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Abstract: FR-PO768

Liquid-Liquid Phase Separation of Lmx1b Contributes to Its Gene-Regulatory Effects, Including Noncanonical Wnt Signaling in Podocytes

Session Information

Category: Glomerular Diseases

  • 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology

Authors

  • Chen, He, Universitatsklinikum Koln, Koln, Nordrhein-Westfalen, Germany
  • Tillmann, Richard Alexander, Universitatsklinikum Koln, Koln, Nordrhein-Westfalen, Germany
  • Papadakis, Antonios, Exzellenzcluster CECAD in der Universitat zu Koln, Koln, Nordrhein-Westfalen, Germany
  • Wiesner, Eva, Universitatsklinikum Koln, Koln, Nordrhein-Westfalen, Germany
  • Beyer, Andreas, Exzellenzcluster CECAD in der Universitat zu Koln, Koln, Nordrhein-Westfalen, Germany
  • Benzing, Thomas, Universitatsklinikum Koln, Koln, Nordrhein-Westfalen, Germany
  • Kann, Martin, Universitatsklinikum Koln, Koln, Nordrhein-Westfalen, Germany

Group or Team Name

  • Nephrolab.
Background

Lmx1b is a transcription factor (TF) that is essential for podocyte maintenance. However, the function and mechanism of action of Lmx1b in podocytes remain poorly understood. Liquid-liquid phase separation (LLPS) has gained attention as a key mechanism for regulating transcription via the formation of nuclear transcriptional condensates, which is mediated by multivalent interaction between TFs, coactivators and general transcriptional machinery. Here, we investigate if Lmx1b undergoes LLPS as a biophysical mechanism of action and elucidate gene-regulatory effects of Lmx1b loss of function in podocytes in vivo.

Methods

Tamoxifen inducible conditional podocyte-specific Lmx1b knockout mice (Lmx1b-ipko) and cell lines were used as model systems. LLPS was investigated using recombinant proteins in vitro as well as in cell culture by overexpression. Lmx1b-ipko mice were phenotyped by STED microscopy. The gene-regulatory effects of Lmx1b in vivo were assessed in a time-series approach by glomerular Multiome (snRNAseq & snATACseq). The effect of Wnt ligands on the level of calcium and the actin cytoskeleton in podocytes was investigated ex vivo using acute kidney slices from transgenic mice expressing fluorescent actin reporter and calcium sensor.

Results

Tamoxifen induction in Lmx1b-ipko mice resulted in a progressive, time-dependent FSGS phenotype. Here, Lmx1b staining in podocyte nuclei showed speckled pattern in STED, suggesting nuclear condensation formation in vivo. Recombinant Lmx1b can undergo LLPS in vitro. Live cell imaging of eGFP.Lmx1b demonstrated that an intrinsic disordered region in Lmx1b is essential for driving LLPS in cells. Transcriptional effects of Lmx1b revealed by Multiome analysis indicated a progressive dysregulation of the non-canonical Wnt signaling pathway, in particular an upregulation of Wnt5b. In an ex vivo podocyte reporter system, the stimulation of glomeruli using Wnt5b resulted in increased calcium signals and altered actin cytoskeleton indicative of podocyte damage.

Conclusion

We determined that LLPS of Lmx1b is a key mechanism of its gene-regulatory action. Lmx1b-dependent gene regulation can be linked to non-canonical Wnt signaling in podocytes as novel downstream effects for mediating podocyte damage.

Funding

  • Government Support – Non-U.S.