ROLE OF IRSP53 IN EPITHELIAL POLARITY ESTABLISHMENT AND LUMEN MORPHOGENESIS

Polarity establishment is essential for the normal development and morphogenesis of virtually all epithelial tissues. Loss of epithelial polarity has been implicated in many disorders including tumorigenesis and metastasis. Epithelial polarity is established by a multifaceted signalling cross-talk between a network of polarity determinants that include cell adhesion molecules, trafficking machinery, polarity complexes and the spindle orientation apparatus. Despite tremendous advance in defining the molecular details of how these processes are controlled, much remains to be understood as to how the coordination between all these components is achieved. IRSp53, a versatile molecule operating at the membrane-actin interface, has been reported to be involved in the process of epithelial polarity but the mechanisms through which it operates has, however, remained completely elusive. Here, we studied the processes through which IRSp53 plays a role in the establishment of epithelial polarity and lumen morphogenesis in MDCK and Caco-2 derived 3-D cysts. We further extended the relevance of our in vitro findings to the kidney morphogenesis in our genetically modified animal models. We found that IRSP53 is apically restricted at the luminal side of various epithelial tubular and glandular human, murine and zebrafish tissues. Further, IRSp53 is early recruited after the first cell division along the AMIS and essential for the formation of a single apical domain and the localized recruitment of aPKC and Podocalyxin (PDX). Molecularly, IRSp53 exerts this function by binding directly through a positively charged patch on its I-BAR-domain to the inactive form of RAB35, which co-localizes with IRSp53 at the onset of AMIS, and by controlling PDX trafficking. Additionally, membrane- and SH3- mediated interactions are required for proper localization and, thus, for the function of IRSp53. IRSp53 is also found to localize to the spindle poles and to aid in the proper orientation of the mitotic spindle during cystogenesis. Consistently, IRSp53 depletion causes spindle misorientation, leading to aberrant cell divisions and multi-lumen phenotype during epithelial cystogenesis. The critical physiological role of IRSp53 in epithelial morphogenesis is corroborated by the finding that genetic deletion of IRSp53 results in abnormal renal tubulogenesis, with profound defects in tubular polarity, architectural organization and lumen formation during both kidney development and in the adult kidneys either in IRSp53 zebrafish mutant lines and in IRSp53 KO murine models. We propose that the proper localisation of IRSp53 is pivotal for the assembly of macromolecular complexes on the apical domain of tubular and glandular epithelia which is, in turn, necessary for the proper kidney development by regulating cell polarity and lumen morphogenesis.