discussion Identification of Plk1-sensitive midbody components Previous studies have indicated that Plk1 has unexplained functions in the late stages of cytokinesis (Lindon and Pines, 2004; Santamaria et al., 2007). To confirm this idea, the rapid-acting Plk1 inhibitor BI2536 was used (Lnrt et al., 2007; Steegmaier et al., 2007). When Plk1 was inhibited as chromosome segregation initiated in early anaphase, cleavage furrow formation failed (Fig. 1.