The coding region was PCR amplified with extended primers that added 5 TEV protease and NheI sites and 3 NotI sites for cloning into pET-28a+ vector (Novagen). propose a model in which the Myo10CWee1 interaction coordinates attainment of spindle position and orientation with anaphase onset. Introduction Mitotic spindles in epithelia typically attain a characteristic position and orientation before anaphase (Baena-Lpez et al., 2005; Lechler and Fuchs, 2005; da Silva and Vincent, 2007; Mao et al., 2011). In the most common example, symmetric division, the spindle is positioned in the approximate middle of the xCy IL1R2 plane and is oriented parallel to the epithelial layer (Gillies and Cabernard, 2011; Morin and Bella?che, 2011; Bergstralh et al., 2017). This ensures that cytokinesis, which divides the cell between the separating chromosomes, maintains epithelial architecture by directing formation of two equal-sized daughter cells in the plane of the epithelium. It is now clear that the spindle achieves its final position and orientation during symmetric division via a combination of cytoskeletal motor-dependent movement and cortical anchoring complexes (Woolner and Papalopulu, 2012; Kiyomitsu and Cheeseman, 2013; di Pietro et al., 2016). It is also clear that failure of proper symmetric positioning results in a variety of pathological consequences, including disrupted tissue architecture and promotion of metastasis (Vasiliev et al., 2004; Fish et al., 2006; Quyn et al., 2010). What remains unclear is whether or how epithelial cells link spindle position to cell cycle progression. In principle, such a mechanism may be unnecessary if the achievement of metaphase takes longer than spindle positioning and if both occur simultaneously. However, in a variety of intact epithelia, spindle positioning and orientation do not commence until after metaphase and, further, the interval from metaphase to anaphase can be many minutes (Adams, 1996; Haydar et al., 2003; Woolner et al., 2008; Peyre et al., 2011; Larson and Bement, 2017), suggesting that epithelial cells can delay anaphase until the spindle has achieved the correct position and orientation. Consistent with this hypothesis, automated analysis of mitotic dynamics in 100 embryonic epithelial cells revealed that spindles execute a stereotyped, two-part dance after achieving metaphase. First, spindles undergo a slow rotational movement until they are parallel to the long axis of the cell; second, they undergo rapid oscillatory movements to and from the cortex, which culminate in KU-60019 xCy plane centering (Larson and Bement, 2017). Strikingly, anaphase onset is temporally correlated with on target cortical contacts by the spindle KU-60019 poles (i.e., contact KU-60019 with cortical positions on the same axis as that defined by the final orientation of the spindle). Based on these results, it was proposed that the spindle dance is part of a mechanism that epithelial cells use to link mitotic progression to proper spindle positioning and orientation. Myosin-10 (Myo10), a microtubule-binding, actin-based motor protein that has been previously implicated in spindle dynamics and mitotic progression in embryonic epithelia, is a strong candidate contributor to the mechanism suggested above (see previous paragraph). Depletion of Myo10 results in spindle lengthening, pole fragmentation, and metaphase delay (i.e., an increase in the amount of time the cells spend between metaphase and anaphase; Woolner et al., 2008), whereas dominant-negative expression of the isolated Myo10 MyTH4 domain, which mediates Myo10s interaction with microtubules (Hirano et al., 2011), produces only some of these phenotypes. Specifically, whereas a high level MyTH4 expression results in pole fragmentation and a metaphase delay, moderate expression produces only the delay (Sandquist et al., 2016), indicating that this fragment produces more limited phenotypes than Myo10 depletion by competing with endogenous Myo10 for binding to some unidentified target. This target is not, apparently, microtubules in that expression of the MyTH4-DD mutant, which is deficient in microtubule binding (Hirano et al., 2011), is at least as efficient in causing metaphase delay as wild-type MyTH4 and is apparently more specific in so doing because it does not result in spindle pole fragmentation, even at higher expression levels (Sandquist et al., 2016). Here we identify Wee1, a.