Mechanics of suspended cells probed by dual optical traps in a confocal microscope
by Florian Schlosser
Date of Examination:2015-07-15
Date of issue:2015-12-02
Advisor:Dr. Florian Rehfeldt
Referee:Dr. Florian Rehfeldt
Referee:Prof. Dr. Sarah Köster
Referee:Prof. Dr. Christoph F. Schmidt
Persistent Address:
http://dx.doi.org/10.53846/goediss-5398
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Abstract
English
Cells are sensitive to mechanical cues from their environment and at the same time generate and transmit forces to their surroundings. In this thesis, we quantitatively measured forces and elastic spring constants of suspended cells. We used a dual optical trap to attach micrometer-sized fibronectin-coated beads to opposite sides of a rounded cell and detected the position fluctuations of the beads with high temporal and spatial resolution. Using a force-feedback mechanism and an acousto-optical deflector we could apply constant or oscillatory external forces to the cell. We found that the elastic response and force generation of cells are strongly governed by their acto-myosin cortex. Cell stiffness decreased substantially with both myosin inhibition by blebbistatin and serum-starvation, but not with microtubule depolymerization by nocodazole. Experiments using giant unilamellar vesicles as a model system showed that the contribution of the lipid envelope to the elastic response of cells is negligible. Force fluctuation experiments showed that cortical forces generated by non-muscle myosin II (NMM II) are present in the range from 0.1 to 10 Hz. While blebbistatin treatment and transfer to serum-free medium strongly reduced the force fluctuations, microtubule depolymerization did not affect them. To be able to track changes in the acto-myosin cytoskeleton, we built a novel setup incorporating a dual optical trap into a Leica SP5 X confocal microscope. We showed that it is possible to detect the force fluctuations that a LifeAct-RFP and non-muscle myosin II-GFP co-transfected cell transmits to two attached beads, and image its acto-myosin cytoskeleton at the same time for up to one hour. The observed filamentous actin network of fibroblast cells in suspension is highly diverse, ranging from cells with only a flat actin cortex to cells that have actin bundles spanning through their whole interior cytosol. We found that cardiac fibroblasts in control medium have a 40 % thicker actin cortex than cardiac fibroblasts treated with 100 µM blebbistatin. We further investigated active and passive mechanical properties of cells used in engineered heart muscle. We found that primary fibroblasts originating from heart, skin and gingiva have comparable spring constants. Cardiac fibroblasts proved to have much higher force fluctuations than the other primary fibroblasts. The amplitude of those force fluctuations is even more strongly relying on force generation of myosin II motors compared to NIH 3T3 cells indicated by a roughly fifty-fold reduction after blebbistatin treatment. We could also prove that it is possible to use the dual optical trap setup to measure contraction forces and beating frequency of cardiomyocyte cells.
Keywords: cell mechanics; optical trap; cell cortex; force fluctuations; active matter
