GFP- coated microparticles to quantify and compare wild- type desmin with known desmin mutations in human heart disease via quantitative live-cell fluorescence Imaging

Al-Aridhi T. GFP- coated microparticles to quantify and compare wild- type desmin with known desmin mutations in human heart disease via quantitative live-cell fluorescence Imaging . Bielefeld: Universität Bielefeld; 2022.Fluorescence imaging and reporter gene technique permit direct non-destructive estimation of many biological processes in living cells. Revealing the full potency of modern microscopy in biology systems requires extracting quantitative information from fluorescence microscopy. There is a persistent need for an unbiased method for reproducible processing and quantifying by fluorescent imaging. The current work provides a novel approach to facilitate the quantification in-vivo using conventional fluorescence microscopy combined with customized Poly (methyl methacrylate) (PMMA) microparticles developed for this purpose. The surface of the microparticles is functionalized with a carboxyl group matrix, which was coupled with four concentrations (10%, 25%, 50%, and 100%) of enhanced green fluorescent protein (EGFP); and the total amount of EGFP bound per single microparticle was determined. EGFP- coated microparticles were detected and imaged by a standard epi-fluorescence inverted microscope combined withOlympus Scan^R automated image acquisition software; it was demonstrated that the amount of coupled EGFP is proportional to a gray intensity value acquired from fluorescence microscopy imaging. A simple algorithm was extracted from a calibration curve generated from a serial illuminance of EGFP- coated microparticles by utilizing the intensity of the four fluorescent gradient microparticles. This novel approach enables quantifying the fluorescently tagged protein within intact live cells. Within the scope of this work, the new technique was tested and applied to direct quantification the abundance of desmin protein in wild-type and three DES missense mutants variants known as DES-p.Ala120Asp (p.A120D), DES-p.Leu136Pro (p.L136P), and DES-p.Asp312Ala (p.D312A), desmin is an essential protein of the intermediate filaments in myocytes, and its mutations are associated with many cardiac and skeletal muscle disorders. C2C12 myoblast cells were transiently transfected with tagged desmin constructs carrying enhanced green fluorescent protein (EGFP) to mimic desmin expression patterns in the striated muscle cell cytoskeleton. I Abstract The microscopy analysis revealed quantitative alterations in the protein expression level, including drastically reduced amounts of desmin protein in the mutants desmin; DES- p.Ala120Asp and DES-p.Leu136Pro. The microscopy results were verified and evaluated by Western blot assay, which illustrates the reliability of the current method as an unbiased, reproducible approach to image processing and quantification. Additionally, cell culture was an ideological model to explore and monitor the effect of the DES-p.Asp312Ala mutation (is not described yet) on the intermediate filament proteins assembly in myocytes. Confocal laser scanning microscope presented a filamentous pattern resembling the DES- wild-type