LOCALIZED WNT3A-SOURCES INSTRUCT MITOTIC ORIENTATION: DISSECTING THE WNT3A-TRANSDUCTION MECHANISM COUPLED WITH NUMA MACHINERY

In multicellular organisms, morphogenesis and homeostasis of epithelial tissues require oriented cell divisions, which rely on the proper positioning of the mitotic spindle. Consistently, defects in epithelial polarity and spindle orientation are often coupled with loss of tissue architecture and altered proliferation. Since mitotic spindle orientation determines the axis of cell division, in stem cells it plays a pivotal role in the symmetric or asymmetric fate acquired by the daughter cells after cytokinesis. During asymmetric cell division, the spindle aligns parallel to the polarity axis to ensure asymmetric inheritance of cell fate determinants and to expose daughter cells to different niche microenvironment. Mechanistically, microtubules (MTs) motors and actin regulators have been implicated in the response to extracellular cues that instruct the orientation of the division by binding to adhesion molecules including cadherins and integrins, or to players of diverse signalling pathways. The synergistic link between the external and internal cues instructing division orientation converge on the evolutionary conserved pathway consisting of dynein/NuMA/LGN/Gai cortical complexes that define the spindle alignment by exerting pulling forces on dynamic plus-ends of astral MTs to control spindle position and orientation. How these force generators respond to extracellular signal remains poorly characterised. In numerous stem cells niches, Wnt ligands assume a restricted spatial localisation that contribute to regulate the mitotic spindle alignment toward the niche. However how cells sense Wnt signals to orient the division axis is largely unknown. A major goal of my PhD project was to dissect genetically and biochemically the mechanism that transduce localised Wnt3a signals to the spindle apparatus during mitosis in order to instruct division orientation. Live-cell imaging experiments conducted in HeLa cells dividing in contact with an in vitro niche system such as Wnt3a-coated nanospheres, revealed that canonical Wnt effectors including LRP6, β-catenin, DVL2 and Cav1 are implicated in the spindle axis alignment towards the Wnt3a source. In addition, we demonstrate that Gai/LGN/NuMA complexes at the cell cortex together with astral MTs, are essential for Wnt3a-dependent spindle orientation. Proximity labelling assay revealed that during mitosis the Wnt3a co-receptor LRP6 is in proximity of NuMA and Gai subunits. Moreover, performing immunoprecipitation assays we demonstrate that Wnt3a stimulation triggers the formation of macromolecular complexes between NuMA and the Wnt effectors β-catenin, Axin1, APC, and LRP6. To identify the protein complexes implicated in mitotic Wnt3a-signal transduction, we set up a novel biochemical assay allowing isolation and identification by proteomic analysis of the macromolecular complexes recruited at the localised Wnt3a contact site. This approach confirmed that NuMA, Gai subunits and Caveolin are part of the cytoplasmic interactome recruited at the Wnt3a-bead contact site, further corroborating their role in the Wnt3a-spindle transduction mechanism. Collectively, these findings indicate that in metaphase NuMA acts as Wnt3a transducer to the mitotic spindle by physically bridging between Wnt3a-activated LRP6 co-receptors and dynein-based astral MT motors. Importantly, our study reveals that the cross-talk between mitotic spindle orientation and Wnt3a signalling is centred biochemically on the interaction between NuMA and canonical Wnt effectors. Considering the spatial distribution of Wnt signals in niche microenvironment, we propose that this newly identified mechanism of mitotic Wnt3a response might be essential for the correct execution of asymmetric stem cell divisions.