Functional characterization of Myl6B: a protein required for spindle and midbody orientation in HeLa cell

Eukaryotic cells undergo dramatic changes in cell shape during cell division. Although tremendous advances have been made in our understanding of the mechanisms of how chromosomes are segregated during animal cell division, how the impressive cortical rearrangements occurring during mitosis and cytokinesis are coordinated with chromosomal segregation remains poorly understood in animal cells dividing symmetrically. Previous work, performed in my laboratory showed that an essential myosin light chain (ELC), named Mlc1p, by coordinating different myosin motors with the action of the IQGAP-like protein Iqg1, is essential for cytokinesis in the budding yeast Saccharomyces cerevisiae (Wagner et al., 2002). Mlc1 is haploinsufficient in budding yeast cells and its binding to IQ motifs of Myo2 is essential for viability (Stevens and Davis, 1999; Wagner et al., 2002). Although, class V and II non-conventional myosins and IQGAP-like proteins are expressed and their function has been well conserved throughout the evolution (Shannon, 2012), very little is known about the existence or role of ELCs similar to the budding yeast Mlc1p and to the fission yeast Cdc4 in animal cells. IQGAPs are evolutionary conserved multimodular proteins containing IQ motifs that act as scaffolds for several components of the mitogen-activated protein kinases (MAPKs) and Rho-like small G proteins regulating cytoskeleton rearrangement during motility and tumorigenesis in animal cells (Hedman et al., 2015). Since ELCs act by binding to the IQ motifs of target proteins and by this means regulate target protein activity in response to calcium signalling, we hypothesised that ELCs might have a similar role in regulating Myo II and IQGAP1 proteins during animal cell division. Based on this idea Dr. Ragnini identified two human Mlc1-like proteins, namely Myl6 and Myl6B using bioinformatics tools. Myl6 and Myl6B, differ by 56 amino acids at their N-terminus and were previously indicated to bind to smooth muscle and non-muscle myosins. However, their function in non-muscle cells was never investigated. We showed that Myl6 and Myl6B ELCs are the human orthologues of the budding and fission yeast Mlc1like ELCs (Mosca PhD Thesis Open University (UK) 2011). Moreover, we demonstrated that Myl6 complements the yeast mlc1 ts mutant and that Myl6B interacts with IQ motifs of yeast and human Myosin II, Myosin V and IQGAP-like proteins. Silencing of these human Mlc1-like proteins causes spindle and midbody orientation defects (Mosca PhD Thesis Open University (UK) 2011). The molecular basis of this defect has been investigate in this study. Here, I show that Myl6B preferentially binds to IQGAP1 in HeLa cells and that it is required for Cdc42 activation during mitosis and thereby for spindle orientation in HeLa cells. The data presented in this thesis uncover an unexpected role of IQGAP1 during spindle orientation in animal symmetric cell division and provides a novel view on how IQGAP deregulation might lead to aneuploidy and cancer development. Givin the fact that down-regulation of IQGAP1 have been proposed for its anti-tumour activity, our findings reveal a novel potential target for anticancer therapy.