Lucas H. Ting

Decoupling Substrate Stiffness, Spread Area, and Micropost Density: A Close Spatial Relationship between Traction Forces and Focal Adhesions

Mechanical cues can influence the manner in which cells generate traction forces and form focal adhesions. The stiffness of a cells substrate and the available area on which it can spread can influence its generation of traction forces, but to what extent these factors are intertwined is unclear. In this study, we used microcontact printing and micropost arrays to control cell spreading, substrate stiffness, and post density to assess their effect on traction forces and focal adhesions. We find that both the spread area and the substrate stiffness influence traction forces in an independent manner, but these factors have opposite effects: cells on stiffer substrates produce higher average forces, whereas cells with larger spread areas generate lower average forces. We show that post density influences the generation of traction forces in a manner that is more dominant than the effect of spread area. Additionally, we observe that focal adhesions respond to spread area, substrate stiffness, and post density in a manner that closely matches the trends seen for traction forces. This work supports the notion that traction forces and focal adhesions have a close relationship in their response to mechanical cues.

Flow mechanotransduction regulates traction forces, intercellular forces, and adherens junctions

Endothelial cells respond to fluid shear stress through mechanotransduction responses that affect their cytoskeleton and cell-cell contacts. Here, endothelial cells were grown as monolayers on arrays of microposts and exposed to laminar or disturbed flow to examine the relationship among traction forces, intercellular forces, and cell-cell junctions. Cells under laminar flow had traction forces that were higher than those under static conditions, whereas cells under disturbed flow had lower traction forces. The response in adhesion junction assembly matched closely with changes in traction forces since adherens junctions were larger in size for laminar flow and smaller for disturbed flow. Treating the cells with calyculin-A to increase myosin phosphorylation and traction forces caused an increase in adherens junction size, whereas Y-27362 cause a decrease in their size. Since tugging forces across cell-cell junctions can promote junctional assembly, we developed a novel approach to measure intercellular forces and found that these forces were higher for laminar flow than for static or disturbed flow. The size of adherens junctions and tight junctions matched closely with intercellular forces for these flow conditions. These results indicate that laminar flow can increase cytoskeletal tension while disturbed flow decreases cytoskeletal tension. Consequently, we found that changes in cytoskeletal tension in response to shear flow conditions can affect intercellular tension, which in turn regulates the assembly of cell-cell junctions.

Micropatterning on Micropost Arrays

Micropatterning of cells can be used in combination with microposts to control cell shape or cell-to-cell interaction while measuring cellular forces. The protocols in this chapter describe how to make SU8 masters for stamps and microposts, how to use soft lithography to replicate these structures in polydimethylsiloxane, and how to functionalize the surface of the microposts for cell attachment.

Effect of Silanization Film Thickness in Soft Lithography of Nanoscale Features

Soft lithography was used to replicate nanoscale features made using electron beam lithography on a polymethylmethacrylate (PMMA) master. The PMMA masters were exposed to fluorinated silane vapors to passivate its surfaces so that polydimethylsiloxane (PDMS) did not permanently bond to the master. From scanning electron microscopy, the silanization process was found to deposit a coating on the master that was a few hundreds of nanometers thick. These silane films partially concealed the nanoscale holes on the PMMA master, causing the soft lithography process to produce PDMS features with dimensions that were significantly reduced. The thickness of the silane films was directly measured on silicon or PMMA masters and was found to increase with exposure time to silane vapors. These findings indicate that the thickness of the silane coatings is a critical parameter when using soft lithography to replicate nanoscale features, and caution should be taken on how long a master is exposed to silane vapors. [DOI: 10.1115/1.4005665]

Platelet Retraction Forces Induced Under High Shear Gradient Activation

Hemodynamics play an important role in the activity of platelets. High shear rate gradients can occur at locations where blood vessels bend, branch, or narrow and can arise at a vascular stent or artificial valve. These shear gradients have been observed to cause platelets to adhere to the vessel wall, leading to their activation and aggregation [1, 2]. High shear gradients can cause a self-sustaining process where platelet aggregation increases the local shear gradient, further causing platelets to adhere and aggregate. This process can become dangerous if the aggregate grows large enough to obstruct the vessel or if it detaches and clogs a vessel downstream.Copyright

Flow Mechanotransduction Regulates Traction Forces, Intercellular Forces, and Adherens Junctions

Endothelial cells react to shear stresses with mechanotransduction responses that modify the cytoskeleton and cell-cell contacts. Cultures of endothelial cells were patterned as monolayers on micropost arrays and different shear flow profiles were applied to investigate the interplay between traction forces, intercellular forces, and adherens junctions (A–C). Cells exposed to laminar flow had elevated traction forces compared to static conditions while cells experiencing unsteady or disturbed flow exhibited lower traction forces (F). Similarly, the size of cell adherens junctions increased after laminar flow and decreased after disturbed flow. Decreasing cytoskeletal tension with Y-27632 decreased the size of adherens junctions (D), while increasing tension through Calyculin-A increased their size (E). A novel approach to measure intercellular forces between cells in the monolayers was developed (G) and these forces were found to be significantly higher for laminar flow than for static or disturbed conditions (H) with adherens junction size reflecting these tension changes.View Large Image | View Hi-Res Image | Download PowerPoint Slide These results indicate that laminar flow can increase cytoskeletal tension while disturbed flow decreases cytoskeletal tension. The corresponding change in cytoskeletal tension under shear can produce intercellular forces that can potentially affect the assembly of adherens junction.

Hemodynamic Shear Regulates Intercellular Forces and Permeability of Endothelial Cell Monolayers

In the cardiovascular system, the flow of blood within the complex vascular network creates hemodynamic shear forces experienced by cells. Situated between the circulating blood and the bulk vascular tissue, the endothelium is a cell monolayer acting as a barrier that protects the underlying arterial tissue. Shear forces have been seen to interact with and regulate endothelial cells through mechanotransduction induced cytoskeletal changes within the cell [1]. Shear forces can be beneficial in cases of laminar flow, which are thought to be atheroprotective by aligning and organizing endothelial cells [2]. However, disturbances to a smooth flow field, caused by vessel bifurcations or obstructions like an implanted stent or a bulging atherosclerotic lesion, can cause recirculation zones to form downstream. In these flow regions, detrimental monolayer remodeling occurs which breaks down the endothelial barrier function [3]. Biochemically, it has been seen that shear forces drive signaling cascades in the Rho/Rac pathways that cascade into morphological changes in the cytoskeleton [4].Copyright

Endothelial cell permeability and alignment quantification under shear flow

Atherosclerosis develops when a breach in the protective endothelium allows macrophages and fatty lipids to enter into the intima. Atherosclerotic plaque material can harden the vessel or constrict blood flow through the vessel. In some cases, the plaque can detach and initiate a cardiac event (1). Hemodynamic shear can act as a mechanical factor that regulates the endothelial barrier by initiating a cellular mechanotransduction response that remodels the structure of individual endothelial cells (2).Copyright