Monitoring individual cell-signaling activity using combined metal-clad waveguide and surface-enhanced fluorescence imaging.

Evanescent field based biosensing systems such as surface plasmon resonance (SPR), diffraction gratings, or metal-clad waveguides (MCWGs) are powerful tools for label-free real-time monitoring of signaling activity of living cells exposed to hormones, pharmacological agents, and toxins. In particular, MCWG-based imaging is well suited for studying relatively thick objects such as cells due to its greater depth of penetration into the sensing medium, compared to SPR. Label-free methods, however, provide only indirect measurements in that the measured signal arises from local changes in material properties rather than from specific biomolecular targets. In the case of cells, the situation is especially complex as the measured label-free signal may result from a combination of very diverse sources: morphological changes, intra-cellular reorganization, cascaded molecular events, protein expression etc. Consequently, deconvolving the contributions of specific sources to a particular cell response profile can be challenging. In the following, we present a cell imaging platform that combines two distinct sensing modalities, namely label-free MCWG imaging and label-based surface enhanced fluorescence (SEF), designed to facilitate the identification of the underlying molecular and structural contributions to the label-free MCWG images. We demonstrate the bimodal capabilities of this imaging platform in experiments designed to visualize actin cytoskeleton organization in vascular smooth muscle cells. We then monitored the real-time response of HEK293 cells expressing the Angiotensin 1 receptor (AT1R), when stimulated by the receptor agonist Angiotensin II (AngII). The analysis of the simultaneous label-free signal obtained by MCWG and the intracellular calcium signal resulting form AT1R activation, measured by SEF, allows relating label-free signal features to specific markers of receptor activation. Our results show that the intracellular calcium levels normally observed following AT1R activation are not required for the initial burst of cellular activity observed in the MCWG signal but rather indicates signaling activity involving the intracellular kinase ROCK.

Metal clad waveguide (MCWG) based imaging using a high numerical aperture microscope objective.

Evanescent-field based methods such as surface plasmon resonance (SPR) have been used very effectively for label-free imaging of microscopic biological material in close proximity to a sensing surface. However, the shallow probing depth of SPR (typically less than ~200 nm) can be problematic when imaging relatively thick biological objects such as cells or bacteria. In this paper, we demonstrate how metal-clad waveguides (MCWG) can be used to achieve deeper probing depth compared to SPR while maintaining good imaging spatial resolution. Comparative numerical simulations of imaging spatial resolution versus probing depth are shown for a number of common SPR, long-range SPR, and MCWG configurations, demonstrating that MCWG offer the best compromise between resolution and depth for imaging thick biological objects. Experimental results of synthetic target and live cell imaging are shown that validate the numerical simulations and demonstrate the capabilities of the method.

Improved resolution in SPR and MCWG microscopy by combining images acquired with distinct mode propagation directions.

In high-resolution surface plasmon (SPR) imaging, lateral resolution is limited along the direction of plasmon propagation by the longitudinal decay length. Though SPR systems can achieve sub-micrometer resolution, the decay length causes a degradation in the images in the direction of plasmon propagation akin to a blurring artifact, with ringing along resonant to nonresonant transition edges. We present a method to significantly reduce this effect based on combining images of a sample acquired with distinct guided-mode propagation directions. As SPR is a special case of the class of optical structures known as metal-clad waveguides (MCWG) that are also affected by the decay length, this work is broadly applicable.

Changes in water chemistry can disable plankton prey defenses.

The effectiveness of antipredator defenses is greatly influenced by the environment in which an organism lives. In aquatic ecosystems, the chemical composition of the water itself may play an important role in the outcome of predator-prey interactions by altering the ability of prey to detect predators or to implement defensive responses once the predator's presence is perceived. Here, we demonstrate that low calcium concentrations (<1.5 mg/L) that are found in many softwater lakes and ponds disable the ability of the water flea, Daphnia pulex to respond effectively to its predator, larvae of the phantom midge, Chaoborus americanus. This low-calcium environment prevents development of the prey's normal array of induced defenses, which include an increase in body size, formation of neck spines, and strengthening of the carapace. We estimate that this inability to access these otherwise effective defenses results in a 50-186% increase in the vulnerability of the smaller juvenile instars of Daphnia, the stages most susceptible to Chaoborus predation. Such a change likely contributes to the observed lack of success of daphniids in most low-calcium freshwater environments, and will speed the loss of these important zooplankton in lakes where calcium levels are in decline.

Label-free visualization and quantification of single cell signaling activity using metal-clad waveguide (MCWG)-based microscopy.

Label-free biosensing methods are very effective for studying cell signaling cascade activation induced by external stimuli. Assays generally involve a large number of cells and rely on the underlying assumption that cell response is homogeneous within a cell population. However, there is an increasing body of evidence showing that cell behavior may vary significantly even among genetically identical cells. In this paper, we demonstrate the use of metal-clad waveguide (MCWG)-based microscopy for label-free real-time monitoring of signaling activity and morphology changes in a small population of cells, with the ability to resolve individual cells. We demonstrate the potential of this approach by quantifying apoptosis-induced intracellular activity in individual cells following exposure to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and by visualizing and quantifying extracellular changes in endothelial cell layer integrity following the activation of the proteinase-activated receptor 1 (PAR1) by thrombin. Results show that averaged signals obtained from a cell population may incorrectly reflect the actual distribution of morphology and kinetics parameters across a cell population by a significant margin.

Tenomodulin is Required for Tendon Endurance Running and Collagen I Fibril Adaptation to Mechanical Load.

Tendons are dense connective tissues that attach muscles to bone with an indispensable role in locomotion because of their intrinsic properties of storing and releasing muscle- generated elastic energy. Tenomodulin (Tnmd) is a well-accepted gene marker for the mature tendon/ligament lineage and its loss-of -function in mice leads to a phenotype with distinct signs of premature aging on tissue and stem/progenitor cell levels. Based on these findings, we hypothesized that Tnmd might be an important factor in the functional performance of tendons. Firstly, we revealed that Tnmd is a mechanosensitive gene and that the C-terminus of the protein co-localize with collagen I-type fibers in the extracellular matrix. Secondly, using an endurance training protocol, we compared Tnmd knockout mice with wild types and showed that Tnmd deficiency leads to significantly inferior running performance that further worsens with training. In these mice, endurance running was hindered due to abnormal response of collagen I cross-linking and proteoglycan genes leading to an inadequate collagen I fiber thickness and elasticity. In sum, our study demonstrates that Tnmd is required for proper tendon tissue adaptation to endurance running and aids in better understanding of the structural-functional relationships of tendon tissues.

Correction: Increased Stiffness in Aged Skeletal Muscle Impairs Muscle Progenitor Cell Proliferative Activity.

[This corrects the article DOI: 10.1371/journal.pone.0136217.].

Erratum to: Increased microenvironment stiffness in damaged myofibers promotes myogenic progenitor cell proliferation.

[This corrects the article DOI: 10.1186/s13395-015-0030-1.].

Receptor for Advanced Glycation End-Products Signaling Interferes with the Vascular Smooth Muscle Cell Contractile Phenotype and Function.

Increased blood glucose concentrations promote reactions between glucose and proteins to form advanced glycation end-products (AGE). Circulating AGE in the blood plasma can activate the receptor for advanced end-products (RAGE), which is present on both endothelial and vascular smooth muscle cells (VSMC). RAGE exhibits a complex signaling that involves small G-proteins and mitogen activated protein kinases (MAPK), which lead to increased nuclear factor kappa B (NF-κB) activity. While RAGE signaling has been previously addressed in endothelial cells, little is known regarding its impact on the function of VSMC. Therefore, we hypothesized that RAGE signaling leads to alterations in the mechanical and functional properties of VSMC, which could contribute to complications associated with diabetes. We demonstrated that RAGE is expressed and functional in the A7r5 VSMC model, and its activation by AGE significantly increased NF-κB activity, which is known to interfere with the contractile phenotype of VSMC. The protein levels of the contraction-related transcription factor myocardin were also decreased by RAGE activation with a concomitant decrease in the mRNA and protein levels of transgelin (SM-22α), a regulator of VSMC contraction. Interestingly, we demonstrated that RAGE activation increased the overall cell rigidity, an effect that can be related to an increase in myosin activity. Finally, although RAGE stimulation amplified calcium signaling and slightly myosin activity in VSMC challenged with vasopressin, their contractile capacity was negatively affected. Overall, RAGE activation in VSMC could represent a keystone in the development of vascular diseases associated with diabetes by interfering with the contractile phenotype of VSMC through the modification of their mechanical and functional properties.

Uncovering ultrastructural defences in Daphnia magna--an interdisciplinary approach to assess the predator-induced fortification of the carapace.

The development of structural defences, such as the fortification of shells or exoskeletons, is a widespread strategy to reduce predator attack efficiency. In unpredictable environments these defences may be more pronounced in the presence of a predator. The cladoceran Daphnia magna (Crustacea: Branchiopoda: Cladocera) has been shown to develop a bulky morphotype as an effective inducible morphological defence against the predatory tadpole shrimp Triops cancriformis (Crustacea: Branchiopoda: Notostraca). Mediated by kairomones, the daphnids express an increased body length, width and an elongated tail spine. Here we examined whether these large scale morphological defences are accompanied by additional ultrastructural defences, i.e. a fortification of the exoskeleton. We employed atomic force microscopy (AFM) based nanoindentation experiments to assess the cuticle hardness along with tapping mode AFM imaging to visualise the surface morphology for predator exposed and non-predator exposed daphnids. We used semi-thin sections of the carapace to measure the cuticle thickness, and finally, we used fluorescence microscopy to analyse the diameter of the pillars connecting the two carapace layers. We found that D. magna indeed expresses ultrastructural defences against Triops predation. The cuticle in predator exposed individuals is approximately five times harder and two times thicker than in control daphnids. Moreover, the pillar diameter is significantly increased in predator exposed daphnids. These predator-cue induced changes in the carapace architecture should provide effective protection against being crushed by the predator's mouthparts and may add to the protective effect of bulkiness. This study highlights the potential of interdisciplinary studies to uncover new and relevant aspects even in extensively studied fields of research.