Relevance of Crumbs homolog 2 in the Glomerular filtration barrier

Abstract

Mammalian podocytes not only form the final layer within the glomerular filtration barrier (GFB) but also withstand enormous pressure that essentially drives renal ultrafiltration. Their effacement has been linked with and usually marks the onset for several glomerular diseases. As is evident from their role, expression of polarity determinants during the early stages of kidney development help determine podocyte morphogenesis. This eventually helps to regulate inter– and intra-cellular transport processes and establish essential cell-cell communications required for efficient barrier formation. The Crumbs complex including the name-giving single-pass transmembrane protein Crumbs (CRB) is an evolutionarily conserved polarity determinant that help establish apico-basal polarity within many epithelia.<br /> In this study, I investigated the role of two mammalian CRB orthologues, namely CRB2 and CRB3A with greater emphasis on the localization and functioning mechanism of CRB2. Utilizing the newly introduced method of expansion microscopy, we were able to identify the localization of CRB2 within the slit diaphragm (SD) of murine glomerulus and compare it with other known SD markers. Our discovery of CRB2 forming homotypic clusters led us to further investigate the role of its extra-long extracellular domain (ECD) within immortalized human kidney podocytes. Employing a multitude of advanced microscopic tools I was then able to further elucidate the mode of action of CRB2s ECD. Results from fluorescence recovery after photobleaching (FRAP) experiments demonstrated a slower mobility for CRB2 at podocyte-podocyte contact sites when compared with CRB3A. Additionally, a peculiar mobility pattern was observed for the ECD-specific CRB2 mutants, with the mutant lacking the entire ECD being particularly immobile. Data obtained using traction force microscopy revealed that CRB2 interacts in trans. However, these interactions are dynamic with CRB2 basically acting as the podocytes’ environment sensor as evident from our live-cell imaging data. Upon contact with other CRB2-expressing cells, there is a massive re-organization of focal adhesion (FA) sites and actin cytoskeleton which ultimately governs cellular mobility and adhesion. A similar cytoskeletal effect could also be observed with CRB3A but to a much lesser extent. The findings from this study, provide for a basic understanding for the role of CRB2s ECD within the early embryonic stages of kidney development. The results presented here could be used as a guide for further research into podocyte cytology and/or development of therapeutics governing podocyte related glomerular diseases.