Deformable 96-well cell culture plate compatible with high-throughput screening platforms.

Adherent cells such as endothelial cells sense applied mechanical stretch to adapt to changes in their surrounding mechanical environment. Despite numerous studies, signaling pathways underlying the cellular mechanosensing and adaptation remain to be fully elucidated partly because of the lack of tools that allow for a comprehensive screening approach. Conventionally, multi-well cell culture plates of standard configurations are used for comprehensive analyses in cell biology study to identify key molecules in a high-throughput manner. Given that situation, here we design a 96-well cell culture plate made of elastic silicone and mechanically stretchable using a motorized device. Computational analysis suggested that highly uniform stretch can be applied to each of the wells other than the peripheral wells. Elastic image registration-based experimental evaluation on stretch distributions within individual wells revealed the presence of larger variations among wells compared to those in the computational analysis, but a stretch level of 10%-that has been employed in conventional studies on cellular response to stretch-was almost achieved with our setup. We exposed vascular smooth muscle cells to cyclic stretch using the device to demonstrate morphological repolarization of the cells, i.e. typical cellular response to cyclic stretch. Because the deformable multi-well plate validated here is compatible with other high-throughput screening-oriented technologies, we expect this novel system to be utilized for future comprehensive analyses of stretch-related signaling pathways

Representative images of spatially selective cell adhesions by µCPe.

<p>(a) Cells adhere to unpeeled regions (left) but not to peeled regions (right) created by physical contact with an aluminum sheet. (b) Demonstration of the selective cell adhesion at a micro-scale using a copper EM grid with a square mesh length of 54 µm. Cells are restricted to spread within square islands. (c) A high magnification view of the cells on the square micropatterns. (d) Cells outside the micropatterns (allowed to spread freely) observed with the same magnification as that of c. (e) Coating with fluorescent fibrinogen visualizes square adhesive regions. (f) Intensity profile along the line A–B in e. Asterisks represent 54 µm. F-actin and nuclei are shown in green and blue, respectively. Scale bars, 200 µm (a and b); 25 µm (c and d); 200 µm (e).</p

Repeatable use with identical silicon wafers.

<p>(a) Cells adhere to unpeeled regions (left) but not to peeled regions (right) created by physical contact with a silicon wafer. 1st, 2nd, and 10th peelings represent selective cell adhesions using the same silicon stamp without ultrasonic cleaning, after 1st cleaning, and after 9th cleaning, respectively. F-actin and nuclei are shown in green and blue, respectively. Scale bar, 100 µm. (b) Contact angles for the same silicon wafers measured in sequence before the first peeling-off (Initial), after the first peeling-off (1st peeling), after the subsequent cleaning (1st cleaning), after the subsequent second peeling-off (2nd peeling), after the ninth cleaning (9th cleaning), after the tenth peeling-off (10th peeling), and after the tenth cleaning (10th cleaning). Data are represented by mean ± SD (<i>n</i> = 3).</p

Solo, a RhoA-targeting guanine nucleotide exchange factor, is critical for hemidesmosome formation and acinar development in epithelial cells.

Cell-substrate adhesions are essential for various physiological processes, including embryonic development and maintenance of organ functions. Hemidesmosomes (HDs) are multiprotein complexes that attach epithelial cells to the basement membrane. Formation and remodeling of HDs are dependent on the surrounding mechanical environment; however, the upstream signaling mechanisms are not well understood. We recently reported that Solo (also known as ARHGEF40), a guanine nucleotide exchange factor targeting RhoA, binds to keratin8/18 (K8/K18) intermediate filaments, and that their interaction is important for force-induced actin and keratin cytoskeletal reorganization. In this study, we show that Solo co-precipitates with an HD protein, β4-integrin. Co-precipitation assays revealed that the central region (amino acids 330-1057) of Solo binds to the C-terminal region (1451-1752) of β4-integrin. Knockdown of Solo significantly suppressed HD formation in MCF10A mammary epithelial cells. Similarly, knockdown of K18 or treatment with Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed HD formation. As Solo knockdown or Y-27632 treatment is known to disorganize K8/K18 filaments, these results suggest that Solo is involved in HD formation by regulating K8/K18 filament organization via the RhoA-ROCK signaling pathway. We also showed that knockdown of Solo impairs acinar formation in MCF10A cells cultured in 3D Matrigel. In addition, Solo accumulated at the site of traction force generation in 2D-cultured MCF10A cells. Taken together, these results suggest that Solo plays a crucial role in HD formation and acinar development in epithelial cells by regulating mechanical force-induced RhoA activation and keratin filament organization

Piezoelectric actuator-based cell microstretch device with real-time imaging capability

Cellular response to physical stretch has been extensively studied as a regulator of various physiological functions. For live cell microscopy combined with stretch experiments, cells are typically seeded on an extensible elastomer sheet. In this case, the position of the cells of interest tends to shift out of the field of view upon stretch, making real-time imaging of identical cells difficult. To circumvent this situation, here we describe a robust methodology in which these cell shifts are minimized. Cells are plated in a custom-designed stretch chamber with an elastomer sheet of a small cell culture area. The cell-supporting chamber is stretched on an inverted microscope by using a piezoelectric actuator that provides small, but precisely controlled displacements. Even under this small displacement within the filed of view, our device allows the cells to undergo physiologically relevant levels of stretch. Identical cells can thus be continuously observed during stretching, thereby potentially enabling imaging of stretch-triggered fast dynamics

Solo binds to β4-integrin.

<p>(A) Co-immunoprecipitation assays. YFP-Solo was expressed in MCF10A cells and the cell lysates were immunoprecipitated (IP) with an anti-GFP antibody and analyzed by immunoblotting with anti-GFP and anti-β4 antibodies. (B–E) Mapping of the binding regions of Solo and β4. (B) Schematic domain structure of β4 and its deletion mutants used in this study. Numbers denote amino acid residues flanking each region. The binding ability of each fragment to FLAG-Solo is indicated in the right column. Conserved domains are denoted as: vWFA, von Willebrand factor type A; EGF, EGF-like; Calx, Calx-beta; FNIII, fibronectin type III; CS, connecting segment. (C) Co-immunoprecipitation assays of β4 fragments with Solo. YFP-tagged β4 fragment (β4-YFP) and FLAG-tagged Solo-WT were co-expressed in COS-7 cells, and the cell lysates were immunoprecipitated with an anti-FLAG antibody and analyzed by immunoblotting with anti-FLAG and anti-GFP antibodies. Arrowheads indicate the expected positions of YFP-tagged β4 fragments. (D) Schematic domain structure of Solo and its deletion mutants used in this study. The binding ability of each fragment to β4 (1451–1752)-YFP is indicated in the right column. Conserved domains are indicated as Solo, CRAL/TRIO, SPEC (spectrin repeats), DH, and PH domains. (E) Co-immunoprecipitation assays of Solo fragments with β4. FLAG-Solo or its fragments were co-expressed with β4 (1451–1752)-YFP in COS-7 cells, and the cell lysates were immunoprecipitated with an anti-FLAG antibody and analyzed by immunoblotting with anti-FLAG and anti-GFP antibodies. (A, C, and E) These experiments were repeated more than three times and reproducible results were obtained.</p

Localization of Solo at the sites of traction force generation and a model for the role of Solo in hemidesmosome remodeling.

<p>(A) Schematic illustration of the side view of the cell on silicone substrates. Wrinkles appear on the substrate depending on the forces exerted by the cells. (B) Wrinkle formation assay. MCF10A cells were transfected with YFP or YFP-Solo, seeded on a thin Matrigel-coated silicone substrate, and cultured for 24 h. Ventral images of YFP (green) and phase-contrast images were acquired with a confocal microscopy. Red arrows indicate ventral localization of Solo, particularly along the wrinkles. Scale bar, 20 μm. (C) A model for Solo-mediated HD remodeling. Solo localizes at the site of force generation on the ventral surface of epithelial cells and promotes HD formation by activating RhoA signaling and reorganizing keratin networks.</p