Biology subjects

Knepper, M.

Publications and source records attributed to Knepper, M..

3 recordsLinked to original sources

bioRxiv2025

GFP Reporter System Reveals Cell-to-Cell Variability in Aquaporin-2 Expression

Vasopressin regulates transcription of the aquaporin-2 gene (Aqp2) in collecting duct principal cells. To investigate regulatory mechanisms in Aqp2 gene transcription, we engineered an Aqp2 reporter cell line using CRISPR/Cas9 to insert a green fluorescent protein (GFP) cassette at the endogenous Aqp2 gene locus in mpkCCD cells. In the absence of dDAVP, a vasopressin analog, these cells exhibited low or undetectable GFP and Aqp2 expression in all cells. dDAVP stimulation (1nM dDAVP for 48hrs) markedly increased both GFP and Aqp2 expression together with reversal upon dDAVP removal. These observations demonstrate that GFP faithfully tracks Aqp2 expression. Interestingly, fewer than 50% of cells express GFP and Aqp2 after dDAVP or forskolin, indicating significant variability even though they were clonally-derived. We flow-sorted the GFP- cells (Aqp2-) and GFP+ cells (Aqp2+), regrew them, and restimulated them separately with dDAVP. Cells originating from GFP- cells gave rise to both GFP- cells and GFP+ cells, and GFP+ cells similarly regenerated both GFP- and GFP+ populations in the same proportion. Flow cytometry analysis of the DNA content showed variability in cell cycle phases, with most GFP+ cells in G0/G1, and more GFP cells in G2/S. RNA-seq analysis of the GFP- and GFP+ cells revealed increased abundance of cell-cycle related transcripts in the GFP- cells. We conclude that: 1) heterogeneity in Aqp2 expression is related to cell cycle state; and 2) the newly generated reporter cell line will likely serve as a useful tool to study Aqp2 transcriptional regulation. NEW & NOTEWORTHYTo investigate regulatory mechanisms in Aqp2 gene transcription, we engineered an Aqp2 reporter cell line using CRISPR/Cas9 to insert a green fluorescent protein (GFP) cassette at the endogenous Aqp2 gene locus in mpkCCD cells. We demonstrate that the GFP reporter accurately and dynamically tracks the expression and regulation of endogenous Aqp2. We reveal that Aqp2 heterogeneity in mpkCCD cells is at least partly driven by differences in cell cycle phase.

systems biology↗

bioRxiv2025

Vasopressin-Dependent β-Catenin Phosphorylation at Ser552 and Branching Structure of Mouse Collecting Duct System

BackgroundPhosphoproteomics studies in both cultured and native collecting duct (CD) cells showed that vasopressin strongly increases PKA-dependent phosphorylation of {beta}-catenin at Ser552. Relatively little is known about the role of Ser552 phosphorylation. MethodsTo address the role of {beta}-catenin Ser552 phosphorylation in the mature renal CD, we have inserted a Ser552Ala mutation in mice using CRISPR-Cas9. ResultsThe mutation did not affect the renal abundance of the vasopressin-regulated water channel aquaporin-2 (AQP2) or urinary osmolality. However, the structure of the CD system was altered. Specifically, the cortical branching ratio (the number of nephrons that merge to form one cortical CD) was reduced from 6.18 {+/-} 0.66 in control mice to 3.33 {+/-} 0.82 in Ser552Ala mutant mice. This was associated with a greater number of cortical and medullary CDs with smaller average diameter. The total number of nephrons (glomerular counts) was not different between control and Ser552Ala mutant mice (both [~]13,500 per kidney). RNA-seq in microdissected cortical CDs of the mice revealed a highly significant enrichment of genes involved in regulation of mitosis and the cell cycle, along with decreases in mRNAs coding for two CDK inhibitor proteins, Cdkn1b and Cdkn1c. At the same time, there were no changes in abundances of major transporter mRNAs, indicative of sustained CD differentiation. A subset of cortical CD cells showed an increase in DNA content, consistent with G2/M cell-cycle arrest. ConclusionsThe observed structural changes in the collecting duct system of adult mice point to a role of vasopressin-mediated post-translational modification of {beta}-catenin at Ser552 in collecting duct development, presumably PKA-mediated Ser552 phosphorylation. We speculate that vasopressin may act to slow or halt branching morphogenesis perinatally and may affect the collecting duct elongation process that normally produces the unbranched region of the CD system in the cortex and outer medulla. Key pointsO_LIVasopressin regulates collecting duct (CD) transport by triggering phosphorylation of multiple proteins including {beta}-catenin at Ser552. C_LIO_LIMutating Ser552 to a non-phosphorylable amino acid in mice resulted in altered CD branching without loss of differentiation in adult CDs. C_LIO_LIThe findings point to a role for {beta}-catenin Ser552 phosphorylation in CD branching and sub-segmental CD elongation. C_LI

physiology↗

bioRxiv2020

Protein kinase A catalytic-α and catalytic-β proteins have non-redundant functions

Vasopressin regulates osmotic water transport in the renal collecting duct by PKA-mediated control of the water channel aquaporin-2 (AQP2). Collecting duct principal cells express two seemingly redundant PKA catalytic subunits, PKA catalytic (PKA-C) and PKA catalytic {beta} (PKA-C{beta}). To identify the roles of these two protein kinases, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA-C or PKA-C{beta} was deleted using CRISPR-Cas9-based genome editing. Controls were cells carried through the genome editing procedure, but without deletion of PKA. TMT mass tagging was used for protein mass spectrometric quantification. Of the 4635 phosphopeptides that were quantified 67 were significantly altered in abundance with PKA-C deletion, while 21 were significantly altered in abundance with PKA-C{beta} deletion. However, only four sites were changed in both. The target proteins identified in PKA-C-null cells were largely associated with cell membranes and membrane vesicles, while target proteins in the PKA-C{beta}-null cells were largely associated with the actin cytoskeleton and cell junctions. In contrast, in vitro incubation of mpkCCD proteins with recombinant PKA-C and PKA-C{beta} resulted in virtually identical phosphorylation changes. In addition, analysis of total protein abundances in the in vivo samples showed that PKA-C deletion resulted in a near disappearance of AQP2 protein, while PKA-C{beta} deletion did not decrease AQP2 abundance. We conclude that PKA-C and PKA-C{beta} serve substantially different functions in renal collecting duct cells and that differences in phosphorylation targets may be due to differences in protein interactions, e.g. mediated by AKAP, C-KAP or PDZ binding.

systems biology↗