.

Monday, January 22, 2018

Design of an optical conveyor for selective separation of a mixture of enantiomers

P. Acebal, L. Carretero, and S. Blaya

Chiral resolution is a fundamental problem in pharmaceutics or agrochemicals, so a great effort has been made to generate experimental techniques capable of producing mechanical separation of a mixture of enantiomers. Unlike other techniques that are usually employed, such as chiral resolving agents or chiral chromatography, we propose a new technique which is directly applicable in solution and without further processing. This technique is based on optical forces, since we show that with the proper design of the polarization states of the incident beams and temporal dephasing, a chiral sensitive optical conveyor can be obtained that is able to transport enantiomers in opposite directions. The implementation of such an optical conveyor with the required focused optical fields produces a well-defined trapping region for each enantiomer, since theoretical simulations over a large number of chiral particle trajectories show that it is possible to reach values of enantiomeric excess of over 99%.

DOI

Single Actin Bundle Rheology

Dan Strehle, Paul Mollenkopf, Martin Glaser, Tom Golde, Carsten Schuldt, Josef A. Käs and Jörg Schnauß

Bundled actin structures play an essential role in the mechanical response of the actin cytoskeleton in eukaryotic cells. Although responsible for crucial cellular processes, they are rarely investigated in comparison to single filaments and isotropic networks. Presenting a highly anisotropic structure, the determination of the mechanical properties of individual bundles was previously achieved through passive approaches observing bending deformations induced by thermal fluctuations. We present a new method to determine the bending stiffness of individual bundles, by measuring the decay of an actively induced oscillation. This approach allows us to systematically test anisotropic, bundled structures. Our experiments revealed that thin, depletion force-induced bundles behave as semiflexible polymers and obey the theoretical predictions determined by the wormlike chain model. Thickening an individual bundle by merging it with other bundles enabled us to study effects that are solely based on the number of involved filaments. These thicker bundles showed a frequency-dependent bending stiffness, a behavior that is inconsistent with the predictions of the wormlike chain model. We attribute this effect to internal processes and give a possible explanation with regard to the wormlike bundle theory.

DOI

Techniques to stimulate and interrogate cell-cell adhesion mechanics

Ruiguo Yang, Joshua A. Broussard, Kathleen J. Green, Horacio D. Espinosa

Cell–cell adhesions maintain the mechanical integrity of multicellular tissues and have recently been found to act as mechanotransducers, translating mechanical cues into biochemical signals. Mechanotransduction studies have primarily focused on focal adhesions, sites of cell-substrate attachment. These studies leverage technical advances in devices and systems interfacing with living cells through cell-extracellular matrix adhesions. As reports of aberrant signal transduction originating from mutations in cell–cell adhesion molecules are being increasingly associated with disease states, growing attention is being paid to this intercellular signaling hub. Along with this renewed focus, new requirements arise for the interrogation and stimulation of cell–cell adhesive junctions. This review covers established experimental techniques for stimulation and interrogation of cell–cell adhesion from cell pairs to monolayers.

DOI

Mfd Dynamically Regulates Transcription via a Release and Catch-Up Mechanism

Tung T. Le, Yi Yang, Chuang Tan, Margaret M. Suhanovsky, Robert M. Fulbright Jr., James T. Inman, Ming Li, Jaeyoon Lee, Sarah Perelman, Jeffrey W. Roberts, Alexandra M. Deaconescu, Michelle D. Wang

The bacterial Mfd ATPase is increasingly recognized as a general transcription factor that participates in the resolution of transcription conflicts with other processes/roadblocks. This function stems from Mfd’s ability to preferentially act on stalled RNA polymerases (RNAPs). However, the mechanism underlying this preference and the subsequent coordination between Mfd and RNAP have remained elusive. Here, using a novel real-time translocase assay, we unexpectedly discovered that Mfd translocates autonomously on DNA. The speed and processivity of Mfd dictate a “release and catch-up” mechanism to efficiently patrol DNA for frequently stalled RNAPs. Furthermore, we showed that Mfd prevents RNAP backtracking or rescues a severely backtracked RNAP, allowing RNAP to overcome stronger obstacles. However, if an obstacle’s resistance is excessive, Mfd dissociates the RNAP, clearing the DNA for other processes. These findings demonstrate a remarkably delicate coordination between Mfd and RNAP, allowing efficient targeting and recycling of Mfd and expedient conflict resolution.

DOI

Single-molecule measurements of the effect of force on Thy-1/αvβ3-integrin interaction using non-purified proteins

Francesca Burgos-Bravo, Nataniel L. Figueroa, Nathalie Casanova-Morales, Andrew F. G. Quest, Christian A. M. Wilson, and Lisette Leyton

Thy-1 and αvβ3 integrin mediate bidirectional cell-to-cell communication between neurons and astrocytes. Thy-1/αvβ3 interactions stimulate astrocyte migration and the retraction of neuronal prolongations, both processes in which internal forces are generated affecting the bimolecular interactions that maintain cell-cell adhesion. Nonetheless, how the Thy-1/αvβ3 interactions respond to mechanical cues is an unresolved issue. In this study, optical tweezers were used as a single-molecule force transducer, and the Dudko-Hummer-Szabo Model was applied to calculate the kinetic parameters of Thy-1/αvβ3 dissociation. A novel experimental strategy was implemented to analyze the interaction of Thy-1-Fc with non-purified αvβ3-Fc integrin, whereby non-specific rupture events were corrected by using a new mathematical approach. This methodology permitted accurately estimating specific rupture forces for Thy-1-Fc/αvβ3-Fc dissociation and calculating the kinetic and transition state parameters. Force exponentially accelerated Thy-1/αvβ3 dissociation, indicating slip bond behavior. Importantly, non-specific interactions were detected even for purified proteins, highlighting the importance of correcting for such interactions. In conclusion, we describe a new strategy to characterize the response of bimolecular interactions to forces even in the presence of non-specific binding events. By defining how force regulates Thy-1/αvβ3 integrin binding, we provide an initial step towards understanding how the neuron-astrocyte pair senses and responds to mechanical cues.

DOI

Study of non-covalent interactions on dendriplex formation: Influence of hydrophobic, electrostatic and hydrogen bonds interactions

María Sánchez-Milla, Isabel Pastor, Marek Maly, M. Jesús Serramía, Rafael Gómez, Javier Sánchez-Nieves, Félix Ritort, M. Ángeles Muñoz-Fernández, F. Javierde la Mata

The interaction of a double stranded small interference RNA (siRNA Nef) with cationic carbosilane dendrimers of generations 1–3 with two different ammonium functions at the periphery ([−NMe2R]+, R = Me, (CH2)2OH) has been studied by experimental techniques (zeta potential, electrophoresis, single molecule pulling experiments) and molecular dynamic calculations. These studies state the presence of different forces on dendriplex formation, depending on generation and type of ammonium group. Whilst for higher dendrimers electrostatic forces mainly drive the stability of dendriplexes, first generation compounds can penetrate into siRNA strands due to the establishment of hydrophobic interactions. Finally, in the particular case of first generation dendrimer [G1O3(NMe2(CH2)2OH))6]6+; the presence of hydroxyl groups reinforces dendriplex stability by hydrogen bonds formation. However, since these small dendrimers do not cover the RNA, only higher generation derivatives protect RNA from degradation.

DOI

Friday, January 19, 2018

Micro/nanofluidics-enabled single-cell biochemical analysis

Ling Lin, Qinghua Chen, Jiashu Sun

The micro/nanofluidic technique has become an important tool for single-cell analysis with the capability to integrate time-consuming and labour-intensive experimental procedures into a small device. Micro/nanofluidics-based biochemical analysis exhibits advantages over conventional methods in terms of small sample volume, rapid turnaround time, straightforward operation, and efficient processing. Single-cell manipulation, therapy, detection and sequencing could be implemented within a sophisticated and multi-functional micro/nanofluidic platform. Here we review recent developments of micro/nanofluidic technologies for single-cell analysis, with emphasis on cell trapping, treatment, and biochemical studies. The potential of micro/nanofluidics-based single-cell analysis is discussed.

DOI

Extended depth of field for single biomolecule optical imaging-force spectroscopy

Minhyeok Chang, Jungsic Oh, Yeonghoon Kim, Sungchul Hohng, and Jong-Bong Lee

Real-time optical imaging combined with single-molecule manipulation broadens the horizons for acquiring information about the spatiotemporal localization and the mechanical details of target molecules. To obtain an optical signal outside the focal plane without unintended interruption of the force signal in single-molecule optical imaging-force spectroscopy, we developed an optical method to extend the depth of field in a high numerical aperture objective (≥ 1.2), required to visualize a single fluorophore. By axial scanning, using an electrically tunable lens with a fixed sample, we were successfully able to visualize the epidermal growth factor receptor (EGFR) moving along the three-dimensionally elongated filamentous actin bundles connecting cells (intercellular nanotube), while another EGFR on the intercellular nanotube was trapped by optical tweezers in living cells. Our approach is simple, fast and inexpensive, but it is powerful for imaging target molecules axially in single-molecule optical imaging-force spectroscopy.

DOI

Testing sub-gravitational forces on atoms from a miniature in-vacuum source mass

Matt Jaffe, Philipp Haslinger, Victoria Xu, Paul Hamilton, Amol Upadhye, Benjamin Elder, Justin Khoury & Holger Müller

Traditional gravity measurements use bulk masses to both source and probe gravitational fields1. Matter-wave interferometers enable the use of probe masses as small as neutrons2, atoms3 and molecular clusters4, but still require fields generated by masses ranging from hundreds of kilograms5,6 to the entire Earth. Shrinking the sources would enable versatile configurations, improve positioning accuracy, enable tests for beyond-standard-model (‘fifth’) forces, and allow observation of non-classical effects of gravity. Here we detect the gravitational force between freely falling caesium atoms and an in-vacuum, miniature (centimetre-sized, 0.19 kg) source mass using atom interferometry. Sensitivity down to gravitational strength forces accesses the natural scale7 for a wide class of cosmologically motivated scalar field models8,9 of modified gravity and dark energy. We improve the limits on two such models, chameleons9 and symmetrons10,11, by over two orders of magnitude. We expect further tests of dark energy theories, and measurements of Newton’s gravitational constant and the gravitational Aharonov–Bohm effect12.

DOI

A minimally invasive optical trapping system to understand cellular interactions at onset of an immune response

David G. Glass, Niall McAlinden, Owain R. Millington, Amanda J. Wright

T-cells and antigen presenting cells are an essential part of the adaptive immune response system and how they interact is crucial in how the body effectively fights infection or responds to vaccines. Much of the experimental work studying interaction forces between cells has looked at the average properties of bulk samples of cells or applied microscopy to image the dynamic contact between these cells. In this paper we present a novel optical trapping technique for interrogating the force of this interaction and measuring relative interaction forces at the single-cell level. A triple-spot optical trap is used to directly manipulate the cells of interest without introducing foreign bodies such as beads to the system. The optical trap is used to directly control the initiation of cell-cell contact and, subsequently to terminate the interaction at a defined time point. The laser beam power required to separate immune cell pairs is determined and correlates with the force applied by the optical trap. As proof of concept, the antigen-specific increase in interaction force between a dendritic cell and a specific T-cell is demonstrated. Furthermore, it is demonstrated that this interaction force is completely abrogated when T-cell signalling is blocked. As a result the potential of using optical trapping to interrogate cellular interactions at the single cell level without the need to introduce foreign bodies such as beads is clearly demonstrated.

DOI