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Aquaporins01:25

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Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics
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A versatile aquaporin-2 cell system for quantitative temporal expression and live cell imaging.

Mikkel R Holst1, Lene N Nejsum1

  • 1Department of Clinical Medicine, Aarhus University , Aarhus , Denmark.

American Journal of Physiology. Renal Physiology
|May 16, 2019
PubMed
Summary

A new cell system allows precise control over Aquaporin-2 (AQP2) expression and its phospho-mimicking mutants. This system enables detailed study of AQP2

Keywords:
Madin-Darby canine kidney cellsaquaporin-2cAMPchannel traffickingenhanced green fluorescent protein-tagged aquaporin-2imaging tubulating endosomesprotein expressiontime-lapse imagingurine concentration

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Renal Physiology

Background:

  • Aquaporin-2 (AQP2) regulates urine concentration via vasopressin and influences cell migration and epithelial morphogenesis.
  • Existing research lacked a system for controlled AQP2 expression and real-time imaging of its dynamics.
  • Understanding AQP2 regulation is crucial for cellular processes and kidney function.

Purpose of the Study:

  • To develop and validate a cell system for temporal and quantitative control of AQP2 and its phospho-mimicking mutants.
  • To enable real-time imaging of fluorescently tagged AQP2 dynamics.
  • To investigate the role of AQP2 phosphorylation in its cellular localization and endosomal trafficking.

Main Methods:

  • Generation and validation of a Flp-In T-REx Madin-Darby canine kidney cell system.
  • Controlled induction of AQP2 and phospho-mimicking mutants (AQP2-S256A, AQP2-S256D).
  • Time-lapse imaging of enhanced green fluorescent protein (EGFP)-tagged AQP2 and mutants following cAMP stimulation.

Main Results:

  • The cell system demonstrated temporal and quantitative control over AQP2 expression.
  • AQP2 translocated to the plasma membrane and phosphorylated at S256 upon cAMP elevation.
  • Time-lapse imaging revealed AQP2-containing endosome tubulation, which decreased post-cAMP stimulation, with differential effects observed for phospho-mimicking mutants.

Conclusions:

  • The developed cell system provides precise control over AQP2 expression and localization.
  • This system facilitates detailed mechanistic studies of AQP2 regulation, including phosphorylation-dependent endosomal dynamics.
  • It offers a valuable platform for investigating AQP2's role in cell migration and epithelial morphogenesis.