Mark F. Murphy

Actin bundling and polymerisation properties of eukaryotic elongation factor 1 alpha (eEF1A), histone H2A-H2B and lysozyme in vitro.

Elongation factor 1 alpha (eEF1A) is a positively charged protein which has been shown to interact with the actin cytoskeleton. However, to date, a specific actin binding site within the eEF1A sequence has not been identified and the mechanism by which eEF1A interacts with actin remains unresolved. Many protein-protein interactions occur as a consequence of their physicochemical properties and actin bundle formation has been shown to result from non-specific electrostatic interaction with basic proteins. This study investigated interactions between actin, eEF1A and two other positively charged proteins which are not regarded as classic actin binding proteins (namely lysozyme and H2A-H2B) in order to compare their actin organising effects in vitro. For the first time using atomic force microscopy (AFM) we have been able to image the interaction of eEF1A with actin and the subsequent bundling of actin in vitro. Interestingly, we found that eEF1A dramatically increases the rate of polymerisation (45-fold above control levels). We also show for the first time that H2A-H2B has remarkably similar effects upon actin bundling (relative bundle size/number) and polymerisation (35-fold increase above control levels) as eEF1a. The presence of lysozyme resulted in bundles which were distinct from those formed due to eEF1A and H2A-H2B. Lysozyme also increased the rate of actin polymerisation above the control level (by 10-fold). Given the striking similarities between the actin bundling and polymerisation properties of eEF1A and H2A-H2B, our results hint that dimerisation and electrostatic binding may provide clues to the mechanism through which eEF1A-actin bundling occurs.

Confocal microscopy segmentation using active contour based on alpha(α)-divergence

This paper describes a novel method for active contour segmentation based on foreground/background alpha-divergence histogram distance measure. In recent years a number of variational segmentation techniques have been proposed for a region based active contour segmentation utilising different distance measures between probability density functions (PDFs) describing foreground and background regions. The most common techniques use χ2, Hellinger/Bhattacharya distances or Kullback-Leibler divergence. In this paper, it is proposed to generalize these methods by using the alpha-divergences distance function. This distance function depending on the selected value of its parameter encompasses mentioned above classical distances. The paper defines a partial differential equation, associated with alpha-divergence variational criterion, that governs the iterative deformations of the active contour. The experimental results on a synthetic data demonstrate that the proposed method outperforms previously proposed histogram based methods in terms of segmentation accuracy and robustness with respect to type and level of noise. The potential of the proposed technique for segmentation of cellular structures in fluorescence confocal microscopy data is also illustrated.

Towards crystal engineering via simulated pulmonary surfactant monolayers to optimise inhaled drug delivery.

PURPOSE To generate theophylline monohydrate crystals underneath Langmuir monolayers composed of material expressed at the alveolar air-liquid interface. Such monolayers can act as nucleation sites to direct crystallisation. The approach offers a novel route to rationally engineer therapeutic crystals and thereby optimise inhaled drug delivery. METHODS Langmuir monolayers consisting of either dipalmitoylphosphatidylcholine (DPPC) or a surfactant mix reflecting pulmonary surfactant were supported on an aqueous theophylline (5.7 mg/ml) subphase. The monolayers were compressed to surface pressures reflecting inhalation and exhalation (i.e. 5 mNm(-1) or 55 mNm(-1)) with a period of 16 h to allow crystallisation. Analysis involved scanning electron microscopy (SEM), atomic force microscopy (AFM) and powder X-ray diffraction (PXRD). RESULTS Condensed isotherms were acquired, which signified surfactant-theophylline interaction. Theophylline monohydrate crystals were obtained and exhibited needle-like morphology. SEM and AFM data highlighted regions of roughened growth along with smooth, stepwise growth on the same crystal face. The surfactant monolayers appeared to influence crystal morphology over time. CONCLUSIONS The data indicate a favourable interaction between each species. The principal mechanism of interaction is thought to be an ion-dipole association. This approach may be applied to generate material with improved complementarity with pulmonary surfactant thus enhancing the interaction between inhaled drug particles and internal lung surfaces.

Evaluation of a nonlinear Hertzian‐based model reveals prostate cancer cells respond differently to force than normal prostate cells

Understanding how the mechanical properties of cells alter with disease may help with the development of novel diagnostics and treatment regimes. The emergence of tools such as the atomic force microscope (AFM) has enabled us to physically measure the mechanical properties of cells. However, suitable models for the analysis of real experimental data are either absent, or fail to provide a simple analysis tool in which experimental data can be analyzed quickly and reliably. The Hertz model has been widely used to study AFM data on living cells, however it makes assumptions that are untrue for cells, namely that cells behave as linear elastic bodies. This article presents and evaluates an alternative nonlinear Hertz model, which allows the Youngs modulus to vary according to a second order polynomial function of indentation depth. Evaluation of the model revealed that prostate cancer cells (PC3) responded more uniformly to force compared to the normal PNT2 cells. Also, more energy (J) was needed to deform the normal prostate cells compared to the prostate cancer cells. Finally, the model described here suggests that overall the normal prostate cells behave in a more linear fashion to applied force compared to the prostate cancer cells. Microsc. Res. Tech., 2013.

The use of abrasive polishing and laser processing for developing polyurethane surfaces for controlling fibroblast cell behaviour.

Studies have shown that surfaces having micro and nano-scale features can be used to control cell behaviours including; cell proliferation, migration and adhesion. The aim of this work was to compare the use of laser processing and abrasive polishing to develop micro/nano-patterned polyurethane substrates for controlling fibroblast cell adhesion, migration and proliferation. Laser processing in a directional manner resulted in polyurethane surfaces having a ploughed field effect with micron-scale features. In contrast, abrasive polishing in a directional and random manner resulted in polyurethane surfaces having sub-micron scale features orientated in a linear or random manner. Results show that when compared with flat (non-patterned) polymer, both the laser processed and abrasive polished surface having randomly organised features, promoted significantly greater cell adhesion, while also enhancing cell proliferation after 72h. In contrast, the abrasive polished surface having linear features did not enhance cell adhesion or proliferation when compared to the flat surface. For cell migration, the cells growing on the laser processed and abrasively polished random surface showed decreased levels of migration when compared to the flat surface. This study shows that both abrasive polishing and laser processing can be used to produce surfaces having features on the nano-scale and micron-scale, respectively. Surfaces produced using both techniques can be used to promote fibroblast cell adhesion and proliferation. Thus both methods offer a viable alternative to using lithographic techniques for developing patterned surfaces. In particular, abrasive polishing is an attractive method due to it being a simple, rapid and inexpensive method that can be used to produce surfaces having features on a comparable scale to more expensive, multi-step methods.

Segmentation of cellular structures in actin tagged fluorescence confocal microscopy images

The paper reports on a novel method for reconstruction of cellular features including cell nuclei and cellular boundaries from actin tagged fluorescence confocal microscopy images. Such reconstruction can provide spatial context for subsequent quantitative analysis of changes to actin organisation and cell morphology in both controlled and stressed cell cultures. The proposed method is fully automatic and is formulated within active contour multiphase level set framework. The derived level set evolution PDEs combine previously proposed curvature and advection flows with propagation flow defined by specially designed set of geodesic distance maps. Additionally the proposed PDEs include additional components to impose known inclusion/exclusion topological constraints between cellular structures. The paper gives an overview of the proposed methodology as well as reports on initial results obtained for monolayer of human prostate cells (PNT2) culture visualised using acting tagged fluorescence confocal microscopy.

Quantifying structure regularity in fluorescence microscopy cell images using a novel multi-dimensional approximate entropy metric

Techniques such as fluorescence microscopy reveal in three dimensions the complex mechanical structure of the cell cytoskeleton, made up of actin filaments, microtubules and intermediate filaments. Methods to quantify the degree of order in this structure could prove useful in classifying cells and may be related to the structural integrity of the cell. Approximate Entropy (ApEn) is a regularity statistic that has previously been used to quantify randomness in 1D time series data. We have extended the ApEn concept to define a multi-dimensional parameter suitable for application to three dimensional image data. This 3D ApEn parameter was applied to fluorescence microscopy images showing actin filaments in normal and cancerous human prostate cells. Differences between the ApEn calculated from the different images were observed, with cancer cells tending to have higher values indicating a lower degree of order.

The effects of acoustic vibration on fibroblast cell migration.

Cells are known to interact and respond to external mechanical cues and recent work has shown that application of mechanical stimulation, delivered via acoustic vibration, can be used to control complex cell behaviours. Fibroblast cells are known to respond to physical cues generated in the extracellular matrix and it is thought that such cues are important regulators of the wound healing process. Many conditions are associated with poor wound healing, so there is need for treatments/interventions, which can help accelerate the wound healing process. The primary aim of this research was to investigate the effects of mechanical stimulation upon the migratory and morphological properties of two different fibroblast cells namely; human lung fibroblast cells (LL24) and subcutaneous areolar/adipose mouse fibroblast cells (L929). Using a speaker-based system, the effects of mechanical stimulation (0-1600Hz for 5min) on the mean cell migration distance (μm) and actin organisation was investigated. The results show that 100Hz acoustic vibration enhanced cell migration for both cell lines whereas acoustic vibration above 100Hz was found to decrease cell migration in a frequency dependent manner. Mechanical stimulation was also found to promote changes to the morphology of both cell lines, particularly the formation of lamellipodia and filopodia. Overall lamellipodia was the most prominent actin structure displayed by the lung cell (LL24), whereas filopodia was the most prominent actin feature displayed by the fibroblast derived from subcutaneous areolar/adipose tissue. Mechanical stimulation at all the frequencies used here was found not to affect cell viability. These results suggest that low-frequency acoustic vibration may be used as a tool to manipulate the mechanosensitivity of cells to promote cell migration.

Comparison of the ability of mammalian eEF1A1 and its oncogenic variant eEF1A2 to interact with actin and calmodulin.

Abstract The question as to why a protein exerts oncogenic properties is answered mainly by well-established ideas that these proteins interfere with cellular signaling pathways. However, the knowledge about structural and functional peculiarities of the oncoproteins causing these effects is far from comprehensive. The 97.5% homologous tissue-specific A1 and A2 isoforms of mammalian translation elongation factor eEF1A represent an interesting model to study a difference between protein variants of a family that differ in oncogenic potential. We propose that the different oncogenic impact of A1 and A2 might be explained by differences in their ability to communicate with their respective cellular partners. Here we probed this hypothesis by studying the interaction of eEF1A with two known partners – calmodulin and actin. Indeed, an inability of the A2 isoform to interact with calmodulin is shown, while calmodulin is capable of binding A1 and interferes with its tRNA-binding and actin-bundling activities in vitro. Both A1 and A2 variants revealed actin-bundling activity; however, the form of bundles formed in the presence of A1 or A2 was distinctly different. Thus, a potential inability of A2 to be controlled by Ca2+-mediated regulatory systems is revealed.

Analysis of dynamic cantilever behavior in tapping mode atomic force microscopy

Tapping mode atomic force microscopy (AFM) provides phase images in addition to height and amplitude images. Although the behavior of tapping mode AFM has been investigated using mathematical modeling, comprehensive understanding of the behavior of tapping mode AFM still poses a significant challenge to the AFM community, involving issues such as the correct interpretation of the phase images. In this paper, the cantilevers dynamic behavior in tapping mode AFM is studied through a three dimensional finite element method. The cantilevers dynamic displacement responses are firstly obtained via simulation under different tip‐sample separations, and for different tip‐sample interaction forces, such as elastic force, adhesion force, viscosity force, and the van der Waals force, which correspond to the cantilevers action upon various different representative computer‐generated test samples. Simulated results show that the dynamic cantilever displacement response can be divided into three zones: a free vibration zone, a transition zone, and a contact vibration zone. Phase trajectory, phase shift, transition time, pseudo stable amplitude, and frequency changes are then analyzed from the dynamic displacement responses that are obtained. Finally, experiments are carried out on a real AFM system to support the findings of the simulations. Microsc. Res. Tech. 78:935–946, 2015.