Measuring Intracellular Mucin Viscosity in Human Bronchial epithelial cells with Cystic Fibrosis




Ponomarchuk, Olga
Requena, Sebastian
Castillo, Marlius
Rebik, Jonathan
Brochiero, Emmanuelle
GrygorczYk, Ryszard
Dzyuba, Sergei
Gryczynski, Ignacy
Gryczynski, Zygmunt
Borejdo, Julian


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Background: Cystic fibrosis (CF) is a genetic disease which causes mucus to be abnormally thick and viscous. The thick mucus harbors bacteria and particulates and is unable to be cleared by the mucociliary system resulting in respiratory disease. Understanding mucus pathology is critical to understanding and treating diseases like CF. While many factors that influence CF mucus to be unusually thick are known, the question remains if there are differences in the viscosity of the mucus before secretion. Before being secreted, mucus exists as granules in the cell known as mucin. In this work, we examine the viscosity of intracellular mucin of human bronchial epithelial cells with and without cystic fibrosis. Methods: We use a simple fluorescent phenyl-BODIPY rotor molecule which is readily uptaken into mucin granules and exhibits dramatic changes in its fluorescent lifetime as a function of its environments viscosity. To measure the distribution of viscosities in intracellular mucin, we use time-resolved fluorescent microscopy to image the non-CF and CF cells and measure the fluorescent lifetime of the probe in intracellular mucin. We employ a machine learning algorithm to analyze the pictures and use a combination of Python and ImageJ to compute the size and viscosity distribution of intracellular mucin granules. Results: Our results show that our molecular rotor is readily uptaken into mucin granules of human epithelial cells. The changes in fluorescent lifetime are substantial enough to determine the apparent viscosity distribution of intracellular mucin granules. The non-CF cells have a single normally distributed peak in the viscosity distribution centered at 560 cP. The CF cells have a bimodal distribution with a peak at 560 cP and an additional peak at 210 cP. The origin and implications this second low viscosity group of mucin granules in is unclear but may provide biophysical insight into CF mucus pathology. Conclusions: Our phenyl-BODIPY molecular rotor in combination with fluorescent lifetime imaging microscopy is a promising method to study the intracellular viscosity distribution of cells. Our results suggest that there is a distinct difference in the viscosity of mucin granules in non-CF cells versus CF cells. We believe our work will provide a new tool for investigators to study intracellular mucin and examine a variety of mucus related diseases.