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Abstract: FR-PO095

Dynamin Regulates Membrane Tension in Renal Epithelial Cells by Establishing Architecture of Actin Cortex

Session Information

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Gu, Changkyu, Massachusetts General Hospital/Harvard Medical School, Charlestown, Massachusetts, United States
  • Hagmann, Henning, University Hospital Cologne, Cologne, Germany
  • Collins, Agnieszka, Massachusetts General Hospital/Harvard Medical School, Charlestown, Massachusetts, United States
  • Mukherjee, Kamalika, Massachusetts General Hospital/Harvard Medical School, Charlestown, Massachusetts, United States
  • Stevens, Monica H., Massachusetts General Hospital/Harvard Medical School, Charlestown, Massachusetts, United States
  • Brinkkoetter, Paul T., University Hospital Cologne, Cologne, Germany
  • Sever, Sanja, Massachusetts General Hospital/Harvard Medical School, Charlestown, Massachusetts, United States
Background

The GTPase dynamin is essential for podocyte structure and function as it plays a role in regulating endocytosis and the actin cytoskeleton. Previously, we showed that dynamin can directly bind to actin and regulate actin polymerization. Importantly, our studies demonstrated that pharmacological targeting of dynamin with a small molecule, Bis-T-23 that promotes formation of dynamin oligomerization (DynOLIGO), ameliorated kidney injury in animal models of human kidney disease by recovering functional actin structures in injured podocytes. Recently, dynamin is also reported to orchestrate the global actomyosin cytoskeleton that underlies renal epithelial cell polarity independent from its role in endocytosis. Here, we expand the role of DynOLIGO to include regulation of membrane tension, and thus endocytosis in renal epithelial cells by establishing architecture of actin cortex.

Methods

Polarized renal epithelial cells (Inner Medular Collecting Duct) were used for this study. Atomic force microscopy was utilized to determine membrane tension. Actin polymerization assays were performed with cell lysates from IMCD cells. The structure of actin filaments was evaluated by electron microscopy of negatively stained specimens. Endocytosis was examined in IMCD cells stably expressing eGFP-clathrin light chain by Total Internal Reflection Fluorescence Microscopy, followed by computer-based image analysis.

Results

Using a combination of distinct dynamin mutants and Bis-T-23 we show that DynOLIGO defines the length of actin filaments in conjunction with gelsolin. In addition, we show that dynamin crosslinks actin filaments into distinct structures in conjunction with its oligomerization state and the length of the actin filaments. Importantly, our data demonstrate that DynOLIGO regulates membrane tension most likely by defining and crosslinking cortical actin. Furthermore, dynamin’s effect on cell surface tension indirectly influences the speed of clathrin coated pit maturation in polarized renal epithelial cells.

Conclusion

Our study defines a novel role for DynOLIGO cycle as a direct regulator of membrane tension and indirect regulator of endocytosis by orchestrating actin cortex architecture in polarized renal epithelial cells.

Funding

  • NIDDK Support