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.

Effects of UV-C Radiation on Common Dandelion and Purple Coneflower: First Results

“…17 Photosynthesis decreases for both stomatal (g s ) and non-stomatal inhibition (A), as already observed on tomato. 16 While photosynthesis being compromised in both species, from the comparison between the two species emerged a greater capacity of purple coneflower in the 120-min treatments to maintain the stomata opened, continuing to exchange CO 2 with the water vapor.…”

A Threshold Shear Force for Calcium Influx in an Astrocyte Model of Traumatic Brain Injury

“…Cells withstand mechanical loading through support from cytoskeleton, a heterogeneous, anisotropic collection of dynamically cross-linked structural proteins. [41][42][43][44][45] The deformation of a cell depends on the intrinsic elastic deformation of fibrous cytoskeletal proteins and plastic deformation involving dynamics of reversible cross-linking and cytoskeleton reorganization. 46,47 Slow shear loading engages the plastic processes that significantly modify the local force around the Ca 2 + transducers acting like dashpots in series with the transducer.…”

“…The device was fabricated using soft lithography following the fabrication process described previously. 27 For inlet and outlet, holes were punched through the PDMS at the ends of the chamber. The chip was mounted on a home-made assembly that allowed fluorescence imaging.…”

Molecular force transduction by ion channels – diversity and unifying principles

“…Time-dependent stresses in single molecules have been measured with atomic-force microscopy (AFM), laser traps and force-sensitive fluorescent Förster resonance energy transfer (FRET) probes (Bustamante et al, 2004;Cornish and Ha, 2007;Meng et al, 2011;Meng and Sachs, 2011a;Meng and Sachs, 2011b). AFM and laser traps can measure the mechanics of single molecules in vitro, but FRET probes function in vivo and in vitro and experiments using these probes have revealed that all filamentous proteins in a cell seem to be under resting tension (Grashoff et al, 2010;Brenner et al, 2011;Meng and Sachs, 2011b;Rahimzadeh et al, 2011;Wang et al, 2011;Meng and Sachs, 2012). If proteinaceous fibers are to be used for signaling, it is important that resting tension is maintained (Ingber, 2008) because fibers under tension can transmit signals close to the speed of sound (Na et al, 2008).…”