Softening of the actin cytoskeleton by inhibition of myosin II
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We have investigated the mechanical properties of fibroblast cells after adding the myosin inhibitor blebbistatin and the Rho-kinase inhibitor Y-27632 by atomic force microscopy (AFM). We have observed a decrease in the elastic modulus from a value of around 20 kPa down to a value around 8 kPa on a time scale of around 30–60 min when applying the myosin inhibitor blebbistatin, whereas the Y-27632 did not show any prominent mechanical effects. From topographic images, we can conclude that, after adding blebbistatin, actin filaments are not visible any more, whereas Y-27632 did not show any prominent effects in cell morphology. This study shows that tension generated by myosin contributes to the cellular stiffness and thus can be observed by measuring the elastic modulus of cells.
KeywordsActin Cytoskeleton Mechanical properties Myosin Fibroblast
The actin cytoskeleton of eukaryotic cells is responsible for cell morphology, but also for many cellular processes including cell migration and cell division. Changes in the architecture and structure of the cytoskeleton, mediated by a large number of actin binding molecules, can result in changes in mechanical properties of this polymeric network. Thus, it has been proposed by Elson et al. to use cellular mechanics as a reporter of cellular processes , which has been implemented first by cell poking . Especially atomic force microscopy (AFM) has been proven to be very helpful in investigating the mechanical response of cells with very high spatial resolution . When taking force curves while scanning the sample (force mapping) , the elastic properties of cells can be probed locally . Especially when studying cellular processes, AFM can help to give new insights into cellular behavior. Examples are the study of the mechanics of cytokinesis  and cell migration [15, 21]. It has been shown that disruption of actin filaments results in a softening of cells .
The mechanical properties of cells are also dependent on cytoskeletal tension generated by myosin, which is important in many cellular processes, e.g., for cell morphology and shape by an interplay of local adhesion (focal adhesion points) and tension between these adhesion points. Cellular tension is mainly produced by non-muscle myosin II, which crosslinks actin filaments and thus can contract the actin cytoskeleton. There are several biochemical pathways to affect the activity of myosin. Blebbistatin directly inhibits myosin function very specifically  and has been used – for instance – to study the function of myosin in cytokinesis . Other drugs that affect the biochemical pathways activating myosin are ML-7 , which is an inhibitor of MLCK (myosin light chain kinase), and Y-27632 , which inhibits Rho-kinase, which also activates myosin. These drugs have been characterized well in biochemical activity assays or biophysical migration assays [5, 13, 13]; however, mechanical data have only been obtained in a few cases , including previous work by AFM, where the influence of ML-7 on cellular stiffness has been investigated . In this study, a decrease of Young’s modulus by a factor of three has been observed in NRK fibroblast cells. These studies  suggest that the two different biochemical pathways activating myosin (via ROCK or via MLCK) are regulated differently, depending on cell type, cell activity, and the cellular process investigated.
Materials and methods
NRK cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Biochrom, Berlin, Germany) with supplemented 10% fetal calf serum (FCS, Biochrom), penicillin and streptomycin (1%), and 1% non-essential-amino acids (NEA). Cells were cultured at 37°C at 5% CO2. Cells were cultured in plastic Petri dishes (Nunclon 947039 & 9407395, Rochester, NY, USA) and plated 1 day before the AFM experiments.
Atomic force microscopy (AFM)
An AFM (MFP3D, Asylum-Research, Santa Barbara, CA, USA) was used to measure the topography and mechanical properties of cells. The AFM was combined with an optical microscope (Axiovert 200, Zeiss, Oberkochen, Germany) to be able to control the position of the AFM tip. We used very soft cantilevers (MLCT, VEECO) with a spring-constant of 0.01 Nm−1. The Petri dishes were mounted in a home-built aluminum holder with vacuum grease, which was fixed by magnets to the base of the AFM. The AFM was put in a polymethylmethacrylate (PMMA) box to maintain 5% CO2. The temperature of the sample was not controlled and was usually around 25°C. The entire microscope was placed on a granite plate suspended with rubber bands from the ceiling to isolate it from vibrations.
Phase-contrast and video microscopy
Long-time control experiments were done by video light microscopy using phase contrast. (Axiovert 135 TV, Zeiss; Time-Lapse-Videorecorder: TL700, Panasonic, Osaka, Japan). A PMMA box was added to the microscope to maintain 5% CO2. Therefore, observations over several hours of live cells were possible. Hence, the effect of the drugs used on cell morphology and the recovery from these drugs could be observed.
We dissolved Rock-Inhibitor (Y-27632, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochlorate) in water at a concentration of 1.04 mM. Blebbistatin (1-phenyl-1,2,3,4-tetrahydro-4-hydroxpyrrolo[2,3-b]-7-methyl-quinolin-4-one) was dissolved in DMSO at a concentration of 3.05 mM. Fifty microliters of either stock solution was added to the medium to the Petri dishes (1 ml), which resulted in a final concentration of 17 μM for Y-27632 and 50 μm for blebbistatin. Drugs were stored in a refrigerator and removed only right before adding them to the Petri dishes because especially blebbistatin is known to be light-sensitive .
Data analysis has been performed using home-built software written in IGOR (Wavemetrics, Lake Oswego, OR, USA). Loading forces were up to 800 pN; however, the range of data analyzed corresponded to loading forces between 100 and 400 pN with typical indentations of 100–200 nm.
Results and discussion
We have evaluated the response to the drugs used here in long-time video phase contrast microscopy over several hours to verify the activity of our drugs and to find a concentration regime where morphological changes could be picked up in most cells. Cell recovery has also been observed after a time period of 1–2 h (data not shown here). In addition, phase contrast microscopy showed similar morphological changes due to the application of drugs, as has been observed in the AFM. Thus, we can conclude that morphological changes in the AFM are not due to the application of mechanical force but due to the drugs applied. These video microscopy studies also confirmed the activity of our drugs, e.g., the activity of blebbistatin has not been modified by the illumination conditions in our setup, although it is known to be light-sensitive and its activity is decreased by light of a wavelength of 488 nm . Because the laser diode of the AFM emitted light in the near infrared, we do not expect any drastic reduction of blebbistatin activity either.
It is very surprising that we have not observed any well-pronounced change in elastic properties after adding the Rho-kinase inhibitor Y-27632 to the medium, although its biochemical activity on myosin activity has been observed in several instances. We have occasionally observed a transient decrease in stiffness, by a factor of 3 to 4; however, this decrease did not last long enough to be measured repeatedly. This may be due to the limited time resolution in our force map measurements (typically 6 min), so that in most cases, we miss these short deviations in stiffness. Another issue is that effects of Y-27632 may be too localized to be detectable. For example, a spatial difference between inhibition of MLCK and ROCK has been observed, especially in focal adhesions . Therefore, the results on Y-27632 should probably be interpreted with some caution here.
The results presented here corroborate results from a previous study, where we have used different drugs (ML7 and KLM) to quantify their influence on cellular stiffness. Because the effects of the drugs used in the previous study are not entirely well understood, we picked two drugs here, where the effects have been characterized very well (blebbistatin) or – in comparison – where an influence via a biochemical pathway can be hypothesized (Y-27632). Because we have observed here a similar decrease in stiffness when adding blebbistatin, we can argue that the reduction in cellular stiffness, in all cases studied, is due to a decrease of myosin activity, resulting in a loss of cytoskeletal tension.
We have investigated the contribution of myosin II activity on the mechanical properties of cells as observed by indentation experiments on live cells by AFM. Blebbistatin, which inhibits myosin II directly, results in a retraction and softening of the cells. Y-27632, which inhibits Rho-kinase and, thus, can decrease myosin activity indirectly, did not show any detectable changes in stiffness. In contrast, in a previous study , inhibition of MLCK by ML-7 resulted in a decrease of stiffness as detected by indentation experiments by AFM. This suggests that myosin activity is regulated differently via MLCK or Rho-kinase dependent on cellular behavior. This difference in activation pathways, which has been found here in mechanical properties of cells, is in agreement with previous studies focusing on membrane protrusion and adhesion of fibroblasts by other methods .