Title

Mapping NKCC1 in the Endocytic Pathway During PKC Activation in Mardin Darby Canine Kidney Cells

Faculty Sponsor

Patrice Bouyer

College

Arts and Sciences

Department/Program

Biology Department

Presentation Type

Poster Presentation

Symposium Date

Spring 5-3-2018

Abstract

Gut clearance represents an important defense mechanism of the gut by flushing out luminal bacteria and toxins. Transepithelial chloride fluid secretion is what drives gut clearance. The basolateral Na-K-2Cl cotransporter 1 (NKCC1) is the main mechanism for loading cells with chloride for its secretion by apical chloride channels (e.g., cystic fibrosis transmembrane regulator). We have previously shown that protein kinase C (PKC) activation causes internalization of NKCC1, thus blunting chloride secretion. However, the fate of NKCC1 remains unknown. To determine if NKCC1 is recycled or degraded, we used Mardin Darby Canine Kidney (MDCK) cells that stably expresses eGFP-NKCC1 to map the endocytic pathway. For immunocytochemistry, MDCK cells were cultured on coverslips in a six-well plate until confluence. Cells were exposed to either phorbol 12-myristate 13-acetate (PMA), an activator of PKC, or DMSO (vehicle). Cells were fixed with 1% paraformaldehyde, incubated with specific primary antibody against endosomal markers, and mounted for immunofluorescence. Images were acquired with an Olympus compound microscope equipped for fluorescence and processed using ImageJ. In these experiments, we did not find colocalization of NKCC1 with Rab5, a marker of the early endosome. We found occasional colocalization of NKCC1 and Rab11, a marker of vesicles recycling to the plasma membrane. Finally, we did not find colocalization of NKCC1 with either LAMP1 or P20S markers of the lysosome and the proteasome. Our results suggest that some NKCC1, after internalization recycles to the membrane in MDCK. Further investigation will be needed to determine the fate on NKCC1 in the endocytic pathway.

Biographical Information about Author(s)

I graduated from Whitko High School as valedictorian in 2016. From 2016 to now, I have attended Valparaiso University where I am majoring in Biology and Chemistry. I plan to graduate from Valpo in May of 2019. I will then further my education by attending dental school. Upon completion, I plan to begin my career as a dentist.

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