Modeling of Noisy Spindle Dynamics Reveals Separable Contributions to Achieving Correct Orientation.

The mechanisms by which the mammalian mitotic spindle is guided to a predefined orientation through microtubule-cortex interactions have recently received considerable interest, but there has been no dynamic model that describes spindle movements toward the preferred axis in human cells. Here, we develop a dynamic model based on stochastic activity of cues anisotropically positioned around the cortex of the mitotic cell and we show that the mitotic spindle does not reach equilibrium before chromosome segregation. Our model successfully captures the characteristic experimental behavior of noisy spindle rotation dynamics in human epithelial cells, including a weak underlying bias in the direction of rotation, suppression of motion close to the alignment axis, and the effect of the aspect ratio of the interphase cell shape in defining the final alignment axis. We predict that the force exerted per cue has a value that minimizes the deviation of the spindle from the predefined axis. The model has allowed us to systematically explore the parameter space around experimentally relevant configurations, and predict the mechanistic function of a number of established regulators of spindle orientation, highlighting how physical modeling of a noisy system can lead to functional biological understanding. We provide key insights into measurable parameters in live cells that can help distinguish between mechanisms of microtubule and cortical-cue interactions that jointly control the final orientation of the spindle.

Microrheology and microstructure of Fmoc-derivative hydrogels.

The viscoelasticity of hydrogel networks formed from the low-molecular-weight hydrogelator Fmoc-tyrosine (Fmoc-Y) is probed using particle-tracking microrheology. Gelation is initiated by adding glucono-δ-lactone (GdL), which gradually lowers the pH with time, allowing the dynamic properties of gelation to be examined. Consecutive plots of probe particle mean square displacement (MSD) versus lag time τ are shown to be superimposable, demonstrating the formation of a self-similar hydrogel network through a percolation transition. The analysis of this superposition yields a gel time t(gel) = 43.4 ± 0.05 min and a critical relaxation exponent n(c) = 0.782 ± 0.007, which is close to the predicted value of 3/4 for semiflexible polymer networks. The generalized Stokes-Einstein relation is applied to the master curves to find the viscoelastic moduli of the critical gel over a wide frequency range, showing that the critical gel is structurally and rheologically fragile. The scaling of G'/G″ as ω(0.795±0.099) ≈ ω(3/4) at high frequencies provides further evidence for semiflexible behavior. Cryogenic scanning electron micrographs depict a loosely connected network close to the gel point with a fibrillar persistence length that is longer than the network mesh size, further indications of semiflexible behavior. The system reported here is one of a number of synthetic systems shown to exhibit semiflexible behavior and indicates the opportunity for further rheological study of other Fmoc derivatives.

A microrheological study of hydrogel kinetics and micro-heterogeneity.

The real-time dynamic heterogeneity of the gelation process of the amino acid derivative Fmoc-tyrosine (Fmoc-Y) is studied using particle tracking microrheology. To trigger gelation, glucono-δ-lactone (GdL) is added, which gradually lowers the p H over several hours. The onset of self-assembly in the system is signified by a sharp drop in the mean-squared displacement of embedded particles, a phenomenon that is found to correlate with the p H of the system reaching the pK(a) of Fmoc-Y. The gel point is identified and found to be dependent on the GdL concentration. Analysis of embedded probe particle dynamics allows the heterogeneity of the sample to be quantified, using three metrics: the heterogeneity ratio (HR), the non-Gaussian parameter of the van Hove correlation function (N and the bin distribution of the mean-squared displacement (MSD) of single particles (f(z)). Results from the three techniques are found to be approximately comparable, with increases in heterogeneity observed in all samples for incubation times t(w) = 0-3 hours. The final heterogeneity in all samples is found to be remarkably low compared to other systems previously reported in the literature.

Electrostatics controls the formation of amyloid superstructures in protein aggregation.

The possibility for proteins to aggregate in different superstructures, i.e. large-scale polymorphism, has been widely observed, but an understanding of the physicochemical mechanisms behind it is still out of reach. Here we present a theoretical model for the description of a generic aggregate formed from an ensemble of charged proteins. The model predicts the formation of multifractal structures with the geometry of the growth determined by the electrostatic interactions between single proteins. The model predictions are successfully verified in comparison with experimental curves for aggregate growth allowing us to reveal the mechanism of formation of such complex structures. The model is general and is able to predict aggregate morphologies occurring both in vivo and in vitro. Our findings provide a framework where the physical interactions between single proteins, the aggregate morphology, and the growth kinetics are connected into a single model in agreement with the experimental data.

Rational design and application of responsive alpha-helical peptide hydrogels.

Biocompatible hydrogels have a wide variety of potential applications in biotechnology and medicine, such as the controlled delivery and release of cells, cosmetics and drugs, and as supports for cell growth and tissue engineering. Rational peptide design and engineering are emerging as promising new routes to such functional biomaterials. Here, we present the first examples of rationally designed and fully characterized self-assembling hydrogels based on standard linear peptides with purely alpha-helical structures, which we call hydrogelating self-assembling fibres (hSAFs). These form spanning networks of alpha-helical fibrils that interact to give self-supporting physical hydrogels of >99% water content. The peptide sequences can be engineered to alter the underlying mechanism of gelation and, consequently, the hydrogel properties. Interestingly, for example, those with hydrogen-bonded networks of fibrils melt on heating, whereas those formed through hydrophobic fibril-fibril interactions strengthen when warmed. The hSAFs are dual-peptide systems that gel only on mixing, which gives tight control over assembly. These properties raise possibilities for using the hSAFs as substrates in cell culture. We have tested this in comparison with the widely used Matrigel substrate, and demonstrate that, like Matrigel, hSAFs support both growth and differentiation of rat adrenal pheochromocytoma cells for sustained periods in culture.

Cracking of drying latex films: an ESEM experiment.

In this study environmental scanning electron microscopy was used to observe the cracking of drying latex films below their glass-transition temperature. By controlling the relative humidity so that it decreases linearly with time, a critical level of humidity at which cracking occurs can be determined and this is measured as a function of film thickness. It was found that the cracking humidity decreases with increases in film thickness for thicknesses in the range of 30 to 100 mum and then remains almost unchanged. A scaling argument can be used to fit the data very well and indicates that cracking occurs as soon as the entire film is consolidated into close packing.

Amyloid fibril-like structure underlies the aggregate structure across the pH range for beta-lactoglobulin.

The protein beta-lactoglobulin aggregates into two apparently distinct forms under different conditions: amyloid fibrils at pH values away from the isoelectric point, and spherical aggregates near it. To understand this apparent dichotomy in behavior, we studied the internal structure of the spherical aggregates by employing a range of biophysical approaches. Fourier transform infrared studies show the aggregates have a high beta-sheet content that is distinct from the native beta-lactoglobulin structure. The structures also bind the amyloidophilic dye thioflavin-T, and wide-angle x-ray diffraction showed reflections corresponding to spacings typically observed for amyloid fibrils composed of beta-lactoglobulin. Combined with small-angle x-ray scattering data indicating the presence of one-dimensional linear aggregates at the molecular level, these findings indicate strongly that the aggregates contain amyloid-like substructure. Incubation of beta-lactoglobulin at pH values increasingly removed from the isoelectric point resulted in the increasing appearance of fibrillar species, rather than spherical species shown by electron microscopy. Taken together, these results suggest that amyloid-like beta-sheet structures underlie protein aggregation over a much broader range of conditions than previously believed. Furthermore, the results suggest that there is a continuum of beta-sheet structure of varying regularity underlying the aggregate morphology, from very regular amyloid fibrils at high charge to short stretches of amyloid-like fibrils that associate together randomly to form spherical particles at low net charge.

Investigation of viability of plant tissue in the environmental scanning electron microscopy.

The advantages of environmental scanning electron microscopy (ESEM) make it a suitable technique for studying plant tissue in its native state. There have been few studies on the effects of ESEM environment and beam damage on the viability of plant tissue. A simple plant tissue, Allium cepa (onion) upper epidermal tissue was taken as the model for study. The change of moisture content of samples was studied at different relative humidities. Working with the electron beam on, viability tests were conducted for samples after exposure in the ESEM under different operating conditions to investigate the effect of electron beam dose on the viability of samples. The results suggested that without the electron beam, the ESEM chamber itself can prevent the loss of initial moisture if its relative humidity is maintained above 90%. With the electron beam on, the viability of Allium cepa (onion) cells depends both on the beam accelerating voltage and the electron dose/unit area hitting the sample. The dose can be controlled by several of the ESEM instrumental parameters. The detailed process of beam damage on cuticle-down and cuticle-up samples was investigated and compared. The results indicate that cuticular adhesion to the cell wall is relatively weak, but highly resistant to electron beam damage. Systematic study on the effect of ESEM operation parameters has been done. Results qualitatively support the intuitive expectations, but demonstrate quantitatively that Allium cepa epidermal cells are able to be kept in a hydrated and viable state under relevant operation condition inside ESEM, providing a basis for further in situ experiments on plant tissues.

Protein aggregation: more than just fibrils.

The aggregation of misfolded proteins into amyloid fibrils, and the importance of this step for various diseases, is well known. However, it is becoming apparent that the fibril is not the only structure that aggregating proteins of widely different types may adopt. Around the isoelectric point, when the net charge is essentially zero, rather monodisperse and quasi-amorphous nanoscale particles form. These particles are found to contain limited runs of beta-sheet structure, but their overall organization is random. These nanoparticles have the potential to be useful for such applications as the slow release of drugs. The amyloid fibrils form away from the isoelectric point, but over certain ranges of, e.g., pH, the fibrils themselves do not exist freely, but form suprafibrillar aggregates termed spherulites. These consist of fibrils radiating from a central nucleus, and form by new species attaching to the ends of growing fibrils, rather than by the aggregation of pre-existing fibrils. Under the polarizing light microscope, they exhibit a Maltese cross shape due to their symmetry. The rate of aggregation is determined by factors involving (at least) protein size, concentration, presence of salt and charge. The occurrence of spherulites, which have been found in vivo as well as in vitro, appears to be generic, although the factors which determine the equilibrium between free fibril and spherulite are not as yet clear.

Passive microrheology of solvent-induced fibrillar protein networks.

Particle tracking microrheology (PTM) has been used to study the sol-gel transition in solvent-induced fibrillar beta-lactoglobulin gels at room temperature and pH 7. The passive nature of microrheology allowed measurements to be made around and below the critical gelation concentration. The method of superposition introduced by Larsen and Furst (Larsen, T. H.; Furst, E. M. Phys. Rev. Lett. 2008, 100, 146001) was applied to the one-particle mean square displacement (MSD), yielding a critical relaxation exponent of n = 0.58 at concentrations close to the measured critical concentration of 4% (w/v). At a higher concentration of 12% (w/v), n was observed to decrease. The pregel and gel master curves were used to find the viscoelastic moduli over 8 decades of frequency. Combined with the measured shift factors, this allowed cure curves at 1 Hz to be constructed for direct comparison with results from bulk rheology. Time-independent modulus superposition was found for all concentrations. Good agreement for concentration scaling was found between the traditional methods for characterizing gels and the recently described microrheological determination of the gel time and critical behavior.

In situ microextraction method to determine the viscosity of biofluid in threadlike structures on the surfaces of mammalian organs.

We report a method to measure the viscosity of microL volumes of biofluid obtained from threadlike structures (NTSs) on the surfaces of mammalian (rabbit) internal organs. The fluid was mechanically microextracted in situ from NTSs on the organ surfaces by a glass capillary connected to an extractor. From the Brownian motion of the 0.8+/-0.1microm diameter granules in the extracted fluid, the fluid viscosity was determined to be 1.4+/-0.1mPa s at room temperature. This viscosity is comparable to the viscosity of rabbit blood plasma.

Aggregation in ß-lactoglobulin.

ß-lactoglobulin is a protein of huge importance to the food industry, and as such it has been extensively studied by the food community sui generis. However, recently there has been an increasing number of studies approaching the protein from a soft matter perspective. Here it is shown how its behaviour can be seen to be generic, in so far as its forms of aggregation are actually typical of many other proteins under comparable conditions, and hence that it is useful to seek unifying mechanisms for its behaviour.

Investigation of the morphology, viability and mechanical properties of yeast cells in environmental SEM.

The mechanical properties of biological cells at nanoscale may be characterized using an environmental scanning electron microscopy (ESEM) combined with a force measurement device. However, the electron beam radiation in an ESEM may damage a specimen. So far, little is known about the radiation damage to biological cells. In this work, single yeast cells were imaged using an ESEM under both high and low vacuum modes. The changes in their morphology and viability were monitored as a function of radiation time for a given beam current of 538 pA corresponding to 10 kV accelerating voltage and spot size 4. Under the two modes, the radiation damage to the morphology of yeast cells became evident after an exposure time of 3 min, but under the low vacuum mode, the damage to their morphology was more severe. However, all cells lost their viability after 5 min under the high vacuum mode with the electron beam off from an initial viability of 95+/-1%. In contrast, the viability of cells under the low vacuum mode was found to be approximately 20% after 20 min. In addition, a newly developed ESEM-based nanomanipulation technique was applied to measure the force imposed on single yeast cells and their deformation, including contact diameter and central lateral diameter for the compression of single yeast cells to a given displacement within a time frame of 1 min, and the data obtained may be used to validate mathematical modeling of the stress-strain relationship for the compression of cells in order to determine their intrinsic mechanical property parameters.

Thermal dependence of thermally induced protein spherulite formation and growth: kinetics of beta-lactoglobulin and insulin.

Amyloid fibril forming proteins have been related to some neurodegenerative diseases and are not fully understood. In some such systems, these amyloid fibrils have been found to form radially oriented spherulite structures. The thermal dependence of formation and growth of these spherulite structures in two model protein systems, beta-lactoglobulin and insulin at low pH aqueous and high temperature conditions, have been monitored with time-lapse optical microscopy and quantified. A population-based polymerization reaction model was developed and applied to the experimental data with excellent agreement. While spherulites in the insulin solutions formed and grew at approximately 25x the rate of spherulites in the beta-lactoglobulin solutions, the temperature dependence and activation energies of both systems were found to be very similar to one another, suggesting that the underlying rate-limiting mechanisms for both formation and growth are consistent across the two systems. The similarity of both of these amyloid fibril forming protein systems provides confidence in their use as model systems for extrapolating understanding to similar systems involved in neurodegenerative diseases.

Microrheology: a review of the method and applications.

A set of local mechanical probes has been developed over the last ten years, allowing a kind of dynamical mechanical testing known as microrheology. This paper provides a short introductory review of these methods of performing rheology, comparing them to conventional rheometry, and highlighting the major advantages. The authors also share their outlook on some of the most promising and fastest developing areas that are being studied though microrheology, in the areas of biophysics and soft matter.

Structure of Spherulites in Insulin, ß-Lactoglobulin, and Amyloid ß.

Under denaturing conditions such as low pH and elevated temperatures, proteins in vitro can misfold and aggregate to form long rigid rods called amyloid fibrils; further self-assembly can lead to larger structures termed spherulites. Both of these aggregates resemble amyloid tangles and plaques associated with Alzheimer's disease in vivo. The ability to form such aggregates in a multitude of different proteins suggests that it is a generic ability in their mechanism to form. Little is known about the structure of these large spherulites ranging from 5 to 100 microns and whether they can reproducibly form in amyloid ß (1-40) (Aß40), a 40-amino acid residue peptide, which is one of the major components of Alzheimer's amyloid deposits. Here, we show that spherulites can readily form in Aß40 under certain monomerization and denaturing conditions. Using polarized and nonpolarized Raman spectroscopy, we analyzed the secondary structure of spherulites formed from three different proteins: insulin, ß-lactoglobulin (BLG), and Aß40. Visually, these spherulites have a characteristic "Maltese Cross" structure under crossed polarizers through an optical microscope. However, our results indicate that insulin and Aß40 spherulites have similar core structures consisting mostly of random coils with radiating fibrils, whereas BLG mostly contains ß-sheets and fibrils that are likely to be spiraling from the core to the edge.

Dependence on material choice of degradation of organic solar cells following exposure to humid air.

Electron microscopy has been used to study the degradation of organic solar cells when exposed to humid air. Devices with various different combinations of commonly used organic solar cell hole transport layers and cathode materials have been investigated. In this way the ingress of water and the effect it has on devices could be studied. It was found that calcium and aluminum in the cathode both react with water, causing voids and delamination within the device. The use of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) was found to increase the degradation by easing water ingress into the device. Replacing these materials removed these degradation features. © 2015 The Authors. Journal of Polymer Science Part B: Polymer Physics published by Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2016, 54, 216-224.

Optical microscopy study for director patterns around disclinations in side-chain liquid crystalline polymer films.

Polarizing optical microscopy is employed to study director fields around disclinations in side-chain liquid crystalline polymer films. Optical black brushes together with stripes around disclinations are observed. The stripes run parallel to the local director and thus decorate overall patterns of nematic director around disclinations. Three director patterns involving radial, spiral, and circular microstructures of a positive integer disclination with s = +1 and one hyperbolic pattern of a negative integer disclination with s = -1 are observed in the thin film. It is found that the specific configurations of a pair of (+1, -1) disclinations form during the late stage of annihilation. Increasing the film thickness leads to disclination instability. We observe that black four-brushes of disclinations with s = +/-1 split into black two-brushes, where two types of director patterns of disclinations with half-integer strengths of s = +/- 1/2 produce. Theoretical analysis is presented to explain this instability.

Sub-nanometre resolution imaging of polymer-fullerene photovoltaic blends using energy-filtered scanning electron microscopy.

The resolution capability of the scanning electron microscope has increased immensely in recent years, and is now within the sub-nanometre range, at least for inorganic materials. An equivalent advance has not yet been achieved for imaging the morphologies of nanostructured organic materials, such as organic photovoltaic blends. Here we show that energy-selective secondary electron detection can be used to obtain high-contrast, material-specific images of an organic photovoltaic blend. We also find that we can differentiate mixed phases from pure material phases in our data. The lateral resolution demonstrated is twice that previously reported from secondary electron imaging. Our results suggest that our energy-filtered scanning electron microscopy approach will be able to make major inroads into the understanding of complex, nano-structured organic materials.

Tensile deformation of bacterial cellulose composites.

The polymeric basis for the mechanical properties of primary plant cell walls has been investigated by forming analogous composites based on fermentation of the bacterium Acetobacter xylinus, either alone or in the presence of xyloglucan or pectin. Simultaneous small-angle X-ray scattering and uniaxial deformation experiments has shown how the cellulose microfibrils reorient during deformation. Despite very different stress/strain curves, the reorientation behaviour is similar, regardless of the presence or absence of xyloglucan or pectin. A simple theory has been developed to predict the orientation behaviour. This is qualitatively similar to the measured behaviour, but differs quantitatively.