Joo Yong Sim

Integrated strain array for cellular mechanobiology studies

We have developed an integrated strain array for cell culture enabling high-throughput mechano-transduction studies. Biocompatible cell culture chambers were integrated with an acrylic pneumatic compartment and microprocessor-based control system. Each element of the array consists of a deformable membrane supported by a cylindrical pillar within a well. For user-prescribed waveforms, the annular region of the deformable membrane is pulled into the well around the pillar under vacuum, causing the pillar-supported region with cultured cells to be stretched biaxially. The optically clear device and pillar-based mechanism of operation enables imaging on standard laboratory microscopes. Straightforward fabrication utilizes off-the-shelf components, soft lithography techniques in polydimethylsiloxane, and laser ablation of acrylic sheets. Proof of compatibility with basic biological assays and standard imaging equipment were accomplished by straining C2C12 skeletal myoblast cells on the device for 6 hours. At higher strains, cells and actin stress fibers realign with a circumferential preference.

Spatial distribution of cell–cell and cell–ECM adhesions regulates force balance while main­taining E-cadherin molecular tension in cell pairs

Cell shape and the spatial distributions of cell–cell and cell–ECM adhesions govern the force balance in cell pairs. Cell–ECM adhesions at the distal ends of cell–cell junctions regulate junction length and the balance of forces across the junction, while molecular tension in E-cadherin remains constant.

Integrated Multifunctional Environmental Sensors

We present the design, microfabrication, and characterization of ten sensors on one silicon die. We demonstrate simultaneous monitoring of multiple environmental parameters, including temperature, humidity, light intensity, pressure, wind speed, wind direction, magnetic field, and acceleration in three axes. Through an integrated design and fabrication process, these ten functions require only six photolithography mask steps. Temperature is measured redundantly using aluminum and doped silicon resistance thermal detectors and a bandgap temperature sensor. Humidity is transduced by the dielectric change of a polymer due to water absorption. Light intensity is measured with a p-n junction photodiode and doped silicon photoresistor. Pressure is transduced using piezoresistor strain gauges on a sealed membrane. Wind speed and direction are measured with two perpendicular hot wire anemometers. Magnetic field strength is measured with a doped Hall effect sensor. Acceleration in three axes is measured using electrostatic comb finger accelerometers, and an additional z-axis accelerometer uses piezoresistor strain gauges. We measured the cross-sensitivity of each function to all other environmental parameters and can use the chips multifunctional capabilities to compensate for these effects. Sensor integration can enable significant cost, size, and power savings over ten individual devices and facilitate deployment in novel applications.

Multi-functional integrated sensors for the environment

We present multi-functional integrated sensors for the environment (MFISEs) combining ten sensor functions on a single silicon die. The purpose of the MFISEs chip is to monitor important environmental parameters such as temperature, humidity and air speed, along with acceleration in three axes. Through a common fabrication process and integrated sensor design, the ten sensing functions required only five photolithography mask steps. To our knowledge, MFISEs demonstrates the highest degree of sensor fusion on a single die. Sensor integration is a key enabler of new applications in mobile electronics and wireless sensor networks, and the potential to use MFISEs for ten sensing functions provides significant cost, size, and power savings over ten individual devices.

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

Significance Tissue morphogenesis requires coordinated regulation of cellular behavior through instructive signals from the local tissue environment, including mechanical forces exerted by neighboring cells. The cell–cell adhesion protein E-cadherin plays an important role in converting tensile forces across the tissue into an intracellular response to regulate actin cytoskeleton organization, reinforce cell–cell adhesion, and activate transcriptional programs. We show E-cadherin also transduces these forces to orient the mitotic spindle, which occurs irrespectively of tension-induced changes in cell shape and instead involves regulation of junctional recruitment of the protein LGN, a core component of the spindle orientation machinery. Because the orientation of cell division controls tissue architecture, these findings support a key role of E-cadherin in mechanical regulation of tissue morphogenesis. 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.

Increasing β-catenin/Wnt3A activity levels drive mechanical strain-induced cell cycle progression through mitosis

Mechanical force and Wnt signaling activate β-catenin-mediated transcription to promote proliferation and tissue expansion. However, it is unknown whether mechanical force and Wnt signaling act independently or synergize to activate β-catenin signaling and cell division. We show that mechanical strain induced Src-dependent phosphorylation of Y654 β-catenin and increased β-catenin-mediated transcription in mammalian MDCK epithelial cells. Under these conditions, cells accumulated in S/G2 (independent of DNA damage) but did not divide. Activating β-catenin through Casein Kinase I inhibition or Wnt3A addition increased β-catenin-mediated transcription and strain-induced accumulation of cells in S/G2. Significantly, only the combination of mechanical strain and Wnt/β-catenin activation triggered cells in S/G2 to divide. These results indicate that strain-induced Src phosphorylation of β-catenin and Wnt-dependent β-catenin stabilization synergize to increase β-catenin-mediated transcription to levels required for mitosis. Thus, local Wnt signaling may fine-tune the effects of global mechanical strain to restrict cell divisions during tissue development and homeostasis. DOI: http://dx.doi.org/10.7554/eLife.19799.001

Controlling cell shape on hydrogels using lift-off protein patterning

Polyacrylamide gels functionalized with extracellular matrix proteins are commonly used as cell culture platforms to evaluate the combined effects of extracellular matrix composition, cell geometry and substrate rigidity on cell physiology. For this purpose, protein transfer onto the surface of polyacrylamide hydrogels must result in geometrically well-resolved micropatterns with homogeneous protein distribution. Yet the outcomes of micropatterning methods have not been pairwise evaluated against these criteria. We report a high-fidelity photoresist lift-off patterning method to pattern ECM proteins on polyacrylamide hydrogels with elastic moduli ranging from 5 to 25 kPa. We directly compare the protein transfer efficiency and pattern geometrical accuracy of this protocol to the widely used microcontact printing method. Lift-off patterning achieves higher protein transfer efficiency, increases pattern accuracy, increases pattern yield, and reduces variability of these factors within arrays of patterns as it bypasses the drying and transfer steps of microcontact printing. We demonstrate that lift-off patterned hydrogels successfully control cell size and shape and enable long-term imaging of actin intracellular structure and lamellipodia dynamics when we culture epithelial cells on these substrates.

Uniaxial cell stretcher enables high resolution live cell imaging

Imaging adherent cells cultured on commercially available stretchable substrates presents challenges for high-magnification objectives. Namely, short working distances between the objective and focal plane, a moving focal plane due to Poissons contraction with stretching, and issues of optical transparency or non-matching refractive indices. Beyond the advantages of visualizing biological structures in detail, we seek to implement specialized high-resolution fluorescence microscopy techniques such as Förster Resonance Energy Transfer (FRET) microscopy while stretching. Enabling FRET imaging of stretched cells provides a powerful tool for modern cell biology and mechanobiology [1]. FRET techniques require high magnification as well as a stable focal plane for imaging. Here, we address this requirement for stretchable substrates with a microfabricated cell strain device suitable for live cell imaging while allowing high-resolution FRET microscopy of cells. We adapt a uniaxial cell stretching concept previously demonstrated by Huh [2] for high magnification imaging by using thin bottom channels and membranes supported above an inverted oil immersion objective. This work presents a major advance for research in cell mechanobiology as it enables direct mechanical actuation combined with imaging of FRET probes engineered to report strained proteins in live cells.

Controlling cell shape on hydrogels using lift-off patterning

Polyacrylamide gels functionalized with extracellular matrix (ECM) proteins are commonly used as cell culture platforms to evaluate the combined effects of ECM composition, cell geometry and substrate rigidity on cell physiology. For this purpose, protein transfer onto the surface of polyacrylamide hydrogels must result in geometrically well-resolved micropatterns with homogeneous protein distribution. Yet the outcomes of micropatterning methods have not been pairwise evaluated against these criteria. We report a high fidelity photoresist lift-off patterning (LOP) method to pattern ECM proteins on polyacrylamide hydrogels ranging from 5 to 25 kPa. We directly compare the protein transfer efficiency and pattern geometrical accuracy of this protocol to the widely used microcontact printing (µCP) method. LOP achieves higher protein transfer efficiency, increases pattern accuracy, and reduces variability of these factors within arrays of patterns as it bypasses the drying and transfer steps of microcontact printing. We demonstrate that lift-off patterned hydrogels successfully control cell size and shape when we culture epithelial cells on these substrates.