Wednesday, July 1, 2015

Factor-dependent processivity in human eIF4A DEAD-box helicase

Cuauhtémoc García-García, Kirsten L. Frieda, Kateryna Feoktistova, Christopher S. Fraser, Steven M. Block

During eukaryotic translation initiation, the small ribosomal subunit, assisted by initiation factors, locates the messenger RNA start codon by scanning from the 5′ cap. This process is powered by the eukaryotic initiation factor 4A (eIF4A), a DEAD-box helicase. eIF4A has been thought to unwind structures formed in the untranslated 5′ region via a nonprocessive mechanism. Using a single-molecule assay, we found that eIF4A functions instead as an adenosine triphosphate–dependent processive helicase when complexed with two accessory proteins, eIF4G and eIF4B. Translocation occurred in discrete steps of 11 ± 2 base pairs, irrespective of the accessory factor combination. Our findings support a memory-less stepwise mechanism for translation initiation and suggest that similar factor-dependent processivity may be shared by other members of the DEAD-box helicase family.


Assessing single upconverting nanoparticle luminescence by optical tweezers

Paloma Rodriguez-Sevilla , Hector Rodriguez-Rodriguez , Marco Pedroni , Adolfo Speghini , Marco Bettinelli , Jose Antonio Garcia Sole , Daniel Jaque , and Patricia Haro-Gonzalez

We report on stable, long-term immobilization and localization of a single colloidal Er3+/Yb3+ codoped upconverting fluorescent nanoparticle (UCNP) by optical trapping with a single infrared laser beam. Contrary to expectations, the single UCNP emission differs from that generated by an assembly of UCNPs. The experimental data reveal that the differences can be explained in terms of modulations caused by radiation-trapping, a phenomenon not considered before but this work reveals to be of great relevance.


The Effect of Short Duration Ultrasound Pulses on the Interaction Between Individual Microbubbles and Fibrin Clots

Christopher Acconcia, Ben Y.C. Leung, Anoop Manjunath, David E. Goertz

In previous work, we examined microscale interactions between microbubbles and fibrin clots under exposure to 1 ms ultrasound pulses. This provided direct evidence that microbubbles were capable of deforming clot boundaries and penetrating into clots, while also affecting fluid uptake and inducing fibrin network damage. Here, we investigate the effect of short duration (15 μs) pulses on microscale bubble-clot interactions as function of bubble diameter (3–9 μm) and pressure. Individual microbubbles (n = 45) were placed at the clot boundary with optical tweezers and exposed to 1 MHz ultrasound. High-speed (10 kfps) imaging and 2-photon microscopy were performed during and after exposure, respectively. While broadly similar phenomena were observed as in the 1 ms pulse case (i.e., bubble penetration, network damage and fluid uptake), substantial quantitative differences were present. The pressure threshold for bubble penetration was increased from 0.39 MPa to 0.6 MPa, and those bubbles that did enter clots had reduced penetration depths and were associated with less fibrin network damage and nanobead uptake. This appeared to be due in large part to increased bubble shrinkage relative to the 1 ms pulse case. Stroboscopic imaging was performed on a subset of bubbles (n = 11) and indicated that complex bubble oscillations can occur during this process.


Microtubule C-terminal tails can change characteristics of motor force production

Mitra Shojania Feizabadi, Babu Reddy J N, Omid Vadpey, Yonggun Jun, Dail Chapman, Steven Rosenfeld and Steven P. Gross

Control of intracellular transport is poorly understood, and functional ramifications of tubulin isoform differences between cell types are mostly unexplored. Motors’ force production and detachment kinetics are critical for their group function, but how microtubule details affect these properties—if at all—are unknown. We investigated these questions using both a vesicular transport human kinesin, Kinesin-1 and also a mitotic kinesin likely optimized for group function, Kinesin-5, moving along either bovine brain or MCF7(breast cancer) microtubules. We found that kinesin-1 functioned similarly on the two sets of microtubules—in particular, its mean force production was approximately the same, though due to its previously reported decreased processivity, the mean duration of Kinesin-1 force production was slightly decreased on MCF7 MTs.
In contrast, Kinesin-5's function changed dramatically on MCF7 microtubules: its average detachment force was reduced and its force-velocity curve was different. In spite of the reduced detachment force, the force-velocity alteration surprisingly improved high-load group function for Kinesin-5 on the cancer-cell microtubules, potentially contributing to functions such as spindle-mediated chromosome separation. Significant differences were previously reported for C-terminal tubulin tails in MCF7 vs bovine brain tubulin. Consistent with this difference being functionally important, elimination of the tails made transport along the two sets of microtubules similar.


Tuesday, June 30, 2015

Optical regulation of cell chain

Xiaoshuai Liu, Jianbin Huang, Yao Zhang & Baojun Li

Formation of cell chains is a straightforward and efficient method to study the cell interaction. By regulating the contact sequence and interaction distance, the influence of different extracellular cues on the cell interaction can be investigated. However, it faces great challenges in stable retaining and precise regulation of cell chain, especially in cell culture with relatively low cell concentration. Here we demonstrated an optical method to realize the precise regulation of cell chain, including removing or adding a single cell, adjusting interaction distance, and changing cell contact sequence. After injecting a 980-nm wavelength laser beam into a tapered optical fiber probe (FP), a cell chain of Escherichia colis (E. colis) is formed under the optical gradient force. By manipulating another FP close to the cell chain, a targeted E. coli cell can be trapped by the FP and removed from the chain. Further, the targeted cell can be added back to the chain at different positions to change the cell contact sequence. The experiments were interpreted by numerical simulations and the impact of cell sizes and shapes on this method was analyzed.


Deformation of phospholipid vesicles in an optical stretcher

Ulysse DELABRE, Kasper Feld, Eleonore Crespo, Graeme Whyte, Cecile Sykes, Udo Seifert and Jochen Guck

Phospholipid vesicles are common model systems for cell membranes. Important aspects of membrane function relate to its mechanical properties. Here we have investigated the deformation behaviour of phospholipid vesicles in a dual-beam laser trap, also called an optical stretcher. This study explicitly makes use of the inherent heating present in such traps to investigate the dependence of vesicle deformation on temperature. By using lasers with different wavelengths, optically induced mechanical stresses and temperature increase can be tuned fairly independently with a single setup. The phase transition temperature of vesicle can be clearly identified by an increase in deformation. In the case of no heating effects, a minimal model for drop deformation in an optical stretcher and a more specific model for vesicle deformation that take explicitly into account the angular dependence of the optical stress are presented to account for the experimental results. Elastic constants are extracted from the fitting procedures, which agree with literature data. This study demonstrates the utility of optical stretching, which is easily combined with microfluidic delivery, for future serial, high-throughput study of the mechanical and thermodynamic properties of phospholipid vesicles.


A combined electrochemical and optical trapping platform for measuring single cell respiration rates at electrode interfaces

Benjamin J. Gross and Mohamed Y. El-Naggar

Metal-reducing bacteria gain energy by extracellular electron transfer to external solids, such as naturally abundant minerals, which substitute for oxygen or the other common soluble electron acceptors of respiration. This process is one of the earliest forms of respiration on earth and has significant environmental and technological implications. By performing electron transfer to electrodes instead of minerals, these microbes can be used as biocatalysts for conversion of diverse chemical fuels to electricity. Understanding such a complex biotic-abiotic interaction necessitates the development of tools capable of probing extracellular electron transfer down to the level of single cells. Here, we describe an experimental platform for single cell respiration measurements. The design integrates an infrared optical trap, perfusion chamber, and lithographically fabricated electrochemical chips containing potentiostatically controlled transparent indium tin oxide microelectrodes. Individual bacteria are manipulated using the optical trap and placed on the microelectrodes, which are biased at a suitable oxidizing potential in the absence of any chemical electron acceptor. The potentiostat is used to detect the respiration current correlated with cell-electrode contact. We demonstrate the system with single cell measurements of the dissimilatory-metal reducing bacterium Shewanella oneidensis MR-1, which resulted in respiration currents ranging from 15 fA to 100 fA per cell under our measurement conditions. Mutants lacking the outer-membrane cytochromes necessary for extracellular respiration did not result in any measurable current output upon contact. In addition to the application for extracellular electron transfer studies, the ability to electronically measure cell-specific respiration rates may provide answers for a variety of fundamental microbial physiology questions.


Optimized three-dimensional trapping of aerosols: the effect of immersion medium

S. Mohammad-Reza Taheri, Ebrahim Madadi, Mohammad Sadeghi, and S. Nader S. Reihani

Optical tweezers have proven to be indispensable micromanipulation tools especially in aqueous solutions. Because of the significantly larger spherical aberration induced by the refractive index mismatch, trapping aerosols has always been cumbersome if not impossible. We introduce a simple but very efficient method for optimized aerosol trapping at a desired depth. We show that a wise selection of the immersion medium and the mechanical tube length not only enables trapping of objects that are known to be untrappable but also provides a way to tune the trappable depth range.


Thursday, June 25, 2015

Synthesis and photo-postmodification of zeolite L based polymer brushes

Tim Buscher, Álvaro Barroso, Cornelia Denz and Armido Studer

A novel type of zeolite L/polymer hybrid material is presented. Polymer brush particles are generated using surface-functionalized zeolite L crystals as macroinitiators in controlled radical polymerization processes. Copolymerization of a photo-cleavable monomer and subsequent spin trapping of functionalized nitroxides under UV irradiation lead to a variety of highly functionalized zeolite L based core–shell particles.


Controlling dispersion forces between small particles with artificially created random light fields

Georges Brügger, Luis S. Froufe-Pérez, Frank Scheffold & Juan José Sáenz

Appropriate combinations of laser beams can be used to trap and manipulate small particles with optical tweezers as well as to induce significant optical binding forces between particles. These interaction forces are usually strongly anisotropic depending on the interference landscape of the external fields. This is in contrast with the familiar isotropic, translationally invariant, van der Waals and, in general, Casimir–Lifshitz interactions between neutral bodies arising from random electromagnetic waves generated by equilibrium quantum and thermal fluctuations. Here we show, both theoretically and experimentally, that dispersion forces between small colloidal particles can also be induced and controlled using artificially created fluctuating light fields. Using optical tweezers as a gauge, we present experimental evidence for the predicted isotropic attractive interactions between dielectric microspheres induced by laser-generated, random light fields. These light-induced interactions open a path towards the control of translationally invariant interactions with tuneable strength and range in colloidal systems.