Monday, February 20, 2017

Single molecule force spectroscopy reveals the effect of BiP chaperone on protein folding

María Paz Ramírez, Maira Rivera, Diego Quiroga-Roger, Andrés Bustamante, Marcela Vega, Mauricio Baez, Elias M. Puchner, Christian A.M. Wilson

BiP (Immunoglobulin Binding Protein) is a member of the Hsp70 chaperones that participates in protein folding in the endoplasmic reticulum. The function of BiP relies on cycles of ATP hydrolysis driving the binding and release of its substrate proteins. It still remains unknown how BiP affects the protein folding pathway and there has been no direct demonstration showing which folding state of the substrate protein is bound by BiP, as previous work has used only peptides. Here, we employ optical tweezers for single molecule force spectroscopy experiments to investigate how BiP affects the folding mechanism of a complete protein and how this effect depends on nucleotides. Using the protein MJ0366 as the substrate for BiP, we performed pulling and relaxing cycles at constant velocity to unfold and refold the substrate. In the absence of BiP, MJ0366 unfolded and refolded in every cycle. However, when BiP was added, the frequency of folding events of MJ0366 significantly decreased, and the loss of folding always occurred after a successful unfolding event. This process was dependent on ATP and ADP, since when either ATP was decreased or ADP was added, the duration of periods without folding events increased. Our results show that the affinity of BiP for the substrate protein increased in these conditions, which correlates with previous studies in bulk. Therefore, we conclude that BiP binds to the unfolded state of MJ0366 and prevents its refolding, and that this effect is dependent on both the type and concentration of nucleotides.


Holographic optical tweezers-based in vivo manipulations in zebrafish embryos

Florian Hörner, Robert Meissner, Sruthi Polali, Jana Pfeiffer, Timo Betz, Cornelia Denz, Erez Raz

Understanding embryonic development requires the characterization of the forces and the mechanical features that shape cells and tissues within the organism. In addition, experimental application of forces on cells and altering cell and organelle shape allows determining the role such forces play in morphogenesis. Here, we present a holographic optical tweezers-based new microscopic platform for in vivo applications in the context of a developing vertebrate embryo that unlike currently used setups allows simultaneous trapping of multiple objects and rapid comparisons of viscoelastic properties in different locations. This non-invasive technique facilitates a dynamic analysis of mechanical properties of cells and tissues without intervening with embryonic development. We demonstrate the application of this platform for manipulating organelle shape and for characterizing the mechanobiological properties of cells in live zebrafish embryos. The method of holographic optical tweezers as described here is of general interest and can be easily transferred to studying a range of developmental processes in zebrafish, thereby establishing a versatile platform for similar investigations in other organisms.


Microfluidics cell sample preparation for analysis: Advances in efficient cell enrichment and precise single cell capture

Liang Huang, Shengtai Bian, Yinuo Cheng, Guanya Shi, Peng Liu, Xiongying Ye, Wenhui Wang

Single cell analysis has received increasing attention recently in both academia and clinics, and there is an urgent need for effective upstream cell sample preparation. Two extremely challenging tasks in cell sample preparation—high-efficiency cell enrichment and precise single cell capture—have now entered into an era full of exciting technological advances, which are mostly enabled by microfluidics. In this review, we summarize the category of technologies that provide new solutions and creative insights into the two tasks of cell manipulation, with a focus on the latest development in the recent five years by highlighting the representative works. By doing so, we aim both to outline the framework and to showcase example applications of each task. In most cases for cell enrichment, we take circulating tumor cells (CTCs) as the target cells because of their research and clinical importance in cancer. For single cell capture, we review related technologies for many kinds of target cells because the technologies are supposed to be more universal to all cells rather than CTCs. Most of the mentioned technologies can be used for both cell enrichment and precise single cell capture. Each technology has its own advantages and specific challenges, which provide opportunities for researchers in their own area. Overall, these technologies have shown great promise and now evolve into real clinical applications.


Elastic back-scattering patterns via particle surface roughness and orientation from single trapped airborne aerosol particles

Richard Fu, Chuji Wang, Olga Muñoz, Gorden Videen, Joshua L. Santarpia, Yong-Le Pan

We demonstrate a method for simultaneously measuring the back-scattering patterns and images of single laser-trapped airborne aerosol particles. This arrangement allows us to observe how the back-scattering patterns change with particle size, shape, surface roughness, orientation, etc. The recoded scattering patterns cover the angular ranges of θ=167.7–180° (including at 180° exactly) and ϕ=0–360° in spherical coordinates. The patterns show that the width of the average speckle intensity islands or rings is inversely proportional to particle size and how the shape of these intensity rings or islands also depends on the surface roughness. For an irregularly shaped particle with substantial roughness, the back-scattering patterns are formed with speckle intensity islands, the size and orientations of these islands depend more on the overall particle size and orientation, but have less relevance to the fine alteration of the surface structure and shapes. The back-scattering intensity at 180° is very sensitive to the particle parameters. It can change from a maximum to a minimum with a change of 0.1% in particle size or refractive index. The method has potential use in characterizing airborne aerosol particles, and may be used to provide back-scattering information for LIDAR applications.


The role of cyclic di-GMP and exopolysaccharide in type IV pilus dynamics

Jan Ribbe, Amy E. Baker, Sebstian Euler, George A. O'Toole and Berenike Maier

For Pseudomonas aeruginosa the levels of cyclic di-GMP govern the transition from the planktonic state to biofilm formation. Type IV pili (T4P) are crucial determinants of biofilm structure and dynamics, but it is unknown how the levels of c-di-GMP affect pilus dynamics. Here, we scrutinized how c-di-GMP affects molecular motor properties and adhesive behavior of T4P. By means of retraction, T4P generated forces of ∼ 30 pN. Deletion mutants in the proteins with known roles in biofilm formation, swarming motility and exopolysaccharide (EPS) production, specifically, the diguanulate cyclases sadC and roeA or the c-di-GMP phosphodiesterase bifA, showed only modest effects on velocity or force of T4P retraction. At high levels of c-di-GMP, the production of exopolysaccharides (EPS) and in particular of Pel is upregulated. We found that Pel production strongly enhances T4P-mediated surface adhesion of P. aeruginosa, suggesting that that T4P — matrix interactions may be involved in biofilm formation by P. aeruginosa. Finally, our data are consistent with the previously proposed sling-shot-like “twitching” motility of P. aeruginosa.


Friday, February 17, 2017

Measurement of laterally induced optical forces at the nanoscale

Fei Huang, Venkata Ananth Tamma, Mohsen Rajaei, Mohammad Almajhadi, and H. Kumar Wickramasinghe

We demonstrate the measurement of laterally induced optical forces using an Atomic Force Microscope (AFM). The lateral electric field distribution between a gold coated AFM probe and a single nano-aperture in a gold film is mapped by measuring the lateral optical force between the apex of the AFM probe and the nano-aperture. The fundamental torsional eigen-mode of an AFM cantilever probe was used to detect the laterally induced optical forces. We engineered the cantilever shape using focused ion beam milling to improve the detected signal to noise ratio. The measured distributions of lateral optical force agree well with electromagnetic simulations of the metal coated AFM probe interacting with the nano-aperture. This technique can be extended to simultaneously detect both lateral and longitudinal optical forces at the nanoscale by using an AFM cantilever as a multi-channel detector. This will enable simultaneous Photon Induced Force Microscopy detection of molecular responses with different incident field polarizations. The technique can be implemented on both cantilever and tuning fork based AFMs.


Force-Dependent Folding and Unfolding Kinetics in DNA Hairpins Reveals Transition-State Displacements along a Single Pathway

Anna Alemany and Felix Ritort

Biomolecules diffusively explore their energy landscape overcoming energy barriers via thermally activated processes to reach the biologically relevant conformation. Mechanically induced unfolding and folding reactions offer an excellent playground to feature these processes at the single-molecule level by monitoring changes in the molecular extension. Here we investigate two-state DNA hairpins designed to have the transition states at different locations. We use optical tweezers to characterize the force-dependent behavior of the kinetic barrier from nonequilibrium pulling experiments by using the continuous effective barrier approach (CEBA). We introduce the mechanical fragility and the molecular transition-state susceptibility, both useful quantities to characterize the response of the transition state to an applied force. Our results demonstrate the validity of the Leffler–Hammond postulate where the transition state approaches the folded state as force increases, implying monotonically decreasing fragility with force and a non-negative transition state susceptibility at all forces.


Photon-phonon Interaction in a Microfiber Induced by Optical and Electrostrictive Forces

Yun-chao Shi, Wei Luo, Fei Xu & Yan-qing Lu

Stimulated Brillouin scattering (SBS) via electrostrictive force is a fundamental interaction between light and sound which limits the power in conventional optical fibers. The emergence of optical microfibers with subwavelength diameter, ultralight mass and an intense light field, provides a new platform for photon–phonon coupling, resulting in the radiation pressure mediated contribution of SBS. This study examines the optomechanical system in cylindrical coordinates, reveals the theoretically radiation pressure induced analogous, and demonstrates contrary effect compared with electrostrictive force in solid or hollow silica microfibers. The finding shows that the photon-phonon coupling, which is related to SBS, can be suppressed in a solid microfiber, and even be completely cancelled in a hollow microfiber.


Unfolding DNA condensates produced by DNA-like charged depletants: A force spectroscopy study

C. H. M. Lima, M. S. Rocha, and E. B. Ramos

In this work, we have measured, by means of optical tweezers, forces acting on depletion-induced DNA condensates due to the presence of the DNA-like charged protein bovine serum albumin (BSA). The stretching and unfolding measurements performed on the semi-flexible DNA chain reveal (1) the softening of the uncondensed DNA contour length and (2) a mechanical behavior strikingly different from those previously observed: the force-extension curves of BSA-induced DNA condensates lack the “saw-tooth” pattern and applied external forces as high as ≈80pN≈80 pN are unable to fully unfold the condensed DNA contour length. This last mechanical experimental finding is in agreement with force-induced “unpacking” detailed Langevin dynamics simulations recently performed by Cortini et al. on model rod-like shaped condensates. Furthermore, a simple thermodynamics analysis of the unfolding process has enabled us to estimate the free energy involved in the DNA condensation: the estimated depletion-induced interactions vary linearly with both the condensed DNA contour length and the BSA concentration, in agreement with the analytical and numerical analysis performed on model DNA condensates. We hope that future additional experiments can decide whether the rod-like morphology is the actual one we are dealing with (e.g. pulling experiments coupled with super-resolution fluorescence microscopy).


Oscillations of absorbing particles at the water-air interface induced by laser tweezers

Min-Cheng Zhong, Zi-Qiang Wang, and Yin-Mei Li

We present an experimental study on oscillation of absorbing particles at the water-air interface. The oscillation is induced by laser tweezers, which are generated with a high numerical aperture objective. When the laser beam is tightly focused at the water-air interface, the optical gradient force attracts the particles to the spot center, and the laser heating of particles results in a strong thermal gradient that drives the particles to leave the spot center. Under the action of thermal and optical gradient force together, the absorbing particles oscillate at the water-air interface.