Estimation of single stress fiber stiffness in cultured aortic smooth muscle cells under relaxed and contracted states: Its relation to dynamic rearrangement of stress fibers |
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| Authors: | Kazuaki Nagayama Takeo Matsumoto |
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| Institution: | 1. Department of Bioengineering, University of California, Berkeley, CA, 94720, USA;2. Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, 94720, USA;3. UC Berkeley-UCSF Graduate Program in Bioengineering, USA;1. Department of Mechanical Systems Engineering, Nagoya University, Nagoya 464-8603, Japan;2. Department of Micro Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8507, Japan;3. Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan;1. The University of Queensland, Delivery of Drugs and Genes Group (D2G2), The Australian Institute for Bioengineering and Nanotechnology, St Lucia, QLD 4072, Australia;2. ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, The University of Queensland, Australia;3. Vaxxas Pty Ltd, TRI, 37 Kent St, Woolloongabba QLD 4102, Australia;4. School of Mechanical and Mining Engineering, Frank White Building 43, The University of Queensland, Brisbane, QLD 4072, Australia;5. The University of Queensland, Faculty of Medicine and Biomedical Sciences, Royal Brisbane and Women’s Hospital, Herston, Queensland 4006, Australia;1. Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA;2. Department of Mechanics and Engineering Science, Peking University, Beijing 100871, China |
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| Abstract: | For a quantitative analysis of intracellular mechanotransduction, it is crucial to know the mechanical properties of actin stress fibers in situ. Here we measured tensile properties of cultured aortic smooth muscle cells (SMCs) in a quasi-in situ tensile test in relaxed and activated states to estimate stiffness of their single stress fibers (SFs). An SMC cultured on substrates was held using a pair of micropipettes and detached from the substrate while maintaining its in situ cell shape and cytoskeletal integrity. Stretching up to ～15% followed by unloading was repeated three times to stabilize their tension–strain curves in the untreated (relaxed) and 10 μM-serotonin-treated (activated) condition. Cell stiffness defined as the average slope of the loading limb of the stable loops was ～25 and ～40 nN/% in relaxed and activated states, respectively. It decreased to ～10 nN/% following SF disruption with cytochalasin D in both states. The number of SFs in each cell measured with confocal microscopy decreased significantly upon serotonin activation from 21.5±3.8 (mean±SD, n=80) to 17.5±3.9 (n=77). The dynamics of focal adhesions (FAs) were observed in adherent cells using surface reflective interference contrast microscopy. FAs aligned and elongated along the cell major axis following activation and then merged with each other, suggesting that the decrease in SFs was caused by their fusion. Average stiffness of single SFs estimated by the average decrease in whole-cell stiffness following SF disruption divided by the average number of SFs in each cell was ～0.7 and ～1.6 nN/% in the relaxed and activated states, respectively. Stiffening of single SFs following SF activation was remarkably higher than stiffening at the whole-cell level. Results indicate that SFs stiffen not only due to activation of the actomyosin interaction, but also due to their fusion, a finding which would not be obtained from analysis of isolated SFs. |
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