| This thesis describes a comprehensive study of understanding the mechanism of rigidity sensing by quantitative analysis using submicron pillar array substrates. From mechanobiology perspective, we explore and study molecular pathways involved in rigidity and force sensing at cell-matrix adhesions with regard to cancer, regeneration, and development by quantification methods.;In Chapter 2 and 3, we developed fabrication and imaging techniques to enhance the performance of a submicron pillar device in terms of spatial and temporal measurement ability, and we discovered a correlation of rigidity sensing forces and corresponding proteins involved in the early rigidity sensing events. In Chapter 2, we introduced optical effect arising from submicron structure imaging, and we described a technique to identify the correct focal plane of pillar tip by fabricating a substrate with designed-offset pillars. From calibration result, we identified the correct focal plane that was previously overlooked, and verified our findings by other imaging techniques. In Chapter 3, we described several techniques to selectively functionalize elastomeric pillars top and compared these techniques in terms of purposes and fabrication complexity. Techniques introduced in this chapter included direct labeling, such as stamping of fluorescent substances (organic dye, nano-diamond, q-dot) to pillars top, as well as indirect labeling that selectively modify the surface of molds with either metal or fluorescent substances.;In Chapter 4, we examined the characteristics of local contractility forces and identified the components formed a sarcomere like contractile unit (CU) that cells use to sense rigidity. CUs were found to be assembled at cell edge, contain myosin II, alpha-actinin, tropomodulin and tropomyosin (Tm), and resemble sarcomeres in size (∼2 mum) and function. Then we performed quantitative analysis of CUs to evaluate rigidity sensing activity over ∼8 hours time course and found that density of CUs decrease with time after spreading on stiff substrate. However addition of EGF dramatically increased local contraction activity such that about 30% of the total contractility was in the contraction units. This stimulatory effect was only observed on stiff substrate not on soft. Moreover, we find that in the early interactions of cells with rigid substrates that EGFR activity is needed for normal spreading and the assembly of local contraction units in media lacking serum and any soluble EGF.;In Chapter 5, we performed high temporal- and spatial-resolution tracking of contractile forces exerted by cells on sub-micron elastomeric pillars. We found that actomyosin-based sarcomere-like CUs simultaneously moved opposing pillars in net steps of ∼2.5 nm, independent of rigidity. What correlated with rigidity was the number of steps taken to reach a force level that activated recruitment of alpha-actinin to the CUs. When we removed actomyosin restriction by depleting tropomyosin 2.1, we observed larger steps and higher forces that resulted in aberrant rigidity sensing and growth of non-transformed cells on soft matrices. Thus, we conclude that tropomyosin 2.1 acts as a suppressor of growth on soft matrices by supporting proper rigidity sensing. |
