Gαi generates multiple Pins activation states to link cortical polarity and spindle orientation in Drosophila neuroblasts

Abstract

Drosophila neuroblasts divide asymmetrically by aligning their mitotic spindle with cortical cell polarity to generate distinct sibling cell types. Neuroblasts asymmetrically localize G␣i, Pins, and Mud proteins; Pins/G␣i direct cortical polarity, whereas Mud is required for spindle orientation. It is currently unknown how G␣i-Pins-Mud binding is regulated to link cortical polarity with spindle orientation. Here, we show that Pins forms a ''closed'' state via intramolecular GoLoco-tetratricopeptide repeat (TPR) interactions, which regulate Mud binding. Biochemical, genetic, and live imaging experiments show that G␣i binds to the first of three Pins GoLoco motifs to recruit Pins to the apical cortex without ''opening'' Pins or recruiting Mud. However, G␣i and Mud bind cooperatively to the Pins GoLocos 2/3 and tetratricopeptide repeat domains, respectively, thereby restricting Pins-Mud interaction to the apical cortex and fixing spindle orientation. We conclude that Pins has multiple activity states that generate cortical polarity and link it with mitotic spindle orientation.cell polarity ͉ cell signaling ͉ differentiation ͉ protein-protein interactions I n complex, multicellular organisms, differentiated cell types are needed to perform diverse functions. One common mechanism for cellular differentiation is asymmetric cell division, in which the mitotic spindle is aligned with the cell polarity axis to generate molecularly distinct sibling cells (1-4). Asymmetric divisions have been proposed to regulate stem cell pool size during development, adult tissue homeostasis, and the uncontrolled proliferation observed in cancer (5). Thus, understanding how the mitotic spindle is coupled to the cell polarity axis is relevant to stem cell and cancer biology. Here, we investigate this question in Drosophila neuroblasts, a model system for studying asymmetric cell division.Drosophila neuroblasts are stem cell-like progenitors that divide asymmetrically to produce a larger self-renewing neuroblast and a smaller ganglion mother cell (GMC) that differentiates into neurons or glia (3). Mitotic neuroblasts segregate factors that promote neuroblast self-renewal to their apical cortex and differentiation factors to their basal cortex. Precise alignment of the mitotic spindle with the neuroblast apical/basal polarity is required for asymmetric cell division and proper brain development: spindle misalignment leads to symmetric cell divisions that expand the neuroblast population and brain size (6-8).

Molecular pathways regulating mitotic spindle orientation in animal cells

“…However, these proteins share a relatively higher conservation in a small C-terminal domain termed the Pinsbinding domain (PBD). The PBD directly interacts with the Pins tetratricopeptide repeat (TPR) domain, and this interaction requires prior Gα protein-mediated relief of an autoinhibitory conformation between the TPR and GoLoco domains of Pins (Du and Macara, 2004;Nipper et al, 2007). This interaction recruits Mud to the Pins cortical crescent, thereby polarizing Mud localization (Fig.…”

Ric-8A Catalyzes Guanine Nucleotide Exchange on Gαi1 Bound to the GPR/GoLoco Exchange Inhibitor AGS3

“…Along with G␣ subunits, GPR1/2 proteins that contain GoLoco/G protein regulatory (GPR) motifs, operating in conjunction with "resistance to inhibitors of cholinesterase" (Ric-8) and RGS7, are necessary and sufficient for regulation of these events (22)(23)(24)(25)(26)(27). In dividing Drosophila neuroblasts, G␣ i subunits complexed with the GPR/GoLoco motif-containing protein PINS (PINS indicates Partner of Inscuteable) binds to Inscuteable or MUD to control asymmetric cell division (28,29). In dividing sensory precursor cells of Drosophila, Ric-8 has been shown to positively regulate G␣ i activity and is implicated in the membrane targeting of both PINS and G␣ i subunits (29 -31).…”

Polarization of Drosophila Neuroblasts During Asymmetric Division

“…The primary connection between the spindle and cortical polarity is the protein Inscuteable, which links the two by binding Baz and the adapter protein Partner of Inscuteable (Pins) (Kraut et al 1996;Shober et al 1999;Yu et al 2000). Pins links the heterotrimeric G-protein subunit Gai to the microtubule and Dynein-binding protein Mud (NuMA in mammals) (Siller et al 2006;Izumi et al 2006;Bowman et al 2006;Nipper et al 2007). In mud mutants, cortical polarity and spindle positioning are unlinked as the spindle position becomes less correlated with the polarity axis.…”