Offre de stage M2, Christelle BENAUD

Unraveling cortical elements regulating mitotic spindle orientation in vertebrates

Establishing a correct axis of cell division, which is defined by the precise positioning of the mitotic spindle, is crucial for development and the maintenance of epithelial tissue integrity. Misregulation of spindle orientation results in disorganized tissue morphology and has been associated with cancer development. The mitotic spindle is positioned within the cell via the anchoring of its astral microtubules to cortical elements. Proper spindle orientation depends thus on their position at the cortex which is influenced both by intracellular and external signals. However, the regulation of the polarization and the organization of the anchoring machinery in a diffusion prone environment are not yet fully understood. Our previous work indicates that annexin A2 is a regulator of prometaphase cortical reorganization and spindle orientation. We have now identified a cortical scaffold protein, ahnak, whose localization is regulated by annexin A2 in mitosis. Furthermore, our results indicate that it is implicated in prometaphase progression and orientation of mammalian cells division. The aim of this study is to decipher the role of ahnak in the establishment of spindle orientation in symmetric cell division of epithelial cells. We will combine both CRISPR approaches, proteomics, 3D cell culture model, and Real Time cell imaging approaches to examine the cortical function of ahnak in prometaphase. We will first address the role of ahnak in regulating mitotic spindle orientation in response to cell adhesion signals and within a multicellular tissue-like context. We will examine ahnak participation in the organization of cortical domains in response to external cue. In particular, we will concentrate on the ability of ahnak to interact with cortical microtubule motors and influence their spatio-temporal organization. In addition, the identification of new protein complexes interacting with ahnak in prometaphase coupled to their functional implication in the establishment of proper division axis will more precisely define ahnak molecular function. Altogether, this study will determine whether ahnak serves as a link between extrinsic cue and the force generators elements governing spindle orientation.