Epithelial cells sense local stiffness via Piezo1 mediated cytoskeletal reorganization.

Local substrate stiffness is one of the major mechanical inputs for tissue organization during its development and remodeling. It is widely recognized that adherent cells use transmembrane proteins (integrins) at focal adhesions to translate ECM mechanical cues into intracellular bioprocess. Here we show that epithelial cells respond to substrate stiffening primarily via actin cytoskeleton organization, that requires activation of mechanosensitive Piezo1 channels. Piezo1 Knockdown cells eliminated the actin stress fibers that formed on stiff substrates, while it had minimal effect on cell morphology and spreading area. Inhibition of Piezo1 channels with GsMTx4 also significantly reduced stiffness-induced F-actin reorganization, suggesting Piezo1 mediated cation current plays a role. Activation of Piezo1 channels with specific agonist (Yoda1) resulted in thickening of F-actin fibers and enlargement of FAs on stiffer substrates, whereas it did not affect the formation of nascent FAs that facilitate spreading on the soft substrates. These results demonstrate that Piezo1 functions as a force sensor that couples with actin cytoskeleton to distinguish the substrate stiffness and facilitate epithelial adaptive remodeling.

Adherent cell remodeling on micropatterns is modulated by Piezo1 channels.

Adherent cells utilize local environmental cues to make decisions on their growth and movement. We have previously shown that HEK293 cells grown on the fibronectin stripe patterns were elongated. Here we show that Piezo1 function is involved in cell spreading. Piezo1 expressing HEK cells plated on fibronectin stripes elongated, while a knockout of Piezo1 eliminated elongation. Inhibiting Piezo1 conductance using GsMTx4 or Gd3+ blocked cell spreading, but the cells grew thin tail-like extensions along the patterns. Images of GFP-tagged Piezo1 showed plaques of Piezo1 moving to the extrusion edges, co-localized with focal adhesions. Surprisingly, in non-spreading cells Piezo1 was located primarily on the nuclear envelope. Inhibiting the Rho-ROCK pathway also reversibly inhibited cell extension indicating that myosin contractility is involved. The growth of thin extrusion tails did not occur in Piezo1 knockout cells suggesting that Piezo1 may have functions besides acting as a cation channel.

Shear stress-induced nuclear shrinkage through activation of Piezo1 channels in epithelial cells.

The cell nucleus responds to mechanical cues with changes in size, morphology and motility. Previous work has shown that external forces couple to nuclei through the cytoskeleton network, but we show here that changes in nuclear shape can be driven solely by calcium levels. Fluid shear stress applied to MDCK cells caused the nuclei to shrink through a Ca2+-dependent signaling pathway. Inhibiting mechanosensitive Piezo1 channels through treatment with GsMTx4 prevented nuclear shrinkage. Piezo1 knockdown also significantly reduced the nuclear shrinkage. Activation of Piezo1 with the agonist Yoda1 caused similar nucleus shrinkage in cells not exposed to shear stress. These results demonstrate that the Piezo1 channel is a key element for transmitting shear force input to nuclei. To ascertain the relative contribution of Ca2+ to cytoskeleton perturbation, we examined F-actin reorganization under shear stress and static conditions, and showed that reorganization of the cytoskeleton is not necessary for nuclear shrinkage. These results emphasize the role of the mechanosensitive channels as primary transducers in force transmission to the nucleus.

E-cadherin mediated lateral interactions between neighbor cells necessary for collective migration.

Collective cell movement is critical in pathological processes such as wound healing and cancer invasion. It entails complex interactions between adjacent cells and between cells-extracellular matrices. Most studies measure the migration patterns and force propagation by placing cells on flat, patterned substrates. The cooperative behavior resulting from cell-cell interactions is not well understood. We have developed a multi-channel microfluidic device that has junctional protein E-cadherin coated onto the sidewalls of the channels that enables the cells' lateral interactions with their neighbors to be studied. Our study reveals that epithelial cells rely on lateral E-cadherin-based adhesions to maintain the cohesion of the group. Cells move faster in narrower channels, but the average velocity along the channels is reduced in E-cadherin coated channels versus non-adhesive channels. We have directly measured the forces in the cross-linking protein, alpha-actinin, using FRET sensors during cell migration, and found that higher tension exists at the cell edges adjacent to the walls coated with E-cadherin, the implication being E-cadherin transmits the shear forces but does not provide a driving force for this migration.

Flow induced adherens junction remodeling driven by cytoskeletal forces.

Adherens junctions (AJs) are a key structural component for tissue organization and function. Under fluid shear stress, AJs exhibit dynamic assembly/disassembly, but how shear stress couples to AJs is unclear. In MDCK cells we measured simultaneously the forces in cytoskeletal α-actinin and the density and length of AJs using a genetically coded optical force sensor, actinin-sstFRET, and fluorescently labeled E-cadherin (E-cad). We found that shear stress of 0.74dyn/cm2 for 3h significantly enhanced E-cad expression at cell-cell contacts and this phenomenon has two phases. The initial formation of segregated AJ plaques coincided with a decrease in cytoskeletal tension, but an increase in tension was necessary for expansion of the plaques and the formation of continuous AJs in the later phase. The changes in cytoskeletal tension and reorganization appear to be an upstream process in response to flow since it occurred in both wild type and dominant negative E-cad cells. Disruption of F-actin with a Rho-ROCK inhibitor eliminated AJ growth under flow. These results delineate the shear stress transduction paths in cultured cells, which helps to understand pathology of a range of diseases that involve dysfunction of E-cadherin.

Vacuum-driven power-free microfluidics utilizing the gas solubility or permeability of polydimethylsiloxane (PDMS).

Suitable pumping methods for flow control remain a major technical hurdle in the path of biomedical microfluidic systems for point-of-care (POC) diagnostics. A vacuum-driven power-free micropumping method provides a promising solution to such a challenge. In this review, we focus on vacuum-driven power-free microfluidics based on the gas solubility or permeability of polydimethylsiloxane (PDMS); degassed PDMS can restore air inside itself due to its high gas solubility or gas permeable nature. PDMS allows the transfer of air into a vacuum through it due to its high gas permeability. Therefore, it is possible to store or transfer air into or through the gas soluble or permeable PDMS in order to withdraw liquids into the embedded dead-end microfluidic channels. This article provides a comprehensive look at the physics of the gas solubility and permeability of PDMS, a systematic review of different types of vacuum-driven power-free microfluidics, and guidelines for designing solubility-based or permeability-based PDMS devices, alongside existing applications. Advanced topics and the outlook in using micropumping that utilizes the gas solubility or permeability of PDMS will be also discussed. We strongly recommend that microfluidics and lab-on-chip (LOC) communities harness vacuum energy to develop smart vacuum-driven microfluidic systems.

Phaseguide-assisted blood separation microfluidic device for point-of-care applications.

We propose a blood separation microfluidic device suitable for point-of-care (POC) applications. By utilizing the high gas permeability of polydimethylsiloxane (PDMS) and phaseguide structures, a simple blood separation device is presented. The device consists of two main parts. A separation chamber with the phaseguide structures, where a sample inlet, a tape-sealed outlet, and a dead-end ring channel are connected, and pneumatic chambers, in which manually operating syringes are plugged. The separation chamber and pneumatic chambers are isolated by a thin PDMS wall. By manually pulling out the plunger of the syringe, a negative pressure is instantaneously generated inside the pneumatic chamber. Due to the gas diffusion from the separation chamber to the neighboring pneumatic chamber through the thin permeable PDMS wall, low pressure can be generated, and then the whole blood at the sample inlets starts to be drawn into the separation chamber and separated through the phaseguide structures. Reversely, after removing the tape at the outlet and manually pushing in the plunger of the syringe, a positive pressure will be created which will cause the air to diffuse back into the ring channel, and therefore allow the separated plasma to be recovered at the outlet on demand. In this paper, we focused on the study of the plasma separation and associated design parameters, such as the PDMS wall thickness, the air permeable overlap area between the separation and pneumatic chambers, and the geometry of the phaseguides. The device required only 2 µl of whole blood but yielding approximately 0.38 µl of separated plasma within 12 min. Without any of the requirements of sophisticated equipment or dilution techniques, we can not only separate the plasma from the whole blood for on-chip analysis but also can push out only the separated plasma to the outlet for off-chip analysis.