Lewis W. Francis

Atomic force microscopy comes of age

AFM (atomic force microscopy) analysis, both of fixed cells, and live cells in physiological environments, is set to offer a step change in the research of cellular function. With the ability to map cell topography and morphology, provide structural details of surface proteins and their expression patterns and to detect pico‐Newton force interactions, AFM represents an exciting addition to the arsenal of the cell biologist. With the explosion of new applications, and the advent of combined instrumentation such as AFM—confocal systems, the biological application of AFM has come of age. The use of AFM in the area of biomedical research has been proposed for some time, and is one where a significant impact could be made. Fixed cell analysis provides qualitative and quantitative subcellular and surface data capable of revealing new biomarkers in medical pathologies. Image height and contrast, surface roughness, fractal, volume and force analysis provide a platform for the multiparameter analysis of cell and protein functions. Here, we review the current status of AFM in the field and discuss the important contribution AFM is poised to make in the understanding of biological systems.

High-resolution imaging using a novel atomic force microscope and confocal laser scanning microscope hybrid instrument: essential sample preparation aspects

The recent data explosion in global gene expression profiling and proteomics has resulted in a need to determine the mechanistic role of biomarker signatures in pathogenicity. Consequently, elaborate technologies are required to assess increasingly smaller sub-cellular compartments and constituents. We describe the development, evaluation and application of an efficient sample preparation methodology to facilitate coupled atomic force microscopy and confocal laser scanning microscopy (AFM–CLSM), providing a novel means of concurrent high-resolution structural and fluorescence imaging. Due to their fragile nature and nanoscale dimensions, filopodia were selected as a model to develop the procedure that maximised fluorescence response, while maintaining epithelial cell ultra-structure. Fixation with ultra-pure methanol-free formaldehyde coupled to quantum dot nanocrystal labelling proved to be vital in achieving high quality AFM–CLSM images. We demonstrated for the first time that filopodia have a “quilted” surface structure. Additionally, high ultra-structural ridges on the apical cell surface resolved by AFM corresponded to punctate moesin clusters, representing direct visualisation of moesin linkages between transmembrane proteins and the cytoskeleton. The capacity of this novel multi-modal imaging technique to probe topography, molecular composition and biophysical properties of ultra-structural features therefore provides unique information that will significantly contribute to our understanding of cellular structure–function relationships.

In vitro growth factor-induced bio engineering of mature articular cartilage

Articular cartilage maturation is the postnatal development process that adapts joint surfaces to their site-specific biomechanical demands. Maturation involves gross morphological changes that occur through a process of synchronised growth and resorption of cartilage and generally ends at sexual maturity. The inability to induce maturation in biomaterial constructs designed for cartilage repair has been cited as a major cause for their failure in producing persistent cell-based repair of joint lesions. The combination of growth factors FGF2 and TGFβ1 induces accelerated articular cartilage maturation in vitro such that many molecular and morphological characteristics of tissue maturation are observable. We hypothesised that experimental growth factor-induced maturation of immature cartilage would result in a biophysical and biochemical composition consistent with a mature phenotype. Using native immature and mature cartilage as reference, we observed that growth factor-treated immature cartilages displayed increased nano-compressive stiffness, decreased surface adhesion, decreased water content, increased collagen content and smoother surfaces, correlating with a convergence to the mature cartilage phenotype. Furthermore, increased gene expression of surface structural protein collagen type I in growth factor-treated explants compared to reference cartilages demonstrates that they are still in the dynamic phase of the postnatal developmental transition. These data provide a basis for understanding the regulation of postnatal maturation of articular cartilage and the application of growth factor-induced maturation in vitro and in vivo in order to repair and regenerate cartilage defects.

Short-term calorie restriction enhances adult hippocampal neurogenesis and remote fear memory in a Ghsr-dependent manner

Graphical abstract

Suppression of Mediator is regulated by Cdk8-dependent Grr1 turnover of the Med3 coactivator

Significance Mediator is a megadalton multisubunit molecular switchboard involved in gene regulation in eukaryotes and is structurally conserved between species. It bridges the general transcription machinery and function-specific DNA binding proteins. It plays a dynamic role in regulating a wide range of processes, involving, for example, thyroid and vitamin D receptors. The role of Mediator appears to be in the fine tuning of the activation and repression of gene expression in many organisms, yet the underlying mechanisms of how its own function is regulated remains to be unraveled. Here we demonstrate how Mediator autoregulates its own function by cross-talk between the tail module and the Cdk8 kinase module in an active process involving priming of the mediator component Med3 for ubiquitin-ligase (Grr1)–mediated degradation by Cdk8 phosphorylation. Mediator, an evolutionary conserved large multisubunit protein complex with a central role in regulating RNA polymerase II–transcribed genes, serves as a molecular switchboard at the interface between DNA binding transcription factors and the general transcription machinery. Mediator subunits include the Cdk8 module, which has both positive and negative effects on activator-dependent transcription through the activity of the cyclin-dependent kinase Cdk8, and the tail module, which is required for positive and negative regulation of transcription, correct preinitiation complex formation in basal and activated transcription, and Mediator recruitment. Currently, the molecular mechanisms governing Mediator function remain largely undefined. Here we demonstrate an autoregulatory mechanism used by Mediator to repress transcription through the activity of distinct components of different modules. We show that the function of the tail module component Med3, which is required for transcription activation, is suppressed by the kinase activity of the Cdk8 module. Med3 interacts with, and is phosphorylated by, Cdk8; site-specific phosphorylation triggers interaction with and degradation by the Grr1 ubiquitin ligase, thereby preventing transcription activation. This active repression mechanism involving Grr1-dependent ubiquitination of Med3 offers a rationale for the substoichiometric levels of the tail module that are found in purified Mediator and the corresponding increase in tail components seen in cdk8 mutants.

Progesterone induces nano-scale molecular modifications on endometrial epithelial cell surfaces.

Background information. The endometrial epithelial cell membrane is a key interface in female reproductive biology. Steroid hormones play a predominant role in cyclic changes which occur at this interface during the female menstrual cycle. Specific changes in the morphology of the endometrial epithelial cell surface become apparent with the epithelial transition that drives the switch from a non‐receptive to receptive surface due to the action of progesterone on an oestrogen primed tissue. AFM (atomic force microscopy) allows the high‐resolution characterization of the endometrial epithelial cell surface. Its contact probe mechanism enables a unique imaging method that requires little sample preparation, yielding topographical and morphological characterization. By stiffening the cell membrane, low concentrations of fixatives allow the surface detail of the cell to be resolved while preserving fine ultra‐structural details for analysis.

Chondroitin Sulfate Immobilized on a Biomimetic Scaffold Modulates Inflammation While Driving Chondrogenesis

Costs associated with degenerative inflammatory conditions of articular cartilage are exponentially increasing in the aging population, and evidence shows a strong clinical need for innovative therapies. Stem cell‐based therapies represent a promising strategy for the treatment of innumerable diseases. Their regenerative potential is undeniable, and it has been widely exploited in many tissue‐engineering approaches, especially for bone and cartilage repair. Their immune‐modulatory capacities in particular make stem cell‐based therapeutics an attractive option for treating inflammatory diseases. However, because of their great plasticity, mesenchymal stem cells (MSCs) are susceptible to different external factors. Biomaterials capable of concurrently providing physical support to cells while acting as synthetic extracellular matrix have been established as a valuable strategy in cartilage repair. Here we propose a chondroitin sulfate‐based biomimetic scaffold that recapitulates the physicochemical features of the chondrogenic niche and retains MSC immunosuppressive potential in vitro, either in response to a proinflammatory cytokine or in the presence of stimulated peripheral blood mononuclear cells. In both cases, a significant increase in the production of molecules associated with immunosuppression (nitric oxide and prostaglandins), as well as in the expression of their inducible enzymes (iNos, Pges, Cox‐2, and Tgf‐β). When implanted subcutaneously in rats, our scaffold revealed a reduced infiltration of leukocytes at 24 hours, which correlated with a greater upregulation of genes involved in inflammatory cell apoptotic processes. In support of its effective use in tissue‐engineering applications of cartilage repair, the potential of the proposed platform to drive chondrogenic and osteogenic differentiation of MSC was also proven.

Optimized sample preparation for high-resolution AFM characterization of fixed human cells

Atomic force microscopy enables the simultaneous acquisition of high‐resolution topographical and biophysical data allowing integrated analysis of cell surfaces during development and pathogenesis, and, critically, can link molecular and biophysical events. Here we used atomic force microscopy to analyse endometrial epithelial cells and neuronally differentiated P19 cells. Optimized reproducible sample preparation techniques enabled micro‐ and nanoscale multi‐parameter analysis. Comparative analysis using atomic force microscopy and scanning electron microscopy demonstrated the utility of atomic force microscopy for examining tissue morphology, and its ability to generate data allowing differentiation of cells from different origins to be monitored. At low resolution atomic force microscopy produced topographic data complementary to scanning electron microscopy images, whilst at high resolution atomic force microscopy captured novel cell surface structural detail for both epithelial and neuronal cell types. Analysis of surface roughness provided biophysical data which enabled qualitative and quantitative differences between samples to be measured. This study provides an important optimization of sample preparation enabling more generalized atomic force microscopy utilization for cellular analysis required for advanced cell surface morphological studies.

Tumor necrosis factor-α and interferon-γ stimulate MUC16 (CA125) expression in breast, endometrial and ovarian cancers through NFκB

Transmembrane mucins (TMs) are restricted to the apical surface of normal epithelia. In cancer, TMs not only are over-expressed, but also lose polarized distribution. MUC16/CA125 is a high molecular weight TM carrying the CA125 epitope, a well-known molecular marker for human cancers. MUC16 mRNA and protein expression was mildly stimulated by low concentrations of TNFα (2.5 ng/ml) or IFNγ (20 IU/ml) when used alone; however, combined treatment with both cytokines resulted in a moderate (3-fold or less) to large (> 10-fold) stimulation of MUC16 mRNA and protein expression in a variety of cancer cell types indicating that this may be a general response. Human cancer tissue microarray analysis indicated that MUC16 expression directly correlates with TNFα and IFNγ staining intensities in certain cancers. We show that NFκB is an important mediator of cytokine stimulation of MUC16 since siRNA-mediated knockdown of NFκB/p65 greatly reduced cytokine responsiveness. Finally, we demonstrate that the 250 bp proximal promoter region of MUC16 contains an NFκB binding site that accounts for a large portion of the TNFα response. Developing methods to manipulate MUC16 expression could provide new approaches to treating cancers whose growth or metastasis is characterized by elevated levels of TMs, including MUC16.

Polyaniline-graphene based α-amylase biosensor with a linear dynamic range in excess of 6 orders of magnitude.

α-amylase is an established marker for diagnosis of pancreatic and salivary disease, and recent research has seen a substantial expansion of its use in therapeutic and diagnostic applications for infection, cancer and wound healing. The lack of bedside monitoring devices for α-amylase detection has hitherto restricted the clinical progress of such applications. We have developed a highly sensitive α-amylase immunosensor platform, produced via in situ electropolymerization of aniline onto a screen-printed graphene support (SPE). Covalently binding an α-amylase specific antibody to a polyaniline (PANI) layer and controlling device assembly using electrochemical impedance spectroscopy (EIS), we have achieved a highly linear response against α-amylase concentration. Each stage of the assembly was characterized using a suite of high-resolution topographical, chemical and mechanical techniques. Quantitative, highly sensitive detection was demonstrated using an artificially spiked human blood plasma samples. The device has a remarkably wide limit of quantification (0.025-1000IU/L) compared to α-amylase assays in current clinical use. With potential for simple scale up to volume manufacturing though standard semiconductor production techniques and subsequently clinical application, this biosensor will enable clinical benefit through early disease detection, and better informed administration of correct therapeutic dose of drugs used to treat α-amylase related diseases.