Selective endocytosis controls slit diaphragm maintenance and dynamics in Drosophila nephrocytes

Abstract

The kidneys generate about 180 liters of primary urine per day by filtration of plasma. An essential part of the filtration barrier is the slit diaphragm, a multiprotein complex containing nephrin as major component. Filter dysfunction typically manifests with proteinuria and mutations in endocytosis regulating genes were discovered as causes of proteinuria. However, it is unclear how endocytosis regulates the slit diaphragm and how the filtration barrier is maintained without either protein leakage or filter clogging. Here we study nephrin dynamics in podocyte-like nephrocytes of Drosophila and show that selective endocytosis either by dynamin- or flotillin-mediated pathways regulates a stable yet highly dynamic architecture. Short-term manipulation of endocytic functions indicates that dynamin-mediated endocytosis of ectopic nephrin restricts slit diaphragm formation spatially while flotillin-mediated turnover of nephrin within the slit diaphragm is needed to maintain filter permeability by shedding of molecules bound to nephrin in endosomes. Since slit diaphragms cannot be studied in vitro and are poorly accessible in mouse models, this is the first analysis of their dynamics within the slit diaphragm multiprotein complex. Identification of the mechanisms of slit diaphragm maintenance will help to develop novel therapies for proteinuric renal diseases that are frequently limited to symptomatic treatment.

Data availability

Transgenic Drosophila lines are available from the corresponding author upon reasonable request. Unprocessed image files were submitted to a public repository (zenodo.org, DOI: 10.5281/zenodo.6418762). Access is not restricted for scientific purposes.

The following data sets were generated

Article and author information

Author details

  1. Konrad Lang

    Department of Medicine, University of Freiburg, Freiburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Julian Milosavljevic

    Department of Medicine, University of Freiburg, Freiburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Helena Heinkele

    Department of Medicine, University of Freiburg, Freiburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Mengmeng Chen

    Department of Medicine, University of Freiburg, Freiburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Lea Gerstner

    Department of Medicine, University of Freiburg, Freiburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Dominik Spitz

    Department of Medicine, University of Freiburg, Freiburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Severine Kayser

    Department of Medicine, University of Freiburg, Freiburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  8. Martin Helmstädter

    Department of Medicine, University of Freiburg, Freiburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  9. Gerd Walz

    Department of Medicine, University of Freiburg, Freiburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  10. Michael Köttgen

    Department of Medicine, University of Freiburg, Freiburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2406-5039
  11. Andrew Spracklen

    Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5550-8595
  12. John Poulton

    Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Tobias Hermle

    Department of Medicine, University of Freiburg, Freiburg, Germany
    For correspondence
    tobias.hermle@uniklinik-freiburg.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0441-7749

Funding

Deutsche Forschungsgemeinschaft (project-ID 431984000)

  • Tobias Hermle

Deutsche Forschungsgemeinschaft (HE 7456/3-1)

  • Tobias Hermle

Deutsche Forschungsgemeinschaft (HE 7456/4-1)

  • Tobias Hermle

Deutsche Gesellschaft für Innere Medizin (Clinician Scientist Fellowship)

  • Tobias Hermle

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Ilse S Daehn, Icahn School of Medicine at Mount Sinai, United States

Version history

  1. Preprint posted: March 2, 2022 (view preprint)
  2. Received: March 28, 2022
  3. Accepted: July 24, 2022
  4. Accepted Manuscript published: July 25, 2022 (version 1)
  5. Version of Record published: August 4, 2022 (version 2)

Copyright

© 2022, Lang et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,393
    views
  • 311
    downloads
  • 15
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Konrad Lang
  2. Julian Milosavljevic
  3. Helena Heinkele
  4. Mengmeng Chen
  5. Lea Gerstner
  6. Dominik Spitz
  7. Severine Kayser
  8. Martin Helmstädter
  9. Gerd Walz
  10. Michael Köttgen
  11. Andrew Spracklen
  12. John Poulton
  13. Tobias Hermle
(2022)
Selective endocytosis controls slit diaphragm maintenance and dynamics in Drosophila nephrocytes
eLife 11:e79037.
https://doi.org/10.7554/eLife.79037

Share this article

https://doi.org/10.7554/eLife.79037

Further reading

    1. Cancer Biology
    2. Cell Biology
    Camille Dantzer, Justine Vaché ... Violaine Moreau
    Research Article

    Immune checkpoint inhibitors have produced encouraging results in cancer patients. However, the majority of ß-catenin-mutated tumors have been described as lacking immune infiltrates and resistant to immunotherapy. The mechanisms by which oncogenic ß-catenin affects immune surveillance remain unclear. Herein, we highlighted the involvement of ß-catenin in the regulation of the exosomal pathway and, by extension, in immune/cancer cell communication in hepatocellular carcinoma (HCC). We showed that mutated ß-catenin represses expression of SDC4 and RAB27A, two main actors in exosome biogenesis, in both liver cancer cell lines and HCC patient samples. Using nanoparticle tracking analysis and live-cell imaging, we further demonstrated that activated ß-catenin represses exosome release. Then, we demonstrated in 3D spheroid models that activation of β-catenin promotes a decrease in immune cell infiltration through a defect in exosome secretion. Taken together, our results provide the first evidence that oncogenic ß-catenin plays a key role in exosome biogenesis. Our study gives new insight into the impact of ß-catenin mutations on tumor microenvironment remodeling, which could lead to the development of new strategies to enhance immunotherapeutic response.

    1. Cell Biology
    Zhongyun Xie, Yongping Chai ... Wei Li
    Research Article

    Asymmetric cell divisions (ACDs) generate two daughter cells with identical genetic information but distinct cell fates through epigenetic mechanisms. However, the process of partitioning different epigenetic information into daughter cells remains unclear. Here, we demonstrate that the nucleosome remodeling and deacetylase (NuRD) complex is asymmetrically segregated into the surviving daughter cell rather than the apoptotic one during ACDs in Caenorhabditis elegans. The absence of NuRD triggers apoptosis via the EGL-1-CED-9-CED-4-CED-3 pathway, while an ectopic gain of NuRD enables apoptotic daughter cells to survive. We identify the vacuolar H+–adenosine triphosphatase (V-ATPase) complex as a crucial regulator of NuRD’s asymmetric segregation. V-ATPase interacts with NuRD and is asymmetrically segregated into the surviving daughter cell. Inhibition of V-ATPase disrupts cytosolic pH asymmetry and NuRD asymmetry. We suggest that asymmetric segregation of V-ATPase may cause distinct acidification levels in the two daughter cells, enabling asymmetric epigenetic inheritance that specifies their respective life-versus-death fates.