Purpose: Haploinsufficiency and overexpression of the Pax6 gene are responsible for defective central nervous system development. The purpose of the current work was to identify and characterize a new potential role for the Pax6 transcription factor in cellular proliferation in addition to its role at the level of gene expression.
Methods: Expression vectors encoding tagged Pax6p46 protein were used to observe directly protein localization during the cell cycle in cells lines. Three dimensional (3-D) fluorescence microscopy imaging was used to observe in vivo mitotic progression and chromosome dynamics to define the mitotic step affected by p46DsRed as well as to validate endogenous p46 localization on chromosomes in quail retinal cells. Video imaging was used to identify the precise moment of onset of effects related to p46 overexpression in living cells. A pulldown assay in HEK cells was used to identify a specific partner of p46.
Results: Pax6p46 protein in transfected cells is localized on the chromosomes, predominantly in a pericentromeric area, and its localization changes as mitosis progresses. Overexpression of p46 protein induces incomplete chromatid separation, resulting in defective mitosis at the onset of the anaphase. A physical interaction between p46 and ESPL1 was identified.
Conclusions: The results suggest that Pax6 exerts an effect on mitosis through protein-protein interactions with proteins localized on chromosomes. Supported by the observation that p46 interacts with separase, an enzyme required for chromatid separation, the authors propose that this interaction is responsible for the mitosis defect observed in cells overexpressing Pax6.