MERON MENGISTU1, H. DANIEL OU-YANG2, AND LINDA LOWE-KRENTZ3
DEPARTMENTS OF BIOLOGICAL SCIENCES1 AND PHYSICS2, LEHIGH UNIVERSITY, BETHLEHEM PA 18015
Mechanical forces in the body have been shown to play an important role in regulating morphologies and functions of cells. Therefore, characterizing the mechanical properties of cells is important for understanding many cellular processes, and even the development of disease. In the cardiovascular system, endothelial cells that form the endothelial lining of blood vessels are constantly exposed to forces from the blood flow, i.e. shear stress and pressure forces, which dictate morphologies and functions of these cells, also playing a significant role in the localization of atherosclerotic plaques caused by endothelial cell dysfunction (Ross, R., 1999[i], Cunningham, K and Gothlieb, A. 2005[ii]).We are interested in studying the mechanical properties of endothelial cells, which will provide a better insight into the molecular basis of atherogenesis.
Like all cells, endothelial cells are viscoelastic, exhibiting both liquid-like and solid-like behaviors (Larson, 1999[iii]), allowing them to be capable of remodeling, moving, and dividing, while maintaining their structural integrity (Tempel et al., 1996[iv]). The cytoskeleton, composed of actin filaments, microtubules, and intermediate filaments, is a cell’s mechanical framework and is responsible for most of its mechanical functions. This filamentous network gives rise to an intracellular space that is inhomogeneous and dynamic, making the study of mechanical properties difficult in cells. To include the inhomogeneous and dynamic cytoskeleton in the study of the mechanical properties of endothelial cells, we used the oscillating optical tweezers approach that enabled us to obtain very local mechanical properties within individual endothelial cells to account for intracellular inhomogeneity. With this methodology, we were able to detect local displacements in the order of 1-nm, and collect as many as 100 data points per second, allowing us to evaluate their dynamics. This approach has been validated in studies of homogeneous polyethylene oxide (PEO) gels (Hough and Ou-Yang, 2006[v], Hough and Ou-Yang, 2002[vi])
Using the optical tweezers methodology, we studied the mechanical properties of bovine aortic endothelial cells (BAECs). We measured the frequency-dependent elastic and viscous moduli, G'(?) and G"(?), over a frequency range of 0.1 to 6,000 Hz. Our results showed that the intracellular space of BAECs remained predominantly elastic rather than having a frequency dependence found in non-biological viscoelastic materials. Although highly inhomogeneous, the elasticity measurements in BAECs fell into only 2 groups of values; softer regions with G'~300 dyn/cm2, or stiffer regions with G'~700 dyn/cm2. These measurements also exhibited fluctuations that were not characteristic of non-biological systems, which we hypothesized were due to cellular dynamics. We verified this hypothesis by depleting BAECs of nutrients, which resulted in the fluctuations disappearing. We also disrupted cytoskeletal distribution in BAECs by depolymerizing actin filaments with cytochalasin B, to study its effect on the viscoelasticity of endothelial cells. With increased exposure to this drug, BAECs lost their integrity and underwent drastic morphological changes, and a relative decrease in cell elasticity. Lastly, we measured G' and G" as a function of time, where we compared intracellular dynamics over 120-seconds between cells grown in non-confluent cultures and those grown to confluence. We found fluctuations in viscoelasticity that were more pronounced in isolated BAECs from the non-confluent cultures, confirming our hypothesis that these cells have more room to move around and divide, possibly causing the larger time-variant fluctuations, whereas confluent cells in contact with one another are spatially constrained, and hence have fewer fluctuations. We also determined that these fluctuations were non-random events, and therefore were caused by intracellular dynamics, by computing the autocorrelation function, <G'G'(t)>, of the elastic modulus, G'(?) (Mengistu, M. et al., in prep).
Meron Mengistu is a fourth-year Ph.D. candidate in Molecular Biology, working under the guidance of Dr. Linda Lowe-Krentz. Her research interests are in the field of cell mechanics, a relatively unknown aspect of Biology today, which she is addressing through collaborations with Dr. H. Daniel Ou-Yang from the Physics department, and Dr. Samir Ghadiali from the department of Mechanical Engineering. She is a member of the American Society for Cell Biology, the American Physical Society, National Society of Collegiate Scholars, and Phi Beta Delta International Honors Society. After Lehigh, Meron plans to seek for a postdoctoral position that will allow her to develop as a scientist, for a career in academia or in the biotechnology industry.