Effects of Substrate Stiffness on Actin Network and Nucleus Size in Human Airway Smooth Muscle Cells

Effects of Substrate Stiffness on Actin Network and Nucleus Size in Human Airway Smooth Muscle Cells Team: Grace Hinds, Kairav Maniar Technical Advisors: Bela Suki, Elizabeth Bartolak-Suki Affiliations: BU BME Effects of Substrate Stiffness on Actin Network and Nucleus Mechanotransduction is Airway a quickly growing fieldMuscle of research which connects mechanical forces Size in Human Smooth Cells seen by the cell to the biological responses of the cell. Areas of research for this include how the physical environment affects intracellular signaling, post-translational modifications, and Mechanotransduction a quickly growing field of research which connects mechanical forces protein folding. However,isthe majority of mechanotransduction research studies the biochemical seen by the cell to the biological responses of the cell. Areas of research for this include how the aspects from an empirical perspective. The goal of this senior design project is to characterize physical environment affects intracellular signaling, modifications, and protein mechanical force transduction into the nucleus. To post-translational this end, we developed a computational folding. However, the majority of mechanotransduction research studies the biochemical aspects model (Matlab) which uses a network of linear springs to mimic structures including the actin from an empirical perspective. The goal of this senior design project is to characterize mechanical cytoskeleton and the nucleus in the cell as well as the extracellular matrix (ECM). The model force transduction into the nucleus. To this end, we developed a computational model (Matlab) can be directly matched with images of the cytoskeleton and the nucleus. Mechanical which uses a network of linear springs to mimic structures including the actin cytoskeleton and the equilibrium configuration of the model is then obtained as a function of ECM stiffness. To test nucleus in the cell as well as the extracellular matrix (ECM). The model can be directly matched the model predictions, we replicated the wide range of physiologically and pathologically with images of the cytoskeleton and the nucleus. Mechanical equilibrium configuration of the model relevant stiffnesses of the ECM through engineering polyacrylamide gels of accurately is then obtained as a function of ECM stiffness. To test the model predictions, we replicated the controlled stiffness. The gels were coated with collagen humanofairway smooth muscle cells wide range of physiologically and pathologically relevant and stiffnesses the ECM through engineeron each Thecontrolled cells were fluorescently withwith NucBlue™ Live were ing cultured polyacrylamide gelsstiffness. of accurately stiffness. The gels stained were coated collagen and ReadyProbes™ Reagent (Hoechst 33342) (blue nucleus), MemGlow 488 fluorescently (green cell human airway smooth muscle cells were cultured on each stiffness. The cells were membrane), and SiR700 (red actin cytoskeleton). Using image processing (ImageJ), we stained with NucBlue™ Live ReadyProbes™ Reagent (Hoechst 33342) (blue nucleus), MemGlow quantified the cell average nuclearand to cell size(red ratioactin withcytoskeleton). respect to substrate stiffness. The image 488 (green membrane), SiR700 Using image processing (Imanalysis-based results the were implemented in cell thesize computational modeltowith the ultimate goal to ageJ), we quantified average nuclear to ratio with respect substrate stiffness. The the relative cell to nuclear size in vitro as a function of substrate stiffness to that compare image analysis-based results were implemented in the computational model with the ultimate goal predicted by the model.size Our single imaging-based to compare the computational relative cell to nuclear in vitro as cell a function of substratecomputational stiffness to thatmodeling predicted by the computational Our single computational modeling provides provides insight into how model. mechanical forcescell areimaging-based transmitted via the actin cytoskeleton to the insightFuture into how mechanical forces are this transmitted via with the actin cytoskeleton to the nucleus. Future nucleus. studies should couple approach biochemical assays and traction force studies should couple this approach with biochemical assays and traction force microscopy to betmicroscopy to better understand mechanotransduction in cells. ter understand mechanotransduction in cells.