A versatile aquaporin-2 cell system for quantitative temporal expression and live cell imaging

Autor: Lene N. Nejsum, Mikkel R. Holst
Rok vydání: 2019
Předmět:
0301 basic medicine
Time Factors
Physiology
Recombinant Fusion Proteins
Green Fluorescent Proteins
Endosomes
Transfection
urologic and male genital diseases
Time-Lapse Imaging
Protein expression
Madin Darby Canine Kidney Cells
Enhanced green fluorescent protein-tagged aquaporin-2
03 medical and health sciences
Dogs
0302 clinical medicine
Live cell imaging
Cyclic AMP
Animals
Phosphorylation
Aquaporin 2
Epithelial morphogenesis
urogenital system
Chemistry
Time-lapse imaging
Cell Membrane
Madin Darby canine kidney cell
Cell migration
Imaging tubulating endosomes
Madin-Darby canine kidney cells
Cell system
Cell biology
Protein Transport
Antidiuretic hormone-vasopressin
030104 developmental biology
Gene Expression Regulation
Microscopy
Fluorescence
Urine concentration
030220 oncology & carcinogenesis
Mutation
CAMP
Channel trafficking
Protein Processing
Post-Translational
Aquaporin-2
Zdroj: Holst, M R & Nejsum, L N 2019, ' A versatile aquaporin-2 cell system for quantitative temporal expression and live-cell imaging ', American Journal of Physiology: Renal Physiology, vol. 317, no. 1, pp. F124-F132 . https://doi.org/10.1152/ajprenal.00150.2019
ISSN: 1522-1466
1931-857X
DOI: 10.1152/ajprenal.00150.2019
Popis: Aquaporin-2 (AQP2) fine tunes urine concentration in response to the antidiuretic hormone vasopressin. In addition, AQP2 has been suggested to promote cell migration and epithelial morphogenesis. A cell system allowing temporal and quantitative control of expression levels of AQP2 and phospho-mimicking mutants has been missing, as has a system allowing expression of fluorescently tagged AQP2 for time-lapse imaging. In the present study, we generated and validated a Flp-In T-REx Madin-Darby canine kidney cell system for temporal and quantitative control of AQP2 and phospho-mimicking mutants. We verified that expression levels can be temporally and quantitatively controlled and that AQP2 translocated to the plasma membrane in response to elevated cAMP, which also induced S256 phosphorylation. The phospho-mimicking mutants AQP2-S256A and AQP2-S256D localized as previously described, primarily intracellular and to the plasma membrane, respectively. Induction of AQP2 expression in combination with transient, low expression of enhanced green fluorescent protein-tagged AQP2 enabled expression without aggregation and correct translocation in response to elevated cAMP. Interestingly, time-lapse imaging revealed AQP2-containing tubulating endosomes and that tubulation significantly decreased 30 min after cAMP elevation. This was mirrored by the phospho-mimicking mutants AQP2-S256A and AQP2-S256D, where AQP2-S256A-containing endosomes tubulated, whereas AQP2-S256D-containing endosomes did not. Thus, this cell system enables a multitude of cell-based assays warranted to provide deeper insights into the mechanisms of AQP2 regulation and effects on cell migration and epithelial morphogenesis.
Databáze: OpenAIRE
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