Laura Pirovano

Mitotic spindle orientation is a prerequisite for the correct completion of mitosis, and is essential for tissue morphogenesis and maintenance. The core constituent of the spindle orientation machinery is represented by Gai:LGN:NuMA complexes, which orient the spindle by generating pulling forces on astral microtubules. Besides several studies identified the minimal binding domains of NuMA and LGN, how such interaction is organized 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. Consistently, an LGN oligomerization-deficient mutant cannot rescue misorientation defects in HeLa cells and Caco-2 three-dimensional cysts. 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 promotes the formation of a large proteins network. Furthermore, we showed 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 movement of dynein along the depolymerizing astral microtubules to promote spindle placement.