Combining mechanical and optical approaches to dissect cellular mechanobiology

Mechanical force modulates a wide array of cell physiological processes. Cells sense and respond to mechanical stimuli using a hierarchy of structural complexes spanning multiple length scales, including force-sensitive molecules and cytoskeletal networks. Understanding mechanotransduction, i.e., the process by which cells convert mechanical inputs into biochemical signals, has required the development of novel biophysical tools that allow for probing of cellular and subcellular components at requisite time, length, and force scales and technologies that track the spatio-temporal dynamics of relevant biomolecules. In this review, we begin by discussing the underlying principles and recent applications of atomic force microscopy, magnetic twisting cytometry, and traction force microscopy, three tools that have been widely used for measuring the mechanical properties of cells and for probing the molecular basis of cellular mechanotransduction. We then discuss how such tools can be combined with advanced fluorescence methods for imaging biochemical processes in living cells in the context of three specific problem spaces. We first focus on fluorescence resonance energy transfer, which has enabled imaging of intra- and inter-molecular interactions and enzymatic activity in real time based on conformational changes in sensor molecules. Next, we examine the use of fluorescence methods to probe force-dependent dynamics of focal adhesion proteins. Finally, we discuss the use of calcium ratiometric signaling to track fast mechanotransductive signaling dynamics. Together, these studies demonstrate how single-cell biomechanical tools can be effectively combined with molecular imaging technologies for elucidating mechanotransduction processes and identifying mechanosensitive proteins.     

Influence of the change in stem length on the load transfer and bone remodelling for a cemented resurfaced femur

The effect of a short-stem femoral resurfacing component on load transfer and potential failure mechanisms has rarely been studied. The stem length has been reduced by approximately 50% as compared to the current long-stem design. Using 3-D FE models of natural and resurfaced femurs, the study is aimed at investigating the influence of a short-stem resurfacing component on load transfer and bone remodelling. Applied loading conditions include normal walking and stair climbing. The mechanical role of the stem along with implant–cement and stem–bone contact conditions was observed to be crucial. Shortening the stem length to half of the current length (long-stem) led to several favourable effects, even though the stress distributions in the implant and the cement were similar in both the cases. The short-stem implant led not only to a more physiological stress distribution but also to bone apposition (increase of 20–70% bone density) in the superior resurfaced head, when the stem–bone contact prevailed. This also led to a reduction in strain concentration in the cancellous bone around the femoral neck–component junction. The normalised peak strain in this region was lower for the short-stem design as compared to that of the long-stem one, thereby reducing the initial risk of neck fracture. The effect of strain shielding (50–75% reduction) was restricted to a small bone volume underlying the cement, which was approximately half of that of the long-stem design. Consequently, bone resorption was considerably less for the short-stem design. The short-stem design offers better prospects than the long-stem resurfacing component.     

The TWEAK–Fn14 system is a critical regulator of denervation-induced skeletal muscle atrophy in mice

Skeletal muscle atrophy occurs in a variety of clinical settings, including cachexia, disuse, and denervation. Inflammatory cytokines have been shown to be mediators of cancer cachexia; however, the role of cytokines in denervation- and immobilization-induced skeletal muscle loss remains unknown. In this study, we demonstrate that a single cytokine, TNF-like weak inducer of apoptosis (TWEAK), mediates skeletal muscle atrophy that occurs under denervation conditions. Transgenic expression of TWEAK induces atrophy, fibrosis, fiber-type switching, and the degradation of muscle proteins. Importantly, genetic ablation of TWEAK decreases the loss of muscle proteins and spared fiber cross-sectional area, muscle mass, and strength after denervation. Expression of the TWEAK receptor Fn14 (fibroblast growth factor–inducible receptor 14) and not the cytokine is significantly increased in muscle upon denervation, demonstrating an unexpected inside-out signaling pathway; the receptor up-regulation allows for TWEAK activation of nuclear factor κB, causing an increase in the expression of the E3 ubiquitin ligase MuRF1. This study reveals a novel mediator of skeletal muscle atrophy and indicates that the TWEAK–Fn14 system is an important target for preventing skeletal muscle wasting.     

Development and characterization of a human articular cartilage‐derived chondrocyte cell line that retains chondrocyte phenotype

Chondrocytes, the only cell type present in articular cartilage, regulate tissue homeostasis by a fine balance of metabolism that includes both anabolic and catabolic activities. Therefore, the biology of chondrocytes is critical for understanding cartilage metabolism. One major limitation when studying primary chondrocytes in culture is their loss of phenotype. To overcome this hurdle, limited attempts have been made to develop human chondrocyte cell lines that retain the phenotype for use as a good surrogate model. In this study, we report a novel approach to the establishment and characterization of human articular cartilage‐derived chondrocyte cell lines. Adenoviral infection followed by culture of chondrocytes in 3‐dimensional matrix within 48 h post‐infection maintained the phenotype prior to clonal selection. Cells were then placed in culture either as monolayer, or in 3‐dimensional matrix of alginate or agarose. The clones were characterized by their basal gene expression profile of chondrocyte markers. Based on type II collagen expression, 21 clones were analyzed for gene expression following treatment with IL‐1 or BMP‐7 and compared to similarly stimulated primary chondrocytes. This resulted in selection of two clones that retained the chondrocyte phenotype as evidenced by expression of type II collagen and other extra‐cellular matrix molecules. In addition, one clone (AL‐4‐17) showed similar responses as primary chondrocytes when treated with IL‐1 or BMP‐7. In summary, this report provides a novel procedure to develop human articular cartilage‐derived chondrocyte cell lines, which preserve important characteristics of articular chondrocytes and represent a useful model to study chondrocyte biology. J. Cell. Physiol. 222: 695–702, 2010. © 2009 Wiley‐Liss, Inc.     

Covalent linkage of CYP101 with the electrode enhances the electrocatalytic activity of the enzyme: Vectorial electron transport from the electrode

Direct electrochemical transfer of electrons to the enzyme provides an excellent method of driving the catalytic reactions of cytochrome P450 enzymes that form a superfamily of vital heme enzymes involved in biological monooxygenation reactions. Covalent attachment of N-(1-pyrenyl) maleimide (pyrene maleimide) to the bacterial cytochrome P450, CYP101 has been carried out and the conjugated enzyme was shown to be specifically immobilized onto the glassy carbon electrode through the pyrene group. The electrode immobilized pyrene-conjugated enzyme showed quasi-reversible electrochemistry with a midpoint potential at −330 ± 10 mV versus Ag/AgCl. The unconjugated enzyme that did not have specific linkage with the pyrene maleimide was non-specifically adsorbed on the electrode surface and the electrochemical response was much weaker than that observed in case of the conjugated enzyme, though the midpoint potential was almost unchanged. The pyrene maleimide bound CYP101 was found to have surface coverage of 1.35 ± 0.3 × 10⁻¹⁰ mol/cm² and the heterogeneous rate of electron transfer was found to be 0.21 ± 0.02 s⁻¹, which is larger than that for the unconjugated enzyme. The pyrene maleimide linked immobilized enzyme was oriented to the electrode so that efficient electron transfer takes place from the electrode to the immobilized enzyme. The oxygenase activity of the immobilized conjugated enzyme was assayed from the enhancement of catalytic current in presence of oxygen and the natural substrate camphor. Mass spectrometric studies also showed enhanced formation of hydroxycamphor by electrochemically driven catalysis in the pyrene maleimide linked immobilized CYP101.     

Structure–toxicity relationship of phenolic analogs as anti-melanoma agents: An enzyme directed prodrug approach

The aim of this study was to identify a phenolic prodrug compound that is minimally metabolized by rat liver microsomes, but yet could form quinone reactive intermediates in melanoma cells as a result of its bioactivation by tyrosinase. In current work, we investigated 24 phenolic compounds for their metabolism by tyrosinase, rat liver microsomes and their toxicity towards murine B16-F0 and human SK-MEL-28 melanoma cells. A linear correlation was found between toxicities of phenolic analogs towards SK-MEL-28 and B16-F0 melanoma cells, suggesting similar mechanisms of toxicity in both cell lines. 4-HEB was identified as the lead compound. 4-HEB (IC₅₀ 48 h, 75 μM) showed selective toxicity towards five melanocytic melanoma cell lines SK-MEL-28, SK-MEL-5, MeWo, B16-F0 and B16-F10, which express functional tyrosinase, compared to four non-melanoma cells lines SW-620, Saos-2, PC3 and BJ cells and two amelanotic SK-MEL-24, C32 cells, which do not express functional tyrosinase. 4-HEB caused significant intracellular GSH depletion, ROS formation, and showed significantly less toxicity to tyrosinase specific shRNA transfected SK-MEL-28 cells. Our findings suggest that presence of a phenolic group in 4-HEB is critical for its selective toxicity towards melanoma cells.     

Flow cytometric screening for anti-leishmanials in a human macrophage cell line

High-throughput drug screening methods against the intracellular stage of Leishmania have been facilitated by the development of in vitro models of infection. The use of cell lines rather than primary cells facilitates these methods. Peripheral blood mononuclear cell (PBMC) derived macrophages and THP-1 cells were infected with stationary phase egfp transfected Leishmania amazonensis parasites and then treated with anti-leishmanial compounds. Drug activity was measured using a flow cytometric approach, and toxicity was assessed using either the MTT assay or trypan blue dye exclusion. Calculated EC₅₀’s for amphotericin B, sodium stibogluconate, and miltefosine were 0.1445 ± 0.0005 μg/ml, 0.1203 ± 0.018 mg/ml, and 26.71 μM using THP-1 cells, and 0.179 ± 0.035 μg/ml, 0.1948 ± 0.0364 mg/ml, and 13.77 ± 10.74 μM using PBMC derived macrophages, respectively. We conclude that a flow cytometric approach using egfp transfected Leishmania species can be used to evaluate anti-leishmanial compounds against the amastigote stage of the parasite in THP-1 cells with excellent concordance to human PBMC derived macrophages.