NUMA:LGN HETERO-HEXAMERS PROMOTE THE ASSEMBLY OF CORTICAL PROTEIN NETWORK TO CONTROL PLANAR CELL DIVISIONS

Mitotic spindle orientation is a prerequisite for the correct completion of mitosis, and is essential for tissue morphogenesis and maintenance. Divisions occurring within the plane of epithelia, or planar divisions, shape the architecture of epithelial sheets, whereas vertical divisions along the apicobasal axis are associated with asymmetric fate specification and stratification. Several studies described the evolutionary conserved trimeric Gi:LGN:NuMA complex as the core constituent of the spindle orientation machinery. In mitosis Gi:LGN:NuMA complexes localize at the cortex and orient the spindle by generating pulling forces on astral microtubules emanating from the spindle poles, via interaction of NuMA with the minus-end directed motor proteins dynein/dynactin. Biochemical and structural studies identified the minimal binding domains of the NuMA:LGN interaction, showing that a 30-residues stretch in the C-terminal part of NuMA binds to the inner groove formed by the eight TPR repeats at the N-terminus of LGN. However, how such interaction is organized at the cell cortex and triggers microtubules-motor activation still remains largely unclear. My PhD project focused on the characterization of the NuMA:LGN interaction and on the analysis of the role of the microtubule-binding domain of NuMA. Studies conducted during this thesis revealed that NuMA and LGN assemble in hetero-hexameric structures organized in a donut-shape architecture. In such arrangement, the LGN helices preceding and following the TPR repeats, and a NuMA motif preceding the shortest LGN-binding motif, are essential for the interaction. Consistently, an LGN oligomerization-deficient mutant cannot rescue misorientation defects caused in HeLa cells and Caco-2 three-dimensional cysts by endogenous LGN ablation. Importantly, in cells expressing the oligomerization-deficient mutant, force generators are correctly localized at the cell cortex. We provided evidence that LGN and NuMA assemble high-order oligomers in cells, and that the 3:3 stoichiometry of the NuMA:LGN complex combined with the dimeric state of NuMA coiled-coils promote the formation of a large proteins network. We also showed that ectopic targeting of an oligomerization-deficient NuMA mutant at the cortex is not sufficient to orient the spindle, indicating that the molecular organization of NuMA in complex with LGN is required to orient the spindle in metaphase. Furthermore, we provided evidence that the NuMA:LGN oligomers are compatible with the direct association of NuMA to microtubules, and that the microtubules-binding domain of NuMA is required to correctly localize NuMA at the poles and at the cortex, and to orient the spindle. Collectively, our findings suggest a model whereby cortical LGN:NuMA hetero-hexamers favor the accumulation of dynein motors at cortical sites. We speculate that direct binding of NuMA to astral microtubule plus-tips assists the processive movement of dynein along the depolymerizing astral microtubules to promote spindle placement.