Correction to: An Easy-to-Fabricate Cell Stretcher Reveals Density-Dependent Mechanical Regulation of Collective Cell Movements in Epithelia.

[This corrects the article DOI: 10.1007/s12195-021-000689-6.].

An Easy-to-Fabricate Cell Stretcher Reveals Density-Dependent Mechanical Regulation of Collective Cell Movements in Epithelia.

INTRODUCTION: Mechanical forces regulate many facets of cell and tissue biology. Studying the effects of forces on cells requires real-time observations of single- and multi-cell dynamics in tissue models during controlled external mechanical input. Many of the existing devices used to conduct these studies are costly and complicated to fabricate, which reduces the availability of these devices to many laboratories. METHODS: We show how to fabricate a simple, low-cost, uniaxial stretching device, with readily available materials and instruments that is compatible with high-resolution time-lapse microscopy of adherent cell monolayers. In addition, we show how to construct a pressure controller that induces a repeatable degree of stretch in monolayers, as well as a custom MATLAB code to quantify individual cell strains. RESULTS: As an application note using this device, we show that uniaxial stretch slows down cellular movements in a mammalian epithelial monolayer in a cell density-dependent manner. We demonstrate that the effect on cell movement involves the relocalization of myosin downstream of Rho-associated protein kinase (ROCK). CONCLUSIONS: This mechanical device provides a platform for broader involvement of engineers and biologists in this important area of cell and tissue biology. We used this device to demonstrate the mechanical regulation of collective cell movements in epithelia. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00689-6.

E-cadherin and LGN align epithelial cell divisions with tissue tension independently of cell shape.

Tissue morphogenesis requires the coordinated regulation of cellular behavior, which includes the orientation of cell division that defines the position of daughter cells in the tissue. Cell division orientation is instructed by biochemical and mechanical signals from the local tissue environment, but how those signals control mitotic spindle orientation is not fully understood. Here, we tested how mechanical tension across an epithelial monolayer is sensed to orient cell divisions. Tension across Madin-Darby canine kidney cell monolayers was increased by a low level of uniaxial stretch, which oriented cell divisions with the stretch axis irrespective of the orientation of the cell long axis. We demonstrate that stretch-induced division orientation required mechanotransduction through E-cadherin cell-cell adhesions. Increased tension on the E-cadherin complex promoted the junctional recruitment of the protein LGN, a core component of the spindle orientation machinery that binds the cytosolic tail of E-cadherin. Consequently, uniaxial stretch triggered a polarized cortical distribution of LGN. Selective disruption of trans engagement of E-cadherin in an otherwise cohesive cell monolayer, or loss of LGN expression, resulted in randomly oriented cell divisions in the presence of uniaxial stretch. Our findings indicate that E-cadherin plays a key role in sensing polarized tensile forces across the tissue and transducing this information to the spindle orientation machinery to align cell divisions.

Cell adhesion. The minimal cadherin-catenin complex binds to actin filaments under force.

Linkage between the adherens junction (AJ) and the actin cytoskeleton is required for tissue development and homeostasis. In vivo findings indicated that the AJ proteins E-cadherin, ß-catenin, and the filamentous (F)-actin binding protein αE-catenin form a minimal cadherin-catenin complex that binds directly to F-actin. Biochemical studies challenged this model because the purified cadherin-catenin complex does not bind F-actin in solution. Here, we reconciled this difference. Using an optical trap-based assay, we showed that the minimal cadherin-catenin complex formed stable bonds with an actin filament under force. Bond dissociation kinetics can be explained by a catch-bond model in which force shifts the bond from a weakly to a strongly bound state. These results may explain how the cadherin-catenin complex transduces mechanical forces at cell-cell junctions.

ClpX(P) generates mechanical force to unfold and translocate its protein substrates.

AAA(+) unfoldases denature and translocate polypeptides into associated peptidases. We report direct observations of mechanical, force-induced protein unfolding by the ClpX unfoldase from E. coli, alone, and in complex with the ClpP peptidase. ClpX hydrolyzes ATP to generate mechanical force and translocate polypeptides through its central pore. Threading is interrupted by pauses that are found to be off the main translocation pathway. ClpX's translocation velocity is force dependent, reaching a maximum of 80 aa/s near-zero force and vanishing at around 20 pN. ClpX takes 1, 2, or 3 nm steps, suggesting a fundamental step-size of 1 nm and a certain degree of intersubunit coordination. When ClpX encounters a folded protein, it either overcomes this mechanical barrier or slips on the polypeptide before making another unfolding attempt. Binding of ClpP decreases the slip probability and enhances the unfolding efficiency of ClpX. Under the action of ClpXP, GFP unravels cooperatively via a transient intermediate.