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Distinct functions of Crumbs regulating slit diaphragms and endocytosis in Drosophila nephrocytes

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Abstract

Mammalian podocytes, the key determinants of the kidney’s filtration barrier, differentiate from columnar epithelial cells and several key determinants of apical–basal polarity in the conventional epithelia have been shown to regulate podocyte morphogenesis and function. However, little is known about the role of Crumbs, a conserved polarity regulator in many epithelia, for slit-diaphragm formation and podocyte function. In this study, we used Drosophila nephrocytes as model system for mammalian podocytes and identified a conserved function of Crumbs proteins for cellular morphogenesis, nephrocyte diaphragm assembly/maintenance, and endocytosis. Nephrocyte-specific knock-down of Crumbs results in disturbed nephrocyte diaphragm assembly/maintenance and decreased endocytosis, which can be rescued by Drosophila Crumbs as well as human Crumbs2 and Crumbs3, which were both expressed in human podocytes. In contrast to the extracellular domain, which facilitates nephrocyte diaphragm assembly/maintenance, the intracellular FERM-interaction motif of Crumbs is essential for regulating endocytosis. Moreover, Moesin, which binds to the FERM-binding domain of Crumbs, is essential for efficient endocytosis. Thus, we describe here a new mechanism of nephrocyte development and function, which is likely to be conserved in mammalian podocytes.

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Acknowledgements

We thank D. Kiehart, E. Knust, A. Nakamura, R. Roepman, the Bloomington Drosophila stock center at the University of Indiana (USA), the National Institute of Genetics, (Shizuoka, Japan), the Vienna Drosophila Resource Center (Austria), and the Developmental Studies Hybridoma Bank at the University of Iowa (USA) for sending reagents. Thanks to Karin Wacker for excellent technical assistance. This work was supported by grants of the German research foundation (DFG) to M. P. K. (DFG3901/2-1, DFG3901/1-2, SFB699-A13) and to T.W. (SCHL1845/2-1, WE2550/2-2).

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Authors and Affiliations

  1. Molecular and Cellular Anatomy, University of Regensburg, Universitätsstr. 31, 93053, Regensburg, Germany

    Florian Hochapfel, Lucia Denk, Gudrun Mendl, Christine Maaßen, Yulia Zaytseva, Reinhard Rachel, Ralph Witzgall & Michael P. Krahn

  2. Medizinische Klinik und Poliklinik D, Universitätsklinikum Münster, Domagkstr. 3a, 48149, Münster, Germany

    Florian Hochapfel, Ulf Schulze, Hermann Pavenstädt, Thomas Weide & Michael P. Krahn

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  1. Florian Hochapfel
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18_2017_2593_MOESM1_ESM.tif

Supplemental Fig. 1. ANP–RFP-accumulation assay. Example of garland nephrocytes with accumulated RFP (RFP channel is depicted in B, D, F, and H). RFP expressed from the Myosin heavy chain promoter was secreted into the hemolymph and taken up by cell garland nephrocytes. The tissue was counterstained with DAPI (without permeabilizing reagents) to visualize cellular structures (A, C, E, and G). A garland nephrocyte was outlined in red in A, B, E, and F. The RFP signal within the encircled area was measured and the signal from unspecific staining (“background”, taken from the tissue of the stomach) was subtracted (encircled in red in C and D and G and H) to obtain a quantification of RFP accumulation within a single nephrocyte. To quantify the overall accumulation efficiency (as described in the methods section), we scored at least 40 nephrocytes from 5 independent larvae (TIFF 1330 kb)

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Supplemental Fig. 2. Downregulation of Moesin affects the ultrastructure and filamentous actin accumulation of nephrocytes. (A) Transmission electron micrographs of nephrocytes expressing Moesin–RNAi show a disturbed ultrastructure similar to Crb–RNAi nephrocytes. (B and C) Phalloidin staining of control (B) and Crb–RNAi (C) garland nephrocytes demonstrates a disturbance of cortical actin in Moesin–RNAi expressing cells. Note that even in control nephrocytes, actin staining is rather weak in comparison with surrounding tissues (e.g., proventriculus, arrows). (D) Immunostaining of garland nephrocytes with Moesin–RNAi visualizes a loss of cortical Crb and Sns (TIFF 3642 kb)

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Supplemental Fig. 3. Overexpression of Crb proteins in garland nephrocytes increases accumulation of RFP. The indicated Crb proteins were overexpressed in garland nephrocytes and the accumulation of secreted RFP in garland nephrocytes was quantified as described in Suppl. Figure 1 (TIFF 208 kb)

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Hochapfel, F., Denk, L., Mendl, G. et al. Distinct functions of Crumbs regulating slit diaphragms and endocytosis in Drosophila nephrocytes. Cell. Mol. Life Sci. 74, 4573–4586 (2017). https://doi.org/10.1007/s00018-017-2593-y

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  • DOI: https://doi.org/10.1007/s00018-017-2593-y

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