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Monday, March 2, 2015

Red Blood Cell Aging During Storage, Studied Using Optical Tweezers Experiment

Justyna Czerwinska, Stefan Michael Wolf, Hanieh Mohammadi, Sylvia Jeney

This paper presents experimental and numerical studies of erythrocyte stretching, with a focus on the aging of red blood cells in an in vitro environment during storage. The experimental studies were performed using optical tweezers. The laser beam was used to pull and stretch a cell sedimented on a flat surface. A force calibration was obtained via a comparison of the experimental data with results from finite element simulations of the cell stretching. The experiments were performed using blood samples from blood bank donations made by three donors. The experiments were performed over 21 days of storage, and the estimate erythrocyte membrane shear modulus during this period increased from 2.5 to 13 μN/m.

DOI

A balance between membrane elasticity and polymerization energy sets the shape of spherical clathrin coats

Mohammed Saleem, Sandrine Morlot, Annika Hohendahl, John Manzi, Martin Lenz & Aurélien Roux

In endocytosis, scaffolding is one of the mechanisms to create membrane curvature by moulding the membrane into the spherical shape of the clathrin cage. However, the impact of membrane elastic parameters on the assembly and shape of clathrin lattices has never been experimentally evaluated. Here, we show that membrane tension opposes clathrin polymerization. We reconstitute clathrin budding in vitro with giant unilamellar vesicles (GUVs), purified adaptors and clathrin. By changing the osmotic conditions, we find that clathrin coats cause extensive budding of GUVs under low membrane tension while polymerizing into shallow pits under moderate tension. High tension fully inhibits polymerization. Theoretically, we predict the tension values for which transitions between different clathrin coat shapes occur. We measure the changes in membrane tension during clathrin polymerization, and use our theoretical framework to estimate the polymerization energy from these data. Our results show that membrane tension controls clathrin-mediated budding by varying the membrane budding energy.

DOI

Ribosome Excursions during mRNA Translocation Mediate Broad Branching of Frameshift Pathways

Shannon Yan, Jin-Der Wen, Carlos Bustamante, Ignacio Tinoco Jr.

Programmed ribosomal frameshifting produces alternative proteins from a single transcript. −1 frameshifting occurs on Escherichia coli’s dnaX mRNA containing a slippery sequence AAAAAAG and peripheral mRNA structural barriers. Here, we reveal hidden aspects of the frameshifting process, including its exact location on the mRNA and its timing within the translation cycle. Mass spectrometry of translated products shows that ribosomes enter the −1 frame from not one specific codon but various codons along the slippery sequence and slip by not just −1 but also −4 or +2 nucleotides. Single-ribosome translation trajectories detect distinctive codon-scale fluctuations in ribosome-mRNA displacement across the slippery sequence, representing multiple ribosomal translocation attempts during frameshifting. Flanking mRNA structural barriers mechanically stimulate the ribosome to undergo back-and-forth translocation excursions, broadly exploring reading frames. Both experiments reveal aborted translation around mutant slippery sequences, indicating that subsequent fidelity checks on newly adopted codon position base pairings lead to either resumed translation or early termination.

DOI

Kinesin-8 Motors Improve Nuclear Centering by Promoting Microtubule Catastrophe

Matko Glunčić, Nicola Maghelli, Alexander Krull, Vladimir Krstić, Damien Ramunno-Johnson, Nenad Pavin, and Iva M. Tolić

In fission yeast, microtubules push against the cell edge, thereby positioning the nucleus in the cell center. Kinesin-8 motors regulate microtubule catastrophe; however, their role in nuclear positioning is not known. Here we develop a physical model that describes how kinesin-8 motors affect nuclear centering by promoting a microtubule catastrophe. Our model predicts the improved centering of the nucleus in the presence of motors, which we confirmed experimentally in living cells. The model also predicts a characteristic time for the recentering of a displaced nucleus, which is supported by our experiments where we displaced the nucleus using optical tweezers.

DOI

Saturday, February 28, 2015

Manipulating ellipsoidal micro-particles by femtosecond vortex tweezers

Changxia Liu, Zhongyi Guo, Yan Li, Xinshun Wang and Shiliang Qu

We study the manipulation of the ellipsoidal micro-particles by the femtosecond vortex tweezer experimentally and theoretically. In our setup the micro-particles can be rotated stably by means of optical torque induced by the femtosecond optical whirl. In order to give insight into observed effects, we establish two adequate theoretical models: one is based upon assumption of full absorption and the other uses the ray tracing method. The numerical simulations agree well with the experimental results.

The lensing effect of trapped particles in a dual-beam optical trap

Steffen Grosser, Anatol W. Fritsch, Tobias R. Kießling, Roland Stange, and Josef A. Käs

In dual-beam optical traps, two counterpropagating, divergent laser beams emitted from opposing laser fibers trap and manipulate dielectric particles. We investigate the lensing effect that trapped particles have on the beams. Our approach makes use of the intrinsic coupling of a beam to the opposing fiber after having passed the trapped particle. We present measurements of this coupling signal for PDMS particles, as well as a model for its dependence on size and refractive index of the trapped particle. As a more complex sample, the coupling of inhomogeneous biological cells is measured and discussed. We show that the lensing effect is well captured by the simple ray optics approximation. The measurements reveal intricate details, such as the thermal lens effect of the beam propagation in a dual-beam trap. For a particle of known size, the model further allows to infer its refractive index simply from the coupling signal.

DOI

The Aqueous Stability of a Mars Salt Analog: Instant Mars

D. L. Nuding, R. D. Davis, R. V. Gough and M. A. Tolbert

Due to their stability in low temperature conditions, aqueous salt solutions are the favored explanation for potential fluid features observed on present-day Mars. A salt analog was developed to closely match the individual cation and anion concentrations at the Phoenix landing site as reported by the Wet Chemistry Laboratory instrument. ’Instant Mars’ closely replicates correct relative concentrations of magnesium, calcium, potassium, sodium, perchlorate, chloride, and sulfate ions. A Raman microscope equipped with an environmental cell probed liquid water uptake and loss by Instant Mars particles in a Mars relevant temperature and relative humidity (RH) environment. Our experiments reveal that Instant Mars particles can form stable, aqueoussolutions starting at 56 ± 5% RH between 235–243 K and persist as a metastable, aqueous solution at or above 13 ± 5% RH. Particle levitation using an optical trap examined the phase state and morphology of suspended Instant Marsparticles exposed to changing water vapor conditions at room temperature. Levitation experiments indicate that water uptake began at 42 ± 8% RH for Instant Mars particles at 293 K. As RH is decreased at 293 K, the aqueous Instant Mars particles transition into a crystalline solid at 18 ± 7% RH. These combined results demonstrate that Instant Mars can take up water vapor from the surrounding environment and transition into a stable, aqueous solution. Furthermore, this aqueous Instant Mars solution can persist as a metastable, supersaturated solution in low RH conditions.

Doing the Waltz with Light

Monika Ritsch-Marte

Light with orbital angular momentum can be compared to a bundle of spaghetti held together firmly and twisted in the middle. Such optical spiral waves have numerous applications. For example, they can be used to selectively sort particles with optical tweezers. In microscopy, they enable edge enhancement, and in quantum cryptography, spiral waves allow for more information to be entangled.

DOI

Wednesday, February 25, 2015

Transitional behavior in hydrodynamically coupled oscillators

S. Box, L. Debono, D. B. Phillips, and S. H. Simpson

In this article we consider the complete set of synchronized and phase-locked states available to pairs of hydrodynamically coupled colloidal rotors, consisting of spherical beads driven about circular paths in the same, and in opposing senses. Oscillators such as these have previously been used as coarse grained, minimal models of beating cilia. Two mechanisms are known to be important in establishing synchrony. The first involves perturbation of the driving force, and the second involves deformation of the rotor trajectory. We demonstrate that these mechanisms are of similar strength, in the regime of interest, and interact to determine observed behavior. Combining analysis and simulation with experiments performed using holographic optical tweezers, we show how varying the amplitude of the driving force perturbation leads to a transition from synchronized to phase-locked states. Analogies with biological systems are discussed, as are implications for the design of biomimetic devices.

DOI

Single-molecule kinetics and footprinting of DNA bis-intercalation: the paradigmatic case of Thiocoraline

Joan Camunas-Soler, Maria Manosas, Silvia Frutos, Judit Tulla-Puche, Fernando Albericio and Felix Ritort

DNA bis-intercalators are widely used in molecular biology with applications ranging from DNA imaging to anticancer pharmacology. Two fundamental aspects of these ligands are the lifetime of the bis-intercalated complexes and their sequence selectivity. Here, we perform single-molecule optical tweezers experiments with the peptide Thiocoraline showing, for the first time, that bis-intercalation is driven by a very slow off-rate that steeply decreases with applied force. This feature reveals the existence of a long-lived (minutes) mono-intercalated intermediate that contributes to the extremely long lifetime of the complex (hours). We further exploit this particularly slow kinetics to determine the thermodynamics of binding and persistence length of bis-intercalated DNA for a given fraction of bound ligand, a measurement inaccessible in previous studies of faster intercalating agents. We also develop a novel single-molecule footprinting technique based on DNA unzipping and determine the preferred binding sites of Thiocoraline with one base-pair resolution. This fast and radiolabelling-free footprinting technique provides direct access to the binding sites of small ligands to nucleic acids without the need of cleavage agents. Overall, our results provide new insights into the binding pathway of bis-intercalators and the reported selectivity might be of relevance for this and other anticancer drugs interfering with DNA replication and transcription in carcinogenic cell lines.

DOI