title: Real-time observation of flow-induced cytoskeletal stress in living cells.
abstract: The mechanical stress due to shear flow has profound effects on cell proliferation, transport, gene expression, and apoptosis. The mechanisms for flow sensing and transduction are unclear, but it is postulated that fluid flow pulls upon the apical surface, and the resulting stress is eventually transmitted through the cytoskeleton to adhesion plaques on the basal surface. Here we report a direct observation of this flow-induced stress in the cytoskeleton in living cells using a parallel plate microfluidic chip with a fluorescence resonance energy transfer (FRET)-based mechanical stress sensor in actinin. The sensing cassette was genetically inserted into the cytoskeletal host protein and transfected into Madin-Darby canine kidney cells. A shear stress of 10 dyn/cm(2) resulted in a rapid increase in the FRET ratio indicating a decrease in stress across actinin with flow. The effect was reversible, and cells were able to respond to repeated stimulation and showed adaptive changes in the cytoskeleton. Flow-induced Ca(2+) elevation did not affect the response, suggesting that flow-induced changes in actinin stress are insensitive to intracellular Ca(2+) level. The reduction in FRET ratio suggests actin filaments are under normal compression in the presence of flow shear stress due to changes in cell shape, and/or actinin is not in series with actin. Treatment with cytochalasin-D that disrupts F-actin reduced prestress and the response to flow. The FRET/flow method is capable of resolving changes of stress in multiple proteins with optical spatial resolution and time resolution >1 Hz. This promises to provide insight into the force distribution and transduction in all cells.
authors.affiliation: Department of Physiology and Biophysics, State University of New York-Buffalo, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Rahimzadeh
authors.forename: Fanjie
authors.initials: F
authors.lastname: Meng
authors.forename: Fredrick
authors.initials: F
authors.lastname: Sachs
authors.forename: Jianbin
authors.initials: J
authors.lastname: Wang
authors.forename: Deepika
authors.initials: D
authors.lastname: Verma
authors.forename: Susan Z
authors.initials: SZ
authors.lastname: Hua
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D000185:Actinin / Q000235:genetics / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D020013:Calcium Signaling / Q000502:physiology; D002460:Cell Line; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D004285:Dogs; D055119:Elastic Modulus / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D046210:Microfluidic Analytical Techniques / Q000379:methods; D044085:Microfluidics; D008856:Microscopy, Fluorescence; D012212:Rheology; D013314:Stress, Mechanical; D014162:Transfection; D014783:Viscosity
chemical_list: D000199:Actins; D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D000185:Actinin; D049452:Green Fluorescent Proteins; D015638:Cytochalasin D