Inscuteable and NuMA proteins bind competitively to Leu-Gly-Asn repeat-enriched protein (LGN) during asymmetric cell divisions

Abstract

Coupling of spindle orientation to cellular polarity is a prerequisite for epithelial asymmetric cell divisions. The current view posits that the adaptor Inscuteable (Insc) bridges between Par3 and the spindle tethering machinery assembled on NuMA∶LGN∶Gαi GDP , thus triggering apico-basal spindle orientation. The crystal structure of the Drosophila ortholog of LGN (known as Pins) in complex with Insc reveals a modular interface contributed by evolutionary conserved residues. The structure also identifies a positively charged patch of LGN binding to an invariant EPE-motif present on both Insc and NuMA. In vitro competition assays indicate that Insc competes with NuMA for LGN binding, displaying a higher affinity, and that it is capable of opening the LGN conformational switch. The finding that Insc and NuMA are mutually exclusive interactors of LGN challenges the established model of force generators assembly, which we revise on the basis of the newly discovered biochemical properties of the intervening components.A symmetric cell divisions regulate the position and the fate choice of daughter cells, with impact on numerous phenotypes of multicellular organisms. During development, asymmetric divisions coordinate cell growth with cell specification to determine tissue morphogenesis, while in adult life they sustain tissue homeostasis and regeneration (1). In asymmetric divisions specific cortical landmarks instruct the orientation of the mitotic spindle to promote unequal partitioning of fate determinants in cellular systems as diverse as Caenorhabditis elegans zygotes, Drosophila neuroblasts, as well as vertebrate skin and neural progenitors (2). Spindle coupling to polarity cues involves the recruitment at cortical sites of molecular devices, known as force generators, whose main task is to capture astral microtubules emanating from the spindle poles and to establish pulling forces. Core components of force generators are the evolutionary conserved NuMA∶LGN∶Gαi complexes, termed Mud∶Pins∶Gαi in flies. Topologically, tetratricopeptide repeats (TPR) present in the N-terminal portion of LGN mediate the interactions with NuMA, while GoLoco motifs at the C terminus serve as a docking platform for four Gαi GDP subunits anchored at the plasma membrane via a myristoyl group (3).LGN exclusively binds to GDPloaded Gαi (4). The association of cortical NuMA with the microtubule motor Dynein/Dynactin (5) provides a sliding anchorage for depolymerizing microtubules, whose shrinkage pulls towards the cortex the connected spindle pole. FRET studies revealed that LGN behaves as a conformational switch held in a closed form in interphase by head-to-tail interactions (3).An issue intimately related to how force generators are assembled is how they are recruited at sites of polarization. In polarized asymmetric divisions, the apico-basal polarity axis is established by the asymmetrical distribution of Par3∶Par6∶aPKC at the apical cortex, which are able to recruit NuMA∶LGN∶Gαi GDP via an adaptor named Inscuteable (Insc) (6). Insc w...

Analysis and modeling of mitotic spindle orientations in three dimensions

“…It has been proposed that lateral localization of LGN recruits NuMA to establish a preferentially horizontal orientation of the mitotic spindle. In this model, mInsc titrates the NuMA binding sites on LGN and thereby inhibits the machinery for horizontal spindle orientation to increase the frequency of oblique spindles (24,25). Once all binding sites on LGN are occupied by mInsc, spindle orientation should be random.…”

“…The NuMA-LGN complex is located around the cell equator and is instructive for orienting the mitotic spindle horizontally (21)(22)(23). More recent biochemical and structural analyses, however, have demonstrated that mInsc and NuMA are mutually exclusive interaction partners of LGN and that mInsc is able to displace NuMA from its LGN binding site (24,25). This competitive interaction for LGN has led to an alternative model where mInsc exerts its effect on spindle orientation by titrating NuMA binding sites on LGN and prevents tethering of the spindle to the cell cortex leading to a randomization of spindle orientation (26,27).…”

Mechanisms of spindle positioning: cortical force generators in the limelight

“…Biophysical and structural analysis of proteins central to the force generating machinery is anticipated to be important. An illustration of such work is given by the recent atomic level characterization of the interaction between parts of LGN and NuMA [65,66]. Coupled with modeling and computer simulations of spindle positioning, this should yield an in depth molecular understanding of the underlying mechanisms.…”

Structural basis for the recognition of the scaffold protein Frmpd4/Preso1 by the TPR domain of the adaptor protein LGN

“…1a) fits into a hydrophobic pocket formed by Leu18, Ala21, Leu22, Thr53, Ala56 and Ile57 in LGN. It should be noted that not only the corresponding residues in the LGN-binding regions of mInsc (Ile52) and its Drosophila homologue (Ile334) (Yuzawa et al, 2011;Culurgioni et al, 2011) but also that in NuMA (Ile1906) are buried in the same hydrophobic pocket (Fig. 2c).…”