Inscuteable regulates the Pins-Mud spindle orientation pathway

Abstract

During asymmetric cell division, alignment of the mitotic spindle with the cell polarity axis ensures that the cleavage furrow separates fate determinants into distinct daughter cells. The protein Inscuteable (Insc) is thought to link cell polarity and spindle positioning in diverse systems by binding the polarity protein Bazooka (Baz; aka Par-3) and the spindle orienting protein Partner of Inscuteable (Pins; mPins or LGN in mammals). Here we investigate the mechanism of spindle orientation by the Insc-Pins complex. Previously, we defined two Pins spindle orientation pathways: a complex with Mushroom body defect (Mud; NuMA in mammals) is required for full activity, whereas binding to Discs large (Dlg) is sufficient for partial activity. In the current study, we have examined the role of Inscuteable in mediating downstream Pins-mediated spindle orientation pathways. We find that the Insc-Pins complex requires Gαi for partial activity and that the complex specifically recruits Dlg but not Mud. In vitro competition experiments revealed that Insc and Mud compete for binding to the Pins TPR motifs, while Dlg can form a ternary complex with Insc-Pins. Our results suggest that Insc does not passively couple polarity and spindle orientation but preferentially inhibits the Mud pathway, while allowing the Dlg pathway to remain active. Insc-regulated complex assembly may ensure that the spindle is attached to the cortex (via Dlg) before activation of spindle pulling forces by Dynein/Dynactin (via Mud).

Figure 1. Inscuteable-mediated orientation of the mitotic spindle requires Gαi.

a, current model of Inscuteable function. Insc serves as a link between the apical PAR complex and the spindle-orienting Pins-Gái complex. b, Ed-Insc (green) transfected S2 cells randomly orient the mitotic spindle (red) with respect to the region that is enriched in Ed. Spindle alignment is measured by drawing a vector from the center of the crescent (arrow) to the center of the mitotic spindle and then along the axis (dashes). c, Expression of Gái (blue) with Ed-Insc (green)is able to confer moderate spindle orienting activity. d, Cumulative percentage plot of spindle angles measured in the S2 Echinoid induced-polarity assay for Ed-Insc and Ed-Insc+Gái compared to previously-published data . In these plots, the cumulative percentage of cells with a spindle angle below a particular value (x-axis) is shown. High spindle orienting activity corresponds to a deflection to lower spindle angles whereas no activity is a line across the diagonal. e, Ed-Insc expression in S2 cells is sufficient to robustly recruit endogenous Pins from the cytoplasm to the region of Ed enrichment. f, Ed alone is unable to polarize endogenous Pins. e, g, Ed-Insc induces colocalization of endogenous Pins with overexpressed Gai. h, Ed-Pins is able to recruit endogenous Dlg. i, Co-expression of Gαi with Ed-Pins results in robust recruitment of endogenous Mud. j,k Ed-GFP is unable to recruit endogenous Dlg or Mud to the induced-polarity cortical domains. l, Ed-Insc is able to recruit Dlg to the cortex, similar to cells expressing Ed-GFP-Pins. m, Ed-Insc is not able to recruit Mud (red) to the Ed-crescent, even in the presence of Gái. Scale bars for all panels represent 5 µm.

Figure 2. Expression of Inscuteable in cells expressing constitutively-active Pins reduces spindle orientation to Dlg-like levels.

a, Co-expression of Ed-Pins 1–466 (green), which robustly orients the mitotic spindle, with Inscuteable (inset), reduces the levels of spindle orientation in adherent, polarized S2 cells. b, Cumulative percentage plot of spindle angles measured in cells co-expressing Ed-Pins 1–466 and Inscuteable compared to published data .

Figure 3. Inscuteable competes with Mud, but not Dlg, for binding to Pins.

a, GST-pulldowns of Inscuteable with different Pins constructs reveals that Inscuteable binds specifically to constructs containing the full array of Pins TPRs. b, Coimmunoprecipitations of endogenous proteins from wild-type L3 brain extracts demonstrate that Inscuteable and Mud form exclusive complexes with Pins. c, GST-pulldown using GST-Pins TPRs incubated with a constant amount of Mud and increasing MBP-Insc reveals effective competition between Mud & Insc for binding to Pins. d, A control titrations of MBP alone do not result in dissociation of Mud from GST-Pins. e, GST-pulldown using GST-Mud incubated with constant 2 uM Pins TPR and increasing amounts of MBP-Insc results in an approximately 1∶1 stoichiometric dissociation of Pins TPRs from GST-Mud. f, A control titration of MBP alone does not result in disruption of Pins-Mud binding. g, Fluorescence anisotropy of TMR-Mud with increasing amounts of Pins TPRs exhibits a robust association profile. h, Addition of Inscuteable to a pre-formed complex of 100 nM TMR-Mud & 1 uM Pins causes a dissociation of the Mud-Pins complex & reduction of TMR-Mud anisotropy. i, GST-pulldown using in vitro Aurora-A phosphorylated Pins 1–466 results in complex formation with the Dlg GK domain. AurA treatment of a pre-formed MBP-Insc/Pins 1–466 complex likewise is able to form a complex with the Dlg GK domain.

Figure 4. Proposed model for Inscuteable regulation of spindle orientation.

a, In early interphase, Inscuteable recruits cortical Gαi-Pins to the apical cortex. Insc-bound Pins can scaffold for Dlg, allowing for early microtubule attachment, but inhibits binding of Mud, preventing ectopic microtubule shortening. b, after nuclear envelope breakdown and trafficking along the mitotic spindle, Mud from astral microtubules competes Pins away from Insc and allows for microtubule shortening.