Fibronectin-coated Substrates Research Articles

LAP2β transmits force to upregulate genes via chromatin domain stretching but not compression.

There is increasing evidence that force impacts almost every aspect of cells and tissues in physiology and disease including gene regulation. However, the molecular pathway of force transmission from the nuclear lamina to the chromatin remain largely elusive. Here we employ two different approaches of a local stress on cell apical surface via an RGD (Arg-Gly-Asp)-coated magnetic bead and whole cell deformation at cell basal surface via uniaxial or biaxial deformation of a fibronectin-coated flexible polydimethylsiloxane substrate. We find that nuclear protein LAP2β mediates force transmission from the nuclear lamina to the chromatin. Knocking down LAP2β increases spontaneous movements of the chromatin by reducing tethering of the chromatin and substantially inhibits the magnetic bead-stress or the substrate-deformation induced chromatin domain stretching and the ensuing dihydrofolate reductase (DHFR) gene upregulation. Analysis of DHFR gene-containing chromatin domain alignments along or perpendicular to the direction of the stretching/compressing reveals that the chromatin domain must be stretched and not compressed in order for the gene to be rapidly upregulated. Together these results suggest that external-load induced rapid transcription upregulation originates from chromatin domain stretching but not compressing and depends on the molecular force transmission pathway of LAP2β. STATEMENT OF SIGNIFICANCE: How force regulates gene expression has been elusive. Here we show that the orientation of the chromatin domain relative to the stress direction is crucial in determining if the chromatin domain will be stretched or compressed in response to a cell surface loading. We also show that nuclear protein Lap2b is a critical molecule that mediates force transmission from the nuclear laminar to the chromatin to regulate gene transcription. This study reveals the molecular force transmission pathway for force-induced gene regulation.

Polarized contact behavior in directionally migrating Xenopus gastrula mesendoderm.

The control of cell-cell adhesion and detachment is essential for collective migration and cell rearrangement. Here, we have used the contact behavior of Xenopus gastrula mesoderm explants migrating directionally on ectoderm conditioned substratum to study the regulation of active cell-cell detachment. When colliding laterally, explants repelled each other, whereas they fused front-to-back when aligned in the direction of migration. For this mesoderm polarization by the substratum, we identified three control modules. First, PDGF-A signaling normally suppresses contact-induced collapse of lamellipodia in a polarized manner. Disruption of PDGF-A function, or of Xwnt6, decreased the polarization of explant contact behavior. Second, the Wnt receptor Xfz7 acted upstream of the kinase Pak1 to control explant fusion independently of PDGF-A-promoted lamellipodia stability. Third, ephrinB1 acted with Dishevelled (Dvl) in front-to-back explant fusion. The second and third modules have been identified previously as regulators of tissue separation at the ectoderm-mesoderm boundary. On non-polarizing, fibronectin-coated substratum, they controlled repulsion between explants in the same way as between tissues during boundary formation. However, explant repulsion/fusion responses were reversed on conditioned substratum by the endogenous guidance cues that also control oriented contact inhibition of lamellipodia. Together, control modules and substratum-bound guidance cues combine preferential front-back adhesion and diminished lateral adhesion of cells to promote collective directional mesoderm migration.

Renieramycin T Inhibits Melanoma B16F10 Cell Metastasis and Invasion via Regulating Nrf2 and STAT3 Signaling Pathways.

As one of marine tetrahydroisoquinoline alkaloids, renieramycin T plays a significant role in inhibiting tumor metastasis and invasion. However, the effect of renieramycin T on inflammation-related tumor metastasis and invasion is still unknown, and its mechanisms remain unclear. Here we established an inflammation-related tumor model by using the supernatant of RAW264.7 cells to simulate B16F10 mouse melanoma cells. The results indicate that renieramycin T suppressed RAW264.7 cell supernatant-reduced B16F10 cell adhesion to a fibronectin-coated substrate, migration, and invasion through the matrigel in a concentration-dependent manner. Moreover, Western blot results reveal that renieramycin T attenuated the phosphorylation of STAT3 and down-regulated the expression of Nrf2. Together, the above findings suggest a model of renieramycin T in suppressing B16F10 cancer cell migration and invasion. It may serve as a promising drug for the treatment of cancer metastasis.

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures.

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

Cranberry and Sumac Extracts Exhibit Antibacterial and Anti-Adhesive Effects Against Streptococcus pyogenes.

Group A Streptococci (GAS) or Streptococcus pyogenes is responsible for acute bacterial pharyngitis in children as well as adults. Streptococcal pharyngitis is initiated by successful attachment and colonization of the bacteria, followed by the establishment of the biofilm in various environments. In this study, we examined the antibacterial activities of in-house prepared aqueous and ethanolic extracts of 10 Atlantic Canada fruits in the context of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill kinetics, and adhesion inhibition properties against S. pyogenes. Per our findings, MIC and MBC for all the tested extracts ranged from 0.25 to 8 mg/mL and from 4 to 64 mg/mL, respectively. Accordingly, at 1⁄2 × MBC, cranberry and sumac extracts also lowered the attachment of GAS to the uncoated and fibronectin-coated substratum. Particularly, cranberry and sumac aqueous extracts were more effective against the adhesion of S. pyogenes ATCC 19615 to the fibronectin-coated surface than a clinical strain. In conclusion, ethanolic and aqueous extracts of cranberry and sumac could potentially be incorporated into natural health products designed for the amelioration of strep throat, yet a detailed understanding of its mode of action (e.g., biofilm inhibition and eradication) could pave its path to the field of antibacterial natural health product discovery, design, and development.

Keloid fibroblasts have elevated and dysfunctional mechanotransduction signaling that is independent of TGF-β

BackgroundFibroblasts found in keloid tissues are known to present an altered sensitivity to microenvironmental stimuli. However, the impact of changes in extracellular matrix stiffness on phenotypes of normal fibroblasts (NFs) and keloid fibroblasts (KFs) is poorly understood. ObjectivesInvestigation the impact of matrix stiffness on NFs and KFs mainly via detecting yes-associated protein (YAP) expression. MethodsWe used fibronectin-coated polyacrylamide hydrogel substrates with a range from physiological to pathological stiffness values with or without TGF-β (fibrogenic inducer). Atomic force microscopy was used to measure the stiffness of fibroblasts. Cellular mechanoresponses were screened by immunocytochemistry, Western blot and Luminex assay. ResultsKFs are stiffer than NFs with greater expression of α-SMA. In NFs, YAP nuclear translocation was induced by increasing matrix stiffness as well as by stimulation with TGF-β. In contrast, KFs showed higher baseline levels of nuclear YAP that was not responsive to matrix stiffness or TGF-β. TGF-β1 induced p-SMAD3 in both KFs and NFs, demonstrating the pathway was functional and not hyperactivated in KFs. Moreover, blebbistatin suppressed α-SMA expression and cellular stiffness in KFs, linking the elevated YAP signaling to keloid phenotype. ConclusionsThese data suggest that whilst normal skin fibroblasts respond to matrix stiffness in vitro, keloid fibroblasts have elevated activation of mechanotransduction signaling insensitive to the microenvironment. This elevated signaling appears linked to the expression of α-SMA, suggesting a direct link to disease pathogenesis. These findings suggest changes to keloid fibroblast phenotype related to mechanotransduction contribute to disease and may be a useful therapeutic target.

Inhibition of Neutrophil Secretion Upon Adhesion as a Basis for the Anti-Inflammatory Effect of the Tricyclic Antidepressant Imipramine.

Recent studies demonstrate the involvement of inflammatory processes in the development of depression and the anti-inflammatory effects of antidepressants. Infiltration and adhesion of neutrophils to nerve tissues and their aggressive secretion are considered as possible causes of inflammatory processes in depression. We studied the effect of the antidepressant imipramine on the adhesion and accompanied secretion of neutrophils under control conditions and in the presence of lipopolysaccharides (LPS). As a model of integrin-dependent neutrophil infiltration into tissues, we used integrin-dependent adhesion of neutrophils to the fibronectin-coated substrate. Imipramine inhibited neutrophil adhesion and concomitant secretion of proteins, including matrix metalloproteinase 9 (MMP-9) and neutrophil gelatinase-associated lipocalin (NGAL), which modify the extracellular matrix and basement membranes required for cell migration. Imipramine also significantly and selectively blocked the release of the free amino acid hydroxylysine, a product of lysyl hydroxylase, an enzyme that affects the organization of the extracellular matrix by modifying collagen lysine residues. In contrast, imipramine enhanced the release of ROS by neutrophils during adhesion to fibronectin and stimulated apoptosis. The anti-inflammatory effect of imipramine may be associated with the suppression of neutrophil infiltration and their adhesion to nerve tissues by inhibiting the secretion of neutrophils, which provides these processes.

First person – Wenhong Li

First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Wenhong Li is first author on 'Differential cellular responses to adhesive interactions with galectin-8- and fibronectin-coated substrates', published in JCS. Wenhong is a PhD student in the lab of Prof. Alexander Bershadsky and Prof. Benjamin Geiger at the Department of Immunology, Weizmann Institute of Science, Israel, investigating cell spreading on an animal lectin, galectin-8.

Differential cellular responses to adhesive interactions with galectin-8- and fibronectin-coated substrates.

ABSTRACTThe mechanisms underlying the cellular response to extracellular matrices (ECMs) that consist of multiple adhesive ligands are still poorly understood. Here, we address this topic by monitoring specific cellular responses to two different extracellular adhesion molecules – the main integrin ligand fibronectin and galectin-8, a lectin that binds β-galactoside residues − as well as to mixtures of the two proteins. Compared with cell spreading on fibronectin, cell spreading on galectin-8-coated substrates resulted in increased projected cell area, more-pronounced extension of filopodia and, yet, the inability to form focal adhesions and stress fibers. These differences can be partially reversed by experimental manipulations of small G-proteins of the Rho family and their downstream targets, such as formins, the Arp2/3 complex and Rho kinase. We also show that the physical adhesion of cells to galectin-8 was stronger than adhesion to fibronectin. Notably, galectin-8 and fibronectin differently regulate cell spreading and focal adhesion formation, yet act synergistically to upregulate the number and length of filopodia. The physiological significance of the coherent cellular response to a molecularly complex matrix is discussed.This article has an associated First Person interview with the first author of the paper.

Fluidic substrate as a tool to probe breast cancer cell adaptive behavior in response to fluidity level

Although the roles of elastic components in breast cancer progression have been widely studied, the importance of matrix dissipative elements in regulating breast cancer behavior is still poorly understood. In this study, we designed viscosity-tunable fluidic substrates to investigate the effects of matrix viscosity on the alteration of breast cancer cellular fate using a hydrophobic molten polymer of poly(e-caprolactone-co-D,L-lactide) [P(CL-co-DLLA)] with different levels of fluidity. The high- and low-fluidity substrates used in this study were shown to behave as viscoelastic liquids at physiological temperature. A nonmetastatic breast cancer cell line (MCF-7) was cultured at the interface of the fibronectin-coated substrate, and its behavior towards the substrate fluidity level was thoroughly characterized. Despite fibronectin-mediated cell-substrate interactions, MCF-7 cells show sensitivity to substrate fluidity levels by forming types aggregates of different sizes and structures over time. Accordingly, MCF-7 cells were undergoing senescence on fluidic substrates, as shown by high metabolic activity over time, suppressed proliferation ability, and positive expression of senescence markers. Moreover, senescence implies more resistance towards anticancer drug treatment. This indicates that a fluidic substrate, as a two-dimensional synthetic matrix system, could demonstrate the importance of mechanical cues in redefining cellular function and cellular fate by changing the viscosity of the pure substrate. A fluidic substrate behaving as a hydrophobic viscoelastic liquid was designed as a tool to investigate the role of matrix viscosity on the alteration of breast cancer cellular fate. The fluidity level of fluidic substrate was tuned by modulating the molecular weight of poly(e-caprolactone-co-D,L-lactide). MCF-7 cells responded to the change in fluidity level of fluidic substrates by forming weak attachment. On high-fluidity substrate, MCF-7 cells formed 3D aggregates, while coalesced on low-fluidity substrate. More importantly, the fluidic substrate mechanically promoted senescence of MCF-7 cells regardless the fluidity level of substrate.

Integrin Binding Dynamics Modulate Ligand-Specific Mechanosensing in Mammary Gland Fibroblasts.

SummaryThe link between integrin activity regulation and cellular mechanosensing of tissue rigidity, especially on different extracellular matrix ligands, remains poorly understood. Here, we find that primary mouse mammary gland stromal fibroblasts (MSFs) are able to spread efficiently, generate high forces, and display nuclear YAP on soft collagen-coated substrates, resembling the soft mammary gland tissue. We describe that loss of the integrin inhibitor, SHARPIN, impedes MSF spreading specifically on soft type I collagen but not on fibronectin. Through quantitative experiments and computational modeling, we find that SHARPIN-deficient MSFs display faster force-induced unbinding of adhesions from collagen-coated beads. Faster unbinding, in turn, impairs force transmission in these cells, particularly, at the stiffness optimum observed for wild-type cells. Mechanistically, we link the impaired mechanotransduction of SHARPIN-deficient cells on collagen to reduced levels of collagen-binding integrin α11β1. Thus integrin activity regulation and α11β1 play a role in collagen-specific mechanosensing in MSFs.

Profiling Cell-Matrix Adhesion Using Digitalized Acoustic Streaming.

Profiling the kinetics of cell-matrix adhesion is of great importance to understand many physiological and pathological processes such as morphogenesis, tissue homeostasis, wound healing, and tumorigenesis. Here, we developed a novel digital acoustofluidic device for parallel profiling cell-matrix adhesion at single-cell level. By introduction of localized and uniform acoustic streaming into an open chamber microfluidic device, the adherent cells within the open chamber can be detached by the streaming-induced Stokes drag force. By digital regulation of pulsed acoustic power from a low level to high levels, the hundreds of adherent cells can be ruptured from the fibronectin-coated substrate accordingly, and their adhesive forces (from several pN to several nN) and kinetics can be determined by the applied power and cell incubation time. As a proof-of-concept application for studying cancer metastasis, we applied this technique to measure the adhesion strength and kinetics of human breast cancer cells to extracellular matrix such as fibronectin and compared their metastatic potentials by measuring the rupture force of cancer cells representing malignant (MCF-7 cells and MDA-MB-231 cells) and nonmalignant (MCF-10A cells) states. Our acoustofluidic device is simple, easy to operate, and capable of measuring, in parallel, hundreds of individual cells' adhesion forces with a resolution at the pN level. Thus, we expect this device could be widely used for both fundamental cell biology research as well as development of cancer diagnostics and tissue engineering technologies.

Extracellular matrix type modulates mechanotransduction of stem cells

Extracellular matrix (ECM) is comprised of different types of proteins, which change in composition and ratios during morphogenesis and disease progression. ECM proteins provide cell adhesion and impart mechanical cues to the cells. Increasing substrate stiffness has been shown to induce Yes-associated protein (YAP) translocation from the cytoplasm to the nucleus, yet these mechanistic studies used fibronectin only as the biochemical cue. How varying the types of ECM modulates mechanotransduction of stem cells remains largely unknown. Using polyacrylamide hydrogels with tunable stiffness as substrates, here we conjugated four major ECM proteins commonly used for cell adhesion: fibronectin, collagen I, collagen IV and laminin, and assessed the effects of varying ECM type and density on YAP translocation in human mesenchymal stem cells (hMSCs). For all four ECM types, increasing ECM ligand density alone can induce YAP nuclear translocation without changing substrate stiffness. The ligand threshold for such biochemical ligand-induced YAP translocation differs across ECM types. While stiffness-dependent YAP translocation can be induced by all four ECM types, each ECM requires a different optimized ligand density for this to occur. Using antibody blocking, we further identified integrin subunits specifically involved in mechanotransduction of different ECM types. Finally, we demonstrated that altering ECM type further modulates hMSC osteogenesis without changing substrate stiffness. These findings highlight the important role of ECM type in modulating mechanotransduction and differentiation of stem cells, and call for future mechanistic studies to further elucidate the role of changes in ECM compositions in mediating mechanotransduction during morphogenesis and disease progression. Statement of SignificanceOur study addresses a critical gap of knowledge in mechanobiology. Increasing substrate stiffness has been shown to induce nuclear YAP translocation, yet only on fibronectin-coated substrates. However, extracellular matrix (ECM) is comprised of different protein types. How varying the type of ECM modulates stem cell mechanotransduction remains largely unknown.We here reveal that the choice of ECM type can directly modulate stem cell mechanotransduction, filling this critical gap.This work has broad impacts in mechanobiology and biomaterials, as it provides the first evidence that varying ECM type can impact YAP translocation independent of substrate stiffness, opening doors for a more rational biomaterials design tuning ECM properties to control cell fate for promoting normal development and for preventing disease progression.

Collagen triple helix repeat containing 1a (Cthrc1a) regulates cell adhesion and migration during gastrulation in zebrafish

Cell adhesion and migration are key cell behaviours during gastrulation in early embryos and metastasis in cancers. Cthrc1 is a secreted protein highly conserved among vertebrates; it is upregulated in injured and diseased arteries, as well as in malignant cancers. There is increasing evidence showing that its expression and activity are associated with cancer progression and inflammatory diseases. However, the mechanism by which it regulates cell migration, and its implication during early development remains unclear. Here we show that zebrafish Cthrc1a is expressed in hypoblast cells, and is required for cell adhesion and migration during gastrulation. Knockdown of cthrc1a in whole embryo inhibits epiboly and convergent extension movements, and reduces the elongation of anteroposterior axis. Cell adhesion assay indicates that Cthrc1a is necessary for mesendoderm cells to interact with fibronectin-coated substratum, and to extend polarised cellular protrusions. Moreover, secreted Cthrc1a proteins diffuse efficiently between blastoderm cells and are recruited by neighbouring cells in an integrin-dependent manner. Consistently, there exists a functional interaction between Cthrc1a and integrin β1 in anteroposterior axis elongation. These results provide insight into the function of Cthrc1a in the regulation of cell adhesion and migration during embryonic axis elongation.

Epithelial cells exert differential traction stress in response to substrate stiffness

Epithelial wound healing is essential for maintaining the function and clarity of the cornea. Successful repair after injury involves the coordinated movements of cell sheets over the wounded region. While collective migration has been the focus of studies, the effects that environmental changes have on this form of movement are poorly understood. To examine the role of substrate compliancy on multi-layered epithelial sheet migration, we performed traction force and confocal microscopy to determine differences in traction forces and to examine focal adhesions on synthetic and biological substrates. The leading edges of corneal epithelial sheets undergo retraction or contraction prior to migration, and alterations in the sheet's stiffness are affected by the amount of force exerted by cells at the leading edge. On substrates of 30 kPa, cells exhibited greater and more rapid movement than on substrates of 8 kPa, which are similar to that of the corneal basement membrane. Vinculin and its phosphorylated residue Y1065 were prominent along the basal surface of migrating cells, while Y822 was prominent between neighboring cells along the leading edge. Vinculin localization was diffuse on a substrate where the basement membrane was removed. Furthermore, when cells were cultured on fibronectin-coated acrylamide substrates of 8 and 50 kPa and then wounded, there was an injury-induced phosphorylation of Y1065 and substrate dependent changes in the number and size of vinculin containing focal adhesions. These results demonstrate that changes in substrate stiffness affected traction forces and vinculin dynamics, which potentially could contribute to the delayed healing response associated with certain corneal pathologies.

Biophysical restriction of growth area using a monodispersed gold sphere nanobarrier prolongs the mitotic phase in HeLa cells.

Gold nanoparticles are widely exploited for biological and biotechnical applications owing to their stability, biocompatibility, and known effects on cellular behaviors. Many studies have focused on nanoparticles that are internalized into cells, but extracellular nanoparticles also can regulate cell behavior, a practice known as in-plane surface nanotopography. We demonstrated that nanobarriers composed of morphologically homogeneous gold nanospheres prolonged the mitotic (M) phase in the cervical cancer cell line HeLa without inducing apoptosis. The nanobarrier was formed by electrostatic deposition of nanospheres on a negatively charged, fibronectin-coated substrate. We tested the effects of differently sized nanospheres. Gold nanospheres 42 nm in diameter were found to be non-toxic, while 111 nm nanospheres induced the production of reactive oxygen species, resulting in apoptotic cell death and arrest of cytokinesis. When exposed to sufficient 83 nm gold nanospheres to fabricate a surface nanobarrier, the M phase was delayed but cells proceeded to cytokinesis and the G1 phase. Live-cell imaging showed that the M phase increased by 2.9 h, 2.4 times longer than in control cells. Biophysical analyses indicated that this could be attributed to the specific size of the nanobarrier that physically limited the growth area around the cell.

Polymeric Nanoparticles Limit the Collective Migration of Cellular Aggregates.

Controlling the propagation of primary tumors is fundamental to avoiding the epithelial to mesenchymal transition process leading to the dissemination and seeding of tumor cells throughout the body. Here we demonstrate that nanoparticles (NPs) limit the propagation of cell aggregates of CT26 murine carcinoma cells used as tumor models. The spreading behavior of these aggregates incubated with NPs is studied on fibronectin-coated substrates. The cells spread with the formation of a cell monolayer, the precursor film, around the aggregate. We study the effect of NPs added either during or after the formation of aggregates. We demonstrate that, in both cases, the spreading of the cell monolayer is slowed down in the presence of NPs and occurs only above a threshold concentration that depends on the size and surface chemistry of the NPs. The density of cells in the precursor films, measured by confocal microscopy, shows that the NPs stick cells together. The mechanism of slowdown is explained by the increase in cell–cell interactions due to the NPs adsorbed on the membrane of the cells. The present results demonstrate that NPs can modulate the collective migration of cells; therefore, they may have important implications for cancer treatment.

Hic-5 expression is a major indicator of cancer cell morphology, migration, and plasticity in three-dimensional matrices.

The focal adhesion proteins Hic-5 and paxillin have been previously identified as key regulators of MDA-MB-231 breast cancer cell migration and morphologic mesenchymal-amoeboid plasticity in three-dimensional (3D) extracellular matrices (ECMs). However, their respective roles in other cancer cell types have not been evaluated. Herein, utilizing 3D cell–derived matrices and fibronectin-coated one-dimensional substrates, we show that across a variety of cancer cell lines, the level of Hic-5 expression serves as the major indicator of the cells primary morphology, plasticity, and in vitro invasiveness. Domain mapping studies reveal sites critical to the functions of both Hic-5 and paxillin in regulating phenotype, while ectopic expression of Hic-5 in cell lines with low endogenous levels of the protein is sufficient to induce a Rac1-dependent mesenchymal phenotype and, in turn, increase amoeboid-mesenchymal plasticity and invasion. We show that the activity of vinculin, when coupled to the expression of Hic-5 is required for the mesenchymal morphology in the 3D ECM. Taken together, our results identify Hic-5 as a critical modulator of tumor cell phenotype that could be utilized in predicting tumor cell migratory and invasive behavior in vivo.

Effects of substrate stiffness on dental pulp stromal cells in culture.

Dental pulp stromal cells (DPSCs) can be differentiated down lineages known to either express bone or dentin specific protein markers. Since the differentiation of cells can be heavily influenced by their environment, it may be possible to influence the osteogenic/odontogenic potential of DPSCs by modulating the mechanical properties of substrate on which they are grown. In this study, human DPSCs were grown with and without hydroxyapatite (HA) microparticles on a range of substrates including fibronectin-coated hydrogels and glass substrates, which represented an elastic moduli range of approximately 3 kPa-50 GPa, over a 21-day period. Alkaline phosphatase activity, osteopontin production, and mineralization were monitored. The presence of HA microparticles increased the relative degree of mineralized matrix produced by the cells relative to those in the same substrate and media condition without the HA microparticles. In addition, cultures with cells grown on stiffer substrates had higher ALP activity and higher degree of mineralization than those grown on softer substrates. This study shows that DPSCs are affected by the mechanical properties of their underlying growth substrate and by the presence of HA microparticles. In addition, relatively stiff substrates (>75 kPa) may be required for significant mineralization of these cultures. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1789-1797, 2018.

Investigating Focal Adhesion Substructures by Localization Microscopy

Cells rely on focal adhesions (FAs) to carry out a variety of important tasks, including motion, environmental sensing, and adhesion to the extracellular matrix. Although attaining a fundamental characterization of FAs is a compelling goal, their extensive complexity and small size, which can be below the diffraction limit, have hindered a full understanding. In this study we have used single-molecule localization microscopy (SMLM) to investigate integrin β3 and paxillin in rat embryonic fibroblasts growing on two different extracellular matrix-representing substrates (i.e., fibronectin-coated substrates and specifically biofunctionalized nanopatterned substrates). To quantify the substructure of FAs, we developed a clustering method based on expectation maximization of a Gaussian mixture that accounts for localization uncertainty and background. Analysis of our SMLM data indicates that the structures within FAs, characterized as a Gaussian mixture, typically have areas between 0.01 and 1 μm2, contain 10–100 localizations, and can exhibit substantial eccentricity. Our approach based on SMLM opens new avenues for studying structural and functional biology of molecular assemblies that display substantial varieties in size, shape, and density.