Structural and molecular interrogation of intact biological systems

Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease

Tuning the Range of Polyacrylamide Gel Stiffness for Mechanobiology Applications

Adjusting the acrylamide monomer and cross-linker content in polyacrylamide gels controls the hydrogel stiffness, yet the reported elastic modulus for the same formulations varies widely and these discrepancies are frequently attributed to different measurement methods. Few studies exist that examine stiffness trends across monomer and cross-linker concentrations using the same characterization platform. In this work, we use Atomic Force Microscopy and analyze force–distance curves to derive the elastic modulus of polyacrylamide hydrogels. We find that gel elastic modulus increases with increasing cross-link concentration until an inflection point, after which gel stiffness decreases with increasing cross-linking. This behavior arises because of the formation of highly cross-linked clusters, which add inhomogeneity and heterogeneity to the network structure, causing the global network to soften even under high cross-linking conditions. We identify these inflection points for three different total polymer formulations. When we alter gelation kinetics by using a low polymerization temperature, we find that gels are stiffer when polymerized at 4 °C compared to room temperature, indicating a complex relationship between gel structure, elasticity, and network formation. We also investigate how gel stiffness changes during storage over 10 days and find that specific gel formulations undergo significant stiffening (1.55 ± 0.13), which may be explained by differences in gel swelling resulting from initial polymerization parameters. Taken together, our study emphasizes the importance of polyacrylamide formulation, polymerization temperature, gelation time, and storage duration in defining the structural and mechanical properties of the polyacrylamide hydrogels

Changes in E-cadherin rigidity sensing regulate cell adhesion.

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin-dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell-cell adhesion assay and live cell imaging of cell-cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell-cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell-cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell-cell adhesion

For whom the cells pull: Hydrogel and micropost devices for measuring traction forces

While performing several functions, adherent cells deform their surrounding substrate via stable adhesions that connect the intracellular cytoskeleton to the extracellular matrix. The traction forces that deform the substrate are studied in mechanotrasduction because they are affected by the mechanics of the extracellular milieu. We review the development and application of two methods widely used to measure traction forces generated by cells on 2D substrates: (i) traction force microscopy with polyacrylamide hydrogels and (ii) calculation of traction forces with arrays of deformable microposts. Measuring forces with these methods relies on measuring substrate displacements and converting them into forces. We describe approaches to determine force from displacements and elaborate on the necessary experimental conditions for this type of analysis. We emphasize device fabrication, mechanical calibration of substrates and covalent attachment of extracellular matrix proteins to substrates as key features in the design of experiments to measure cell traction forces with polyacrylamide hydrogels or microposts. We also report the challenges and achievements in integrating these methods with platforms for the mechanical stimulation of adherent cells. The approaches described here will enable new studies to understand cell mechanical outputs as a function of mechanical inputs and advance the understanding of mechanotransduction mechanisms

3D Microwell Platforms for Control of Single Cell 3D Geometry and Intracellular Organization.

IntroductionCell structure and migration is impacted by the mechanical properties and geometry of the cell adhesive environment. Most studies to date investigating the effects of 3D environments on cells have not controlled geometry at the single-cell level, making it difficult to understand the influence of 3D environmental cues on single cells. Here, we developed microwell platforms to investigate the effects of 2D vs. 3D geometries on single-cell F-actin and nuclear organization.MethodsWe used microfabrication techniques to fabricate three polyacrylamide platforms: 3D microwells with a 3D adhesive environment (3D/3D), 3D microwells with 2D adhesive areas at the bottom only (3D/2D), and flat 2D gels with 2D patterned adhesive areas (2D/2D). We measured geometric swelling and Young's modulus of the platforms. We then cultured C2C12 myoblasts on each platform and evaluated the effects of the engineered microenvironments on F-actin structure and nuclear shape.ResultsWe tuned the mechanical characteristics of the microfabricated platforms by manipulating the gel formulation. Crosslinker ratio strongly influenced geometric swelling whereas total polymer content primarily affected Young's modulus. When comparing cells in these platforms, we found significant effects on F-actin and nuclear structures. Our analysis showed that a 3D/3D environment was necessary to increase actin and nuclear height. A 3D/2D environment was sufficient to increase actin alignment and nuclear aspect ratio compared to a 2D/2D environment.ConclusionsUsing our novel polyacrylamide platforms, we were able to decouple the effects of 3D confinement and adhesive environment, finding that both influenced actin and nuclear structure

For whom the cells pull: Hydrogel and micropost devices for measuring traction forces.

While performing several functions, adherent cells deform their surrounding substrate via stable adhesions that connect the intracellular cytoskeleton to the extracellular matrix. The traction forces that deform the substrate are studied in mechanotrasduction because they are affected by the mechanics of the extracellular milieu. We review the development and application of two methods widely used to measure traction forces generated by cells on 2D substrates: (i) traction force microscopy with polyacrylamide hydrogels and (ii) calculation of traction forces with arrays of deformable microposts. Measuring forces with these methods relies on measuring substrate displacements and converting them into forces. We describe approaches to determine force from displacements and elaborate on the necessary experimental conditions for this type of analysis. We emphasize device fabrication, mechanical calibration of substrates and covalent attachment of extracellular matrix proteins to substrates as key features in the design of experiments to measure cell traction forces with polyacrylamide hydrogels or microposts. We also report the challenges and achievements in integrating these methods with platforms for the mechanical stimulation of adherent cells. The approaches described here will enable new studies to understand cell mechanical outputs as a function of mechanical inputs and advance the understanding of mechanotransduction mechanisms

Quantification of protein transfer efficiency to PAAm gels of varying stiffness.

<p>(A,B) Arrays of 45 μm² square protein patterns on 25 kPa PAAm gels created by LOP and μCP before and after transfer to gel surface. (C) Quantification of protein transfer efficiency from glass coverslips to PAAm gel of varying stiffness. Differences between LOP and μCP for each stiffness are statistically significant (p-value < 2.2E-16, Mann-Whitney-Wilcoxon test). Substantially more protein is transferred from patterns created by photoresist lift-off. Data are represented as box plots. The median, 1^st and 3^rd quartile, and minimum and maximum values are shown, n = 150 for each method and stiffness shown. (D) Overview of μCP method to pattern proteins on PAAm gels.</p

Controlling cell shape on hydrogels using lift-off protein patterning

<div><p>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.</p></div

LOP fabrication of protein patterns on polyacrylamide gels.

<p>(A,B) Photoresist patterns are fabricated by standard contact photolithography on glass coverslips. Inset at right shows array of S1818 photoresist features after development. (C) Unspecific protein adhesion to the resist-patterned coverslip is blocked by incubating with biopassive PLL(20)-g[3.5]-PEG(2) copolymer. (D,E) Following photoresist lift-off, the resulting PLL-g-PEG pattern is backfilled with the ECM protein of interest. Inset at right shows a fluorescence micrograph of labeled gelatin on glass after backfill. (F) To transfer the protein pattern to the PAAm gel, the gel is polymerized between the protein patterned glass coverslip and a silanized coverslip. (G) After gel polymerization, the top coverslip is removed from the PAAm gel. Inset at right shows a fluorescence micrograph of a labeled protein transferred to a PAAm gel. (H) Inset at right shows pairs of epithelial cells on the patterned PAAm gel restricting the geometry of the protein functionalized regions.</p

Comparison of pattern accuracy between LOP and μCP methods.

<p>(A) Average images of 150 binarized protein patterns created by LOP and μCP on 25 kPa gels. (B) Difference images calculated by comparing the average images and the theoretical pattern mask. Edges and corners are resolved substantially better in patterns created by LOP. (C) Theoretical pattern shape with a region highlighted corresponding to where profile column average scans were taken. (D) Profile column average scans across 150 binarized patterns show that the variation in protein signal at the pattern edges is strongly reduced in LOP patterns. Plotted are the median (line), 1^st / 3^rd quartile (box) and 5–95% (whisker) of the probability of protein present across the pattern width.</p