BioProject

Atypical PKCiota/lambda is a key effector of the PAR polarity complex which is considered a master determinant of apico-basal polarity in single-layered epithelial cells. More...

Atypical PKCiota/lambda is a key effector of the PAR polarity complex which is considered a master determinant of apico-basal polarity in single-layered epithelial cells. In polarized epithelial cells aPKC is exquisitely localized by the PAR6 partner to a belt nearby the tight junctions. Surprisingly, tissue specific knock out of the transgenic floxed version with villin-CRE showed a chronic inflammation phenotype. The observations were made in three different independent transgenic mouse lines and published by three different groups including ours (PMID: 27226486; PMID: 30552022; PMID: 21744423 ), which is reassuring for reproducibility. An additional double KO in both aPKC isoforms further confirmed the results (PMID: 30552022 ). The animals were viable and there was not strong diarrheal or constipation phenotype characteristic of polarity defects in intestinal epithelia. With few exceptions such as ezrin localization, apical polarity markers were generally well polarized. There were changes in cytokine expression by epithelial cells and infiltration of CD8+ T cells (PMID: 27226486; PMID: 30552022). The goal of the study was to get an unbiased catalog of the transcriptional changes induced by aPKC iota/lambda defect in intestinal epithelial cells to understand the ontology of gene expression downstream of the PAR polarity complex. The results are consistent with broad changes in NF-kB and IFN dependent transcriptional pathways, possibly mediated by ROCK and Med17 downstream of aPKCiota/lambda (PMID: 2722648; PMID: 33596087). This study was published in PMID: 33596087. Overall design: Mouse intestine was extracted from Prkci flox/flox (control, samples 784(M) 787(F) 794(M)) and co-housed endogamic Prkci flox/flox x Villin-CRE (experimental conditional KO, 826(M) 861(F) 867(M)) according to IACUC-approved protocol. Immediately after euthanasia, 10 cm segments of jejunum-ileum were cut. Several washes of the lumen were performed using PBS supplemented with 10 mM glucose, 30 mM EDTA, 5 mM DTT to remove debris and mucus. Following washes, each segment of intestine was clamped with the same ice-cold solution for 20 minutes. Intestinal mucosae were squeezed by exerting gentle pressure with tweezers and drained by releasing one clamp into sample tube. After a quick spin down, RNA inhibitor (Qiagen inc., Germantown, MD) was immediately added to the pellet. This procedure was repeated at least 5 times until microscopic visualization of the cell suspension showed mostly epithelial cell clumps. Total RNA was extracted using 1 mL TRIzol® Reagent and vigorously homogenized several times using 3 ml syringe (needles 18G11/2 and 20G11/2). Samples were then incubated at room temperature to permit complete dissociation of the nucleoprotein complex before adding chloroform (0.2 mL/mL phenol). Total RNA was then precipitated with isopropanol from the aqueous phase followed by 10 minutes centrifugation 12,000g at 4°C. Pellets were washed with 75% Ethanol, centrifuged for 5 minutes at 7500g, air-dried and finally resuspended in RNase-free water. All RNA samples were subjected to DNase Treatment and Removal Reagents following manufacturer’s instructions (Ambion by Life Technologies provided by Thermofisher Scientific, Waltham, MA). RNA samples were then stored at -80C and submitted for poly-A selection and Illumina NextSeq/NovaSeq NGS technology RNAseq. Less...