After prolonged expression, however, the epidermal progenitors return to dividing along the tissue polarity axis, a scheme in which mInsc and NuMA no longer co-localize. These results indicate that Insc and Mud can be decoupled from one another. We have examined the effect of Insc-Pins complex formation both in an induced polarity Enoxacin hydrate spindle orientation assay and in in vitro binding assays. Our results indicate that Insc plays a more active role in spindle positioning than previously appreciated. Rather than passively coupling polarity and spindle positioning systems, Insc acts to regulate the activity of downstream Pins pathways. We have shown that the Dlg pathway is unaffected by Inscuteable expression while the Mud pathway is inhibited by Insc binding. Recent work on the mammalian versions of these proteins explains the structural mechanism for competition between the Insc-Pins and Pins-Mud complexes. The binding sites on Pins for these two proteins overlap making binding mutually exclusive because of steric considerations. The observation of Insc dissociation of the Pins-Mud complex in Drosophila and mammalian proteins suggests that Insc regulation of Mud-binding is a highly conserved behavior. This competition between Mud and Insc for Pins binding is consistent with previous work done with a chimeric version of Inscuteable/Pins. This protein, in which the Pins TPR domain was replaced with the Inscuteable Ankyrin-repeat domain, bypasses the Insc-Pins recruitment step of apical complex formation. In these cells, the chimeric Insc-Pins protein was able to rescue apical/basal polarity and spindle orientation in metaphase pins mutant neuroblasts. As this protein lacks the Mud-binding TPR domain, Mud binding to Pins is not absolutely 11-hydroxy-sugiol necessary for spindle alignment. Importantly, the PinsLINKER domain is still intact in the Insc-Pins fusion, implying that Dlg, not Mud, function is sufficient for partial activity, as observed in the S2 system. The Mud and Dlg pathways may play distinct roles in spindle positioning. The Dlg pathway, through the activity of the plus-end directed motor Khc73, may function to attach the cortex to the spindle through contacts with astral microtubules. In contrast, the Mud pathway, through the minus-end directed Dynein/ Dynactin generates force to draw the centrosome towards the center of the cortical crescent.