MBI Weekly Meeting Seminar
Time: 9.30am-10.30am
Date: Friday, 29 March 2019
Venue: Level 5 Seminar Room, T-Lab
Spatially restricted proteomics of the polarity proteins Par3 and Pals1 reveals sub-compartmentalization of the epithelial apical-lateral membrane border
In mammalian epithelial cells the Par, Crumbs and Scribble polarity modules occupy distinct apical and lateral membrane compartments that overlap at the level of tight junctions (TJ). However, the precise sub-junctional organization of the polarity proteins remains unclear, and a comprehensive molecular and spatial analysis of the TJ-associated polarity network is lacking. In this talk I will present our recent efforts in defining the organization of the apical polarity proteins Par3 and Pals1 in fully polarized MDCK-II cells using APEX2-mediated quantitative proximity proteomics (QPP), electron microscopy, and RNAi. Our data show that Pals1 defines a distinct membrane compartment apical of TJ highly reminiscent of the invertebrate marginal zone. The formation of the Pals1 domain, which is composed of Pals1, PatJ, Crumbs3 and Lin7, is strictly dependent upon Par3 expression. QPP using a Par3-Pals1 SILAC pair resolved the molecular and spatial organisation of the apical-lateral border and links the Pals1 domain to the HIPPO pathway, GPCR signaling, and apical membrane trafficking regulators. Finally, prompted by our proteomics analysis we have identified a novel TJ-associated Rac1 GAP, ARHGAP12, which is recruited to TJ via Par3 and ZO2. Our current data suggest a model in which ARHGAP12 controls TJ formation and junctional tension via the actin polymerization factors N-WASP and WAVE2. Taken together our work defines the spatial and molecular organisation of the apical-lateral membrane border in mammalian epithelia, uncovers an intriguing spatial conservation of invertebrate and vertebrate cell polarity proteins, and provides a comprehensive resource for the identification of potentially novel regulators of cell polarity and mammalian cell junctions.
