S4 A), and the centriolar marker Cep135 (Fig. Plk1 that is required for correct mitotic spindle positioning. MISP is an actin-associated protein throughout the cell cycle. MISP depletion led to an impaired metaphase-to-anaphase transition, which depended on phosphorylation by Plk1. Loss of MISP induced mitotic defects including spindle misorientation accompanied by shortened astral microtubules. Furthermore, we find that MISP created a complex with and regulated the cortical distribution of the +TIP binding protein p150glued, a subunit of the dyneinCdynactin complex. We propose that Plk1 phosphorylates MISP, thus stabilizing cortical and astral microtubule attachments required for proper mitotic spindle positioning. Introduction The determination of the correct cell division axis is crucial for organism development and is mediated by the precise positioning of the mitotic spindle (Ahringer, 2003; G?nczy, 2008). External positioning signals are transmitted into the cell via the cell cortex (Thry et al., 2005; Toyoshima and Nishida, 2007) and relayed to the mitotic spindle Naspm trihydrochloride through pulling forces acting on astral microtubules (MTs) attached to cortical structures (Grill et al., 2003). These cortical cues are spatially defined by retraction fibers modulating the positioning of actin regulators and therefore force generation (Thry et al., 2005; Fink et al., 2011). Moreover, astral MTs are engaged with these cortical structures through so-called +TIPs including adenomatous polyposis coli (APC), CLASPs, and the dyneinCdynactin complex, which have been shown to regulate spindle orientation and positioning (OConnell and Wang, 2000; Schuyler and Pellman, 2001; Rogers et al., 2002; Mimori-Kiyosue and Tsukita, 2003; Samora et al., 2011). The cortically localized dyneinCdynactin complex is believed to provide pulling forces on astral MTs and is recruited by heterotrimeric G proteins/LGN/NuMA during spindle positioning in embryos, with homologies to proteins of the AKAP family (Fig. S1 C). To analyze the function of MISP, rabbit polyclonal antibodies were raised against the full-length protein. In Western blots, the antibody recognized Naspm trihydrochloride a major band at the expected molecular weight of 75 kD and a slower migrating band, which were largely reduced in MISP-depleted cells using two different siRNAs (Ol1 and Ol2; Fig. S1 D). To investigate whether MISP protein levels are regulated during the cell cycle, HeLa Naspm trihydrochloride cells were synchronized with a double thymidine block at the G1/S boundary and released for different time points. Cell cycle progression was controlled by Western blot analysis of cell cycle marker proteins and monitored by FACS analysis. As shown in Fig. 1 A, MISP was only weakly expressed in G1 and S phases of synchronized HeLa cells, with increasing protein levels and slower migrating bands appearing stepwise in G2/M phases and persisting until the end of mitosis. The slower migrating form of MISP disappeared in response to -phosphatase treatment, indicating that MISP is a phosphoprotein (Fig. 1 B). Open in a separate window Figure 1. MISP is a mitotic phosphoprotein and interacts with Plk1. (A) HeLa cells were synchronized at the G1/S transition by a double thymidine block/release. Samples were analyzed by Western blot with depicted antibodies. FACS analyses of the DNA content by Naspm trihydrochloride propidium iodide (PI) staining from indicated time points were performed. The experiment was repeated three times. (B) Nocodazole-blocked HeLa cells analyzed with indicated antibodies, with and without -phosphatase (-PPase) Naspm trihydrochloride treatment. (C) Plk1 immunoprecipitated from mitotic HeLa cells was analyzed by Western blot with indicated antibodies. (D) HeLa cells were transfected with Flag or Flag-MISP and arrested in metaphase. Immunoprecipitation was performed using Flag M2 antibodies and were analyzed with indicated antibodies. Asterisk indicates IgGs. (E) Flag-MISP was overexpressed and immunoprecipitated from HeLa cells that were either untreated or arrested in mitosis. Far Western blot (FWB) analysis was performed using GST-PBD of Plk1 and GST-Plk1 Polo box mutant (FAM) and Mouse monoclonal to eNOS probed by Western blot. Mitotic lysates were additionally treated with -PPase. To assess the contribution of Plk1 function to the regulation of MISP during mitosis, we first sought to confirm the interaction between MISP and Plk1. As seen in Fig. 1 C, endogenous MISP was present in Plk1 immunoprecipitates. In addition, interactions between ectopically expressed Flag-MISP and Plk1 could also be detected in vivo (Fig. 1 D). Interestingly, Plk1 was found to bind to the highest phosphorylated form of MISP (Fig. 1 C). Plk1 is known to bind to substrates in a phospho-specific manner via its PBD (Elia et al., 2003a,b). To test this, we made use of a far Western blot assay (Neef et al., 2003). The GST-PBD of Plk1 was able to bind to the highest.