Imaging viscosity of intragranular mucin matrix in cystic fibrosis cells




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


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Purpose: Abnormalities of mucus viscosity play a critical role in the athogenesis of several respiratory diseases, including cystic fibrosis (CF). Currently, there are no approaches to assess the rheological properties of mucin granule matrices in live cells. This is the first example of the use of a molecular rotor, a BODIPY dye, to quantitatively visualize the viscosity of intragranular mucin matrices in a large population of individual granules in differentiated primary bronchial epithelial cells using fluorescence lifetime imaging microscopy. 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 fluorescence lifetime microscopy (FLIM) to image the non-CF and CF. 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: In this work, we demonstrate the use of a simple BODIPY rotor to measure the apparent viscosity of intracellular mucin granules in human bronchial epithelial cells with and without CF. The molecular rotor is readily taken up into mucin granules and can be used to quantify the intracellular viscosity of mucin granules. Additionally, as a control, we use a non-rotor analog of the phenyl-BODIPY probe which is demonstrates little or no change in its fluorescent lifetime. Our results indicate the molecular rotor can be a valuable tool to study and quantify mucus pathology in diseased cells. Conclusion: We demonstrated that BODIPY-rotor could probe intragranular viscosities of CF and non-CF cells. Importantly, two different populations of viscosities were identified in the CF granules as opposed to a single population of viscosities in non-CF granules. This indicates a heterogeneous nature of the CF granules, which might be related to the pathology. Overall, our results suggest that BODIPY viscometers could be viable tools for assessing the viscoelastic properties of mucin matrix within intact granules in live cells. Combining FLIM studies with such molecular viscometers should provide valuable insight into various stages of CF mucus pathogenesis, and potentially could aid in the development of efficient therapeutic approaches to combat the disease.