Interaction between the mammalian cell polarity proteins mInsc (mammalian homologue of Inscuteable) and Leu-Gly-Asn repeatenriched protein (LGN), as well as that between their respective Drosophila homologues Inscuteable and Partner of Inscuteable (Pins), plays crucial roles in mitotic spindle orientation, a process contributing to asymmetric cell division. Here, we report a crystal structure of the LGN-binding domain (LBD) of human mInsc complexed with the N-terminal tetratricopeptide repeat (TPR) motifs of human LGN at 2.6-Å resolution. In the complex, mInsc-LBD adopts an elongated structure with three binding modules-an α-helix, an extended region, and a β-sheet connected with a loop-that runs antiparallel to LGN along the concave surface of the superhelix formed by the TPRs. Structural analysis and structure-based mutagenesis define residues that are critical for mInsc-LGN association, and reveal that the activator of G-protein signaling 3 (AGS3)-binding protein Frmpd1 [4.1/ezrin/radixin/moesin (FERM) and PSD-95/Dlg/ZO-1 (PDZ) domain-containing protein 1] and its relative Frmpd4 interact with LGN via a region homologous to a part of mInsc-LBD, whereas nuclear mitotic apparatus protein (NuMA) and the C terminus of LGN recognize the TPR domain in a manner different from that by mInsc. mInsc binds to LGN with the highest affinity (K D ≈ 2.4 nM) and effectively replaces the Frmpd proteins, NuMA, and the LGN C terminus, suggesting the priority of mInsc in binding to LGN. We also demonstrate, using mutant proteins, that mInsc-LGN interaction is vital for stabilization of LGN and for intracellular localization of mInsc. C ell polarization plays an essential role in asymmetric cell division, cell migration, and the proper function of various differentiated cell types (1). In the fruit fly Drosophila melanogaster, neurons and glial cells are produced by asymmetric division of neuroblasts that delaminate as single cells from apical-basal polarized neuroectoderm (2). The asymmetric division requires establishment of apical-basal polarity in neuroblasts; the Bazookacontaining protein complex is specifically localized to the apical cortex for polarity establishment. The mitotic spindle poles, normally arranged in parallel to the plane of the neuroectoderm, are also needed to be oriented along the apical-basal axis, which orientation is driven by another evolutionarily conserved protein complex containing Partner of Inscuteable (Pins), Mud, and the Gαi subunit of trimeric Gi proteins. The adaptor protein Inscuteable (Insc) simultaneously binds to Bazooka and Pins to provide a physical link between the two complexes; the link thereby couples cortical cell polarity and spindle orientation for asymmetric cell division.Homologues of both Insc and Pins are known to exist exclusively in animals (from insects to mammals). The mammalian homologue of Insc (mInsc) also appears to contribute to mitotic spindle orientation and asymmetric cell division. Like Drosophila Insc, mInsc is capable of simultaneously interacting with the mamm...

Successful chemotaxis requires not only increased motility but also sustained directionality. Here, we show that, during neutrophil chemotaxis via receptors coupled with the Gi family of heterotrimeric G proteins, directional movement is regulated by mInsc, a mammalian protein distantly related to the Drosophila polarity-organizer Inscuteable. The GDP-bound, Gβγ-free Gαi subunit accumulates at the front of chemotaxing neutrophils to recruit mInsc-complexed with the Par3-aPKC evolutionarily conserved polarity complex-via LGN/AGS3 that simultaneously binds to Gαi-GDP and mInsc. Both mInsc-deficient and aPKC-blocked neutrophils exhibit a normal motile activity but migrate in an undirected manner. mInsc deficiency prevents neutrophils from efficiently stabilizing pseudopods at the leading edge; the stability is restored by wild-type mInsc, but not by a mutant protein defective in binding to LGN/AGS3. Thus, mInsc controls directional migration via noncanonical G protein signaling, in which Gβγ-free Gαi-GDP, a product from Gαi-GTP released after receptor activation, plays a central role.

Par6–aPKC recruitment to the premature apical membrane through Morg1 interaction with Par6 is required for definition of apical identity of epithelial cells.

Correct cyst morphogenesis of epithelial cells requires apical-basal polarization, which is partly regulated by mitotic spindle orientation, a process dependent on the heterotrimeric G protein subunit Gαi and its binding protein LGN. Here, we show that in three-dimensional culture of mammalian epithelial Madin-Darby canine kidney (MDCK) cells, the Gαi-activating protein Ric-8A is crucial for orientation of the mitotic spindle and formation of normal cysts that comprise a single layer of polarized cells with their apical surfaces lining an inner lumen. Consistent with the involvement of LGN, cystogenesis can be well organized by ADP-ribosylated Gαi, retaining the ability to interact with LGN, but not by the interaction-defective mutant protein Gαi2 (N150I). In monolayer culture of MDCK cells, functional tight junction (TJ) assembly, a process associated with epithelial cell polarization, is significantly delayed in Ric-8A-depleted cells as well as in Gαi-depleted cells in a mitosis-independent manner. Ric-8A knockdown results in a delayed cortical delivery of Gαi and the apical membrane protein gp135, and an increased formation of intercellular lumens surrounded by membranes rich in Gαi3 and gp135. TJ development also involves LGN and its related protein AGS3. Thus, Ric-8A regulates mammalian epithelial cell polarity for TJ assembly and cystogenesis probably in concert with Gαi and LGN/AGS3.

Bipolar spindle assembly in mitotic cells is a prerequisite to ensure correct alignment of chromosomes for their segregation to each daughter cell; spindle microtubules are tethered at plus ends to chromosomes and focused at minus ends to either of the two spindle poles. NuMA (nuclear mitotic apparatus protein) is present solely in the nucleus in interphase cells, but relocalizes during mitosis to the spindle poles to play a crucial role in spindle assembly via focusing spindle microtubules to each pole. In the present study we show that the kinesin-5 family motor Eg5 is a protein that directly interacts with NuMA, using a proteomics approach and various binding assays both in vivo and in vitro. During mitosis Eg5 appears to interact with NuMA in the vicinity of the spindle poles, whereas the interaction does not occur in interphase cells, where Eg5 is distributed throughout the cytoplasm but NuMA exclusively localizes to the nucleus. Slight, but significant, depletion of Eg5 in HeLa cells by RNA interference results in formation of less-focused spindle poles with misaligned chromosomes in metaphase; these phenotypes are similar to those induced by depletion of NuMA. Since NuMA is less accumulated at the spindle poles in Eg5-depleted cells, Eg5 probably contributes to spindle assembly via regulating NuMA localization. Furthermore, depletion of cytoplasmic dynein induces mislocalization of NuMA and phenotypes similar to those observed in NuMA-depleted cells, without affecting Eg5 localization to the spindles. Thus dynein appears to control NuMA function in conjunction with Eg5.