NuMA:LGN hetero-hexamers cluster Dynein motors at the cortex to orchestrate spindle orientation

Oriented cell divisions are essential for tissue development during embryogenesis and for homeostasis of adult organisms. Disruption of orientation mechanisms in stem cells leads to tissue disorganization and tumor-like proliferation [1]. In epithelial cells, key players of spindle orientation are Gαi/LGN/NuMA complexes, which localize at the cortex and generate pulling forces on astral microtubules (MTs) via direct interaction of NuMA with the Dynein/Dynactin MTsmotors [2]. How LGN and NuMA triggers MT-motors activation remains largely unclear. I will present the oligomeric crystal structure of the N-terminal TPR domain of LGN in complex with the C-terminal portion of NuMA determined at 3.7 Å resolution by molecular replacement on a merged dataset. In this hetero-hexameric assembly, the first helix of one LGNTPR molecule hooks on the last TPR repeat of an adjacent LGNTPR domain, while the NuMA chains lines in the inner surface to the TPR domains and between them in an overall donut-like architecture. The combination of NuMA coiled-coil dimers with the 3:3 LGN:NuMA stoichiometry results in the formation of a protein network visible by Cryo-EM, that we think help clustering of Dynein motors at the mitotic cortex. Consistently, oligomerization deficient LGN mutants cannot rescue the misorientation defects observed in HeLa cells and Caco-2 cysts lacking the endogenous protein. Interestingly, the LGN:NuMA complex is compatible with the binding of NuMA-Cterminus to MTs [3]. The NuMA MT-binding domain cosediments with taxol-stabilized MTs, and also binds α,β-tubulin heterodimer in solution in SEC experiments. To understand the structural principles of the interaction of NuMA with MTs, we are crystallizing the NuMA MT-binding domain in the absence and in the presence of tubulin dimers. Collectively, or findings suggest a model whereby in mitosis NuMA:LGN oligomers favour cortical clustering of Dynein/Dynactin, and coordinate processive movement of these motors via direct binding of NuMA to the MT-lattice.