Symmetry breaking signaling mechanisms during cell polarization

Abstract

Breaking of cellular symmetry in order to establish an apico-basal polarity axis initiates de novo formation of cell polarity. However, symmetry breaking provides a formidable challenge from a signaling perspective, because by definition no spatial cues are present to instruct axis establishment. Instead, symmetry breaking is driven by amplification of stochastic fluctuations in signaling activity. A concomitant problem of this is that polarization can occur at multiple sites; a situation that could be detrimental to the cell. Therefore, cells must be able to restrict polarization to a single domain. Throughout evolution, small GTPases, and Cdc42 in particular, are involved in the control of symmetry breaking events. By engaging different GEFs, GAPs and effectors, Cdc42 signaling is orchestrated with pinpoint precision to control the various cellular processes that contribute to cell polarization. In this thesis, novel signaling pathways are revealed that contribute to symmetry breaking during enterocyte polarization. The prime model system for studying these pathways are Ls174T:W4 cells, which break cellular symmetry upon forced polarization. The central research question underlying this work is: how are intracellular signaling pathways organized to enable symmetry breaking? We demonstrate that singularity in apical domain specification during epithelial cell polarization is actively enforced and that this requires Cdc42 signaling. For this Cdc42 mobility at the apical domain is regulated by the disease-associated flippase ATP8B1. Loss of this regulation results in the formation of multiple apical domains and affects apical membrane size in Ls174T:W4 cells. In a 3D context, loss of ATP8B1 affects lumen architecture indicative of an important function for singularity enforcement in tissue morphogenesis. We reveal that upon activation by the Cdc42GEF Tuba, Cdc42 mobility is drastically reduced to ensure localized signaling. The molecular origin of this immobilization is identified and the importance of this regulation is demonstrated. We investigate the role of the tumor suppressor PTEN in apical domain specification and find that PTEN is required to restrict apical domain formation and that for this PTEN requires binding to PTPL1. In chapter five signaling by the Rap2 small GTPases is introduced. An overview is provided on the biochemical mechanisms of Rap2 signaling and the biological functions in which Rap2 signaling is implicated are discussed. In chapter six the mechanisms of isoform specific Rap2 signaling during brush border formation are elucidated. Rap2A is the sole isoform contributing to enterocytic brush border formation despite expression of the highly similar Rap2B and Rap2C isoforms. A combination of specific localization and selective activation explains how highly similar GTPases can signal independently. We therefore identify novel signaling mechanisms that contribute to symmetry breaking by controlling apical membrane clustering, singularity in apical domain specification and brush border formation. Importantly, we reveal that singularity in apical domain specification is actively safeguarded in polarized epithelial cells by Cdc42 signaling and implicate this regulation in human pathologies.