High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-ß on the Epithelial-to-Mesenchymal Transition.

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications, however, require direct quantification of these forces. We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems. In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-ß, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-ß exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

Multiplexed, high-throughput measurements of cell contraction and endothelial barrier function.

Vascular leakage, protein exudation, and edema formation are events commonly triggered by inflammation and facilitated by gaps that form between adjacent endothelial cells (ECs) of the vasculature. In such paracellular gap formation, the role of EC contraction is widely implicated, and even therapeutically targeted. However, related measurement approaches remain slow, tedious, and complex to perform. Here, we have developed a multiplexed, high-throughput screen to simultaneously quantify paracellular gaps, EC contractile forces, and to visualize F-actin stress fibers, and VE-cadherin. As proof-of-principle, we examined barrier-protective mechanisms of the Rho-associated kinase inhibitor, Y-27632, and the canonical agonist of the Tie2 receptor, Angiopoietin-1 (Angpt-1). Y-27632 reduced EC contraction and actin stress fiber formation, whereas Angpt-1 did not. Yet both agents reduced thrombin-, LPS-, and TNFα-induced paracellular gap formation. This unexpected result suggests that Angpt-1 can achieve barrier defense without reducing EC contraction, a mechanism that has not been previously described. This insight was enabled by the multiplex nature of the force-based platform. The high-throughput format we describe should accelerate both mechanistic studies and the screening of pharmacological modulators of endothelial barrier function.

Traction Force Screening Enabled by Compliant PDMS Elastomers.

Actomyosin contractility is an essential element of many aspects of cellular biology and manifests as traction forces that cells exert on their surroundings. The central role of these forces makes them a novel principal therapeutic target in diverse diseases. This requires accurate and higher-capacity measurements of traction forces; however, existing methods are largely low throughput, limiting their utility in broader applications. To address this need, we employ Fourier-transform traction force microscopy in a parallelized 96-well format, which we refer to as contractile force screening. Critically, rather than the frequently employed hydrogel polyacrylamide, we fabricate these plates using polydimethylsiloxane rubber. Key to this approach is that the polydimethylsiloxane used is very compliant, with a lower-bound Young's modulus of ∼0.4 kPa. We subdivide these monolithic substrates spatially into biochemically independent wells, creating a uniform multiwell platform for traction force screening. We demonstrate the utility and versatility of this platform by quantifying the compound and dose-dependent contractility responses of human airway smooth muscle cells and retinal pigment epithelial cells. By directly quantifying the endpoint of therapeutic intent, airway-smooth-muscle contractile force, this approach fills an important methodological void in current screening approaches for bronchodilator drug discovery, and, more generally, in measuring contractile response for a broad range of cell types and pathologies.

Basic Fibroblast Growth Factor 2 Is a Determinant of CD4 T Cell-Airway Smooth Muscle Cell Communication through Membrane Conduits.

Activated CD4 T cells connect to airway smooth muscle cells (ASMCs) in vitro via lymphocyte-derived membrane conduits (LMCs) structurally similar to membrane nanotubes with unknown intercellular signals triggering their formation. We examined the structure and function of CD4 T cell-derived LMCs, and we established a role for ASMC-derived basic fibroblast growth factor 2 (FGF2b) and FGF receptor (FGFR)1 in LMC formation. Blocking FGF2b's synthesis and FGFR1 function reduced LMC formation. Mitochondrial flux from ASMCs to T cells was partially FGF2b and FGFR1 dependent. LMC formation by CD4 T cells and mitochondrial transfer from ASMCs was increased in the presence of asthmatic ASMCs that expressed more mRNA for FGF2b compared with normal ASMCs. These observations identify ASMC-derived FGF2b as a factor needed for LMC formation by CD4 T cells, affecting intercellular communication.