M. J. Tang, J. C. J. Camp, L. Rkiouak, J. McGregor, I. M. Watson, R. A. Cox, M. Kalberer, A. D. Ward and F. D. Pope
Silica (SiO2) is an important mineral present in atmospheric mineral dust particles, and the heterogeneous reaction of N2O5 on atmospheric aerosol is one of the major pathways to remove nitrogen oxides from the atmosphere. The heterogeneous reaction of N2O5 with SiO2 has only been investigated by two studies previously, and the reported uptake coefficients differ by a factor of >10. In this work two complementary laboratory techniques were used to study the heterogeneous reaction of SiO2 particles with N2O5 at room temperature and at different relative humidities (RHs). The uptake coefficients of N2O5, γ(N2O5), were determined to be (7.2 ± 0.6) × 10–3 (1σ) at 7% RH and (5.3 ± 0.8) × 10–3 (1σ) at 40% RH for SiO2 particles, using the aerosol flow tube technique. We show that γ(N2O5) determined in this work can be reconciled with the two previous studies by accounting for the difference in geometric and BET derived aerosol surface areas. To probe the particle phase chemistry, individual micrometer sized SiO2 particles were optically levitated and exposed to a continuous flow of N2O5 at different RHs, and the composition of levitated particles was monitored online using Raman spectroscopy. This study represents the first investigation into the heterogeneous reactions of levitated individual SiO2 particles as a surrogate for mineral dust. Relative humidity was found to play a critical role: while no significant change of particle composition was observed by Raman spectroscopy during exposure to N2O5 at RH of <2%, increasing the RH led to the formation of nitrate species on the particle surface which could be completely removed after decreasing the RH back to <2%. This can be explained by the partitioning of HNO3 between the gas and adsorbed phases. The atmospheric implications of this work are discussed.
DOI
Monday, September 29, 2014
Common intermediates and kinetics, but different energetics, in the assembly of SNARE proteins
Sylvain Zorman, Aleksander A Rebane, Lu Ma, Guangcan Yang, Matthew A Molski, Jeff Coleman, Frederic Pincet, James E Rothman, Yongli Zhang
Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are evolutionarily conserved machines that couple their folding/assembly to membrane fusion. However, it is unclear how these processes are regulated and function. To determine these mechanisms, we characterized the folding energy and kinetics of four representative SNARE complexes at a single-molecule level using high-resolution optical tweezers. We found that all SNARE complexes assemble by the same step-wise zippering mechanism: slow N-terminal domain (NTD) association, a pause in a force-dependent half-zippered intermediate, and fast C-terminal domain (CTD) zippering. The energy release from CTD zippering differs for yeast (13 kBT) and neuronal SNARE complexes (27 kBT), and is concentrated at the C-terminal part of CTD zippering. Thus, SNARE complexes share a conserved zippering pathway and polarized energy release to efficiently drive membrane fusion, but generate different amounts of zippering energy to regulate fusion kinetics.
DOI
Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are evolutionarily conserved machines that couple their folding/assembly to membrane fusion. However, it is unclear how these processes are regulated and function. To determine these mechanisms, we characterized the folding energy and kinetics of four representative SNARE complexes at a single-molecule level using high-resolution optical tweezers. We found that all SNARE complexes assemble by the same step-wise zippering mechanism: slow N-terminal domain (NTD) association, a pause in a force-dependent half-zippered intermediate, and fast C-terminal domain (CTD) zippering. The energy release from CTD zippering differs for yeast (13 kBT) and neuronal SNARE complexes (27 kBT), and is concentrated at the C-terminal part of CTD zippering. Thus, SNARE complexes share a conserved zippering pathway and polarized energy release to efficiently drive membrane fusion, but generate different amounts of zippering energy to regulate fusion kinetics.
DOI
Hierarchical organization of chiral rafts in colloidal membranes
Prerna Sharma, Andrew Ward, T. Gibaud, Michael F. Hagan & Zvonimir Dogic
Liquid–liquid phase separation is ubiquitous in suspensions of nanoparticles, proteins and colloids. It has an important role in gel formation, protein crystallization and perhaps even as an organizing principle in cellular biology. With a few notable exceptions, liquid–liquid phase separation in bulk proceeds through the continuous coalescence of droplets until the system undergoes complete phase separation. But when colloids, nanoparticles or proteins are confined to interfaces, surfaces or membranes, their interactions differ fundamentally from those mediated by isotropic solvents, and this results in significantly more complex phase behaviour. Here we show that liquid–liquid phase separation in monolayer membranes composed of two dissimilar chiral colloidal rods gives rise to thermodynamically stable rafts that constantly exchange monomeric rods with the background reservoir to maintain a self-limited size. We visualize and manipulate rafts to quantify their assembly kinetics and to show that membrane distortions arising from the rods’ chirality lead to long-range repulsive raft–raft interactions. Rafts assemble into cluster crystals at high densities, but they can also form bonds to yield higher-order structures. Taken together, our observations demonstrate a robust membrane-based pathway for the assembly of monodisperse membrane clusters that is complementary to existing methods for colloid assembly in bulk suspensions. They also reveal that chiral inclusions in membranes can acquire long-range repulsive interactions, which might more generally have a role in stabilizing assemblages of finite size.
DOI
Liquid–liquid phase separation is ubiquitous in suspensions of nanoparticles, proteins and colloids. It has an important role in gel formation, protein crystallization and perhaps even as an organizing principle in cellular biology. With a few notable exceptions, liquid–liquid phase separation in bulk proceeds through the continuous coalescence of droplets until the system undergoes complete phase separation. But when colloids, nanoparticles or proteins are confined to interfaces, surfaces or membranes, their interactions differ fundamentally from those mediated by isotropic solvents, and this results in significantly more complex phase behaviour. Here we show that liquid–liquid phase separation in monolayer membranes composed of two dissimilar chiral colloidal rods gives rise to thermodynamically stable rafts that constantly exchange monomeric rods with the background reservoir to maintain a self-limited size. We visualize and manipulate rafts to quantify their assembly kinetics and to show that membrane distortions arising from the rods’ chirality lead to long-range repulsive raft–raft interactions. Rafts assemble into cluster crystals at high densities, but they can also form bonds to yield higher-order structures. Taken together, our observations demonstrate a robust membrane-based pathway for the assembly of monodisperse membrane clusters that is complementary to existing methods for colloid assembly in bulk suspensions. They also reveal that chiral inclusions in membranes can acquire long-range repulsive interactions, which might more generally have a role in stabilizing assemblages of finite size.
DOI
Friday, September 26, 2014
Combined holographic optical trapping and optical image processing using a single diffractive pattern displayed on a spatial light modulator
Alexander Jesacher, Stefan Bernet, and Monika Ritsch-Marte
We demonstrate simultaneous holographic optical trapping and optical image processing using a single-phase diffraction pattern displayed on a liquid crystal spatial light modulator (SLM). The ability of modern SLMs to provide multiorder phase shifts represents a degree of freedom that allows the calculation of diffraction patterns that act in precisely defined but different ways on light beams of different wavelengths. We exploit this property to calculate a single-phase hologram that shapes multiple optical traps at 785 nm while performing double-helix point spread function engineering at 532 nm. Both channels are independent to a large degree and have efficiencies of about 75% compared to the ideal diffractive patterns.
DOI
We demonstrate simultaneous holographic optical trapping and optical image processing using a single-phase diffraction pattern displayed on a liquid crystal spatial light modulator (SLM). The ability of modern SLMs to provide multiorder phase shifts represents a degree of freedom that allows the calculation of diffraction patterns that act in precisely defined but different ways on light beams of different wavelengths. We exploit this property to calculate a single-phase hologram that shapes multiple optical traps at 785 nm while performing double-helix point spread function engineering at 532 nm. Both channels are independent to a large degree and have efficiencies of about 75% compared to the ideal diffractive patterns.
DOI
Kinesin processivity is gated by phosphate release
Bojan Milic, Johan O. L. Andreasson, William O. Hancock, and Steven M. Block
Kinesin-1 is a motor protein central to intracellular transport. Prevailing models of the kinesin mechanochemical cycle—which invoke docking of the neck linker domain upon ATP binding—fail to explain the remarkable processivity of kinesin, which represents a competition between dissociation from the microtubule and continuation of the stepping cycle. We show that kinesin dissociation, which characterizes the end of a processive run, is gated by phosphate release following ATP hydrolysis. The structural change driving kinesin motility, likely neck linker docking, is therefore completed only upon hydrolysis. Our results offer insights into gating mechanisms and necessitate revisions to existing models of the kinesin cycle.
DOI
Kinesin-1 is a motor protein central to intracellular transport. Prevailing models of the kinesin mechanochemical cycle—which invoke docking of the neck linker domain upon ATP binding—fail to explain the remarkable processivity of kinesin, which represents a competition between dissociation from the microtubule and continuation of the stepping cycle. We show that kinesin dissociation, which characterizes the end of a processive run, is gated by phosphate release following ATP hydrolysis. The structural change driving kinesin motility, likely neck linker docking, is therefore completed only upon hydrolysis. Our results offer insights into gating mechanisms and necessitate revisions to existing models of the kinesin cycle.
DOI
Wednesday, September 24, 2014
Rapidly Exploring Random Tree Algorithm-Based Path Planning for Robot-Aided Optical Manipulation of Biological Cells
Tao Ju; Shuang Liu; Jie Yang; Dong Sun
In numerous cellular applications, cells are transported to specific positions or extracted from complex cell solutions. Therefore, an efficient cell transportation path planner for these applications is important for avoiding collisions with other cells or obstacles. In this paper, a path planning approach to transporting cells using a robot-aided optical manipulation system is presented. Optical tweezers functions as a special end-effector in transporting a target cell to the desired position along the generated path. The path planner is designed based on the rapidly exploring random trees (RRT) algorithm for calculating a collision-free path for cell transportation. Both static and dynamic path planners are developed. For the dynamic path planner, an online monitoring strategy is employed to dynamically avoid collisions with randomly appeared obstacles caused by environmental influence such as the Brownian movement of microparticles. Experiments of transporting yeast cells are performed to demonstrate the effectiveness of the proposed approach. Note to Practitioners - Manipulations of cells and other microparticles represent an essential process for most cell-based bioengineering applications, such as cytopathology, cell sociology, and cytotaxonomy. Cell transportation, which is treated as a typical cell manipulation task, has recently received considerable attention because of its wide applications. This paper presents a novel approach to applying RRT-based path planner to cell transportation with a robot-aided optical manipulation system. The research outcome provides a unique solution to achieving cell transportation automatically and efficiently.
DOI
In numerous cellular applications, cells are transported to specific positions or extracted from complex cell solutions. Therefore, an efficient cell transportation path planner for these applications is important for avoiding collisions with other cells or obstacles. In this paper, a path planning approach to transporting cells using a robot-aided optical manipulation system is presented. Optical tweezers functions as a special end-effector in transporting a target cell to the desired position along the generated path. The path planner is designed based on the rapidly exploring random trees (RRT) algorithm for calculating a collision-free path for cell transportation. Both static and dynamic path planners are developed. For the dynamic path planner, an online monitoring strategy is employed to dynamically avoid collisions with randomly appeared obstacles caused by environmental influence such as the Brownian movement of microparticles. Experiments of transporting yeast cells are performed to demonstrate the effectiveness of the proposed approach. Note to Practitioners - Manipulations of cells and other microparticles represent an essential process for most cell-based bioengineering applications, such as cytopathology, cell sociology, and cytotaxonomy. Cell transportation, which is treated as a typical cell manipulation task, has recently received considerable attention because of its wide applications. This paper presents a novel approach to applying RRT-based path planner to cell transportation with a robot-aided optical manipulation system. The research outcome provides a unique solution to achieving cell transportation automatically and efficiently.
DOI
Volatility and Oxidative Aging of Aqueous Maleic Acid Aerosol Droplets and the Dependence on Relative Humidity
Benjamin J. Dennis-Smither, Frances H. Marshall, Rachael E. H. Miles, Thomas C. Preston, and Jonathan P. Reid
The microphysical structure and heterogeneous oxidation by ozone of single aerosol particles containing maleic acid (MA) has been studied using aerosol optical tweezers and cavity enhanced Raman spectroscopy. The evaporation rate of MA from aqueous droplets has been measured over a range of relative humidities and the pure component vapor pressure determined to be (1.7 ± 0.2) × 10–3 Pa. Variation in the refractive index (RI) of an aqueous MA droplet with relative humidity (RH) allowed the subcooled liquid RI of MA to be estimated as 1.481 ± 0.001. Measurements of the hygroscopic growth are shown to be consistent with equilibrium model predictions from previous studies. Simultaneous measurements of the droplet composition, size, and refractive index have been made during ozonolysis at RHs in the range 50–80%, providing insight into the volatility of organic products, changes in the droplet hygroscopicity, and optical properties. Exposure of the aqueous droplets to ozone leads to the formation of products with a wide range of volatilities spanning from involatile to volatile. Reactive uptake coefficients show a weak dependence on ozone concentration, but no dependence on RH or salt concentration. The time evolving RI depends significantly on the RH at which the oxidation proceeds and can even show opposing trends; while the RI increases with ozone exposure at low relative humidity, the RI decreases when the oxidation proceeds at high relative humidity. The variations in RI are broadly consistent with a framework for predicting RIs for organic components published by Cappa et al. ( J. Geophys. Res. 2011, 116, D15204). Once oxidized, particles are shown to form amorphous phases on drying rather than crystallization, with slow evaporation kinetics of residual water.
DOI
The microphysical structure and heterogeneous oxidation by ozone of single aerosol particles containing maleic acid (MA) has been studied using aerosol optical tweezers and cavity enhanced Raman spectroscopy. The evaporation rate of MA from aqueous droplets has been measured over a range of relative humidities and the pure component vapor pressure determined to be (1.7 ± 0.2) × 10–3 Pa. Variation in the refractive index (RI) of an aqueous MA droplet with relative humidity (RH) allowed the subcooled liquid RI of MA to be estimated as 1.481 ± 0.001. Measurements of the hygroscopic growth are shown to be consistent with equilibrium model predictions from previous studies. Simultaneous measurements of the droplet composition, size, and refractive index have been made during ozonolysis at RHs in the range 50–80%, providing insight into the volatility of organic products, changes in the droplet hygroscopicity, and optical properties. Exposure of the aqueous droplets to ozone leads to the formation of products with a wide range of volatilities spanning from involatile to volatile. Reactive uptake coefficients show a weak dependence on ozone concentration, but no dependence on RH or salt concentration. The time evolving RI depends significantly on the RH at which the oxidation proceeds and can even show opposing trends; while the RI increases with ozone exposure at low relative humidity, the RI decreases when the oxidation proceeds at high relative humidity. The variations in RI are broadly consistent with a framework for predicting RIs for organic components published by Cappa et al. ( J. Geophys. Res. 2011, 116, D15204). Once oxidized, particles are shown to form amorphous phases on drying rather than crystallization, with slow evaporation kinetics of residual water.
DOI
Intermediates Stabilized by Tryptophan Pairs Exist in Trpzip Beta-Hairpins
Zhongbo Yu, Sangeetha Selvam, and Hanbin Mao
Transitions of protein secondary structures, such as alpha-helices and beta-hairpins, are often too small and too fast to follow by many single-molecular approaches. Here we describe new population deconvolution methods to investigate the mechanical unfolding/refolding events in Trpzip β-hairpins that are tethered between two optically trapped polystyrene particles through click chemistry. The application of force to the Trpzip peptides shifted population distribution, which allowed us to identify intermediates from regular force–extension curves of the peptides after population deconvolution analysis. Comparison of the intermediates between the Trpzip2 and Trpzip4 peptides suggests the intermediates are likely stabilized by the tryptophan pair stacking. We anticipate the method of population deconvolution described here can offer a unique capability to investigate fast transitions in small biological structures.
DOI
Transitions of protein secondary structures, such as alpha-helices and beta-hairpins, are often too small and too fast to follow by many single-molecular approaches. Here we describe new population deconvolution methods to investigate the mechanical unfolding/refolding events in Trpzip β-hairpins that are tethered between two optically trapped polystyrene particles through click chemistry. The application of force to the Trpzip peptides shifted population distribution, which allowed us to identify intermediates from regular force–extension curves of the peptides after population deconvolution analysis. Comparison of the intermediates between the Trpzip2 and Trpzip4 peptides suggests the intermediates are likely stabilized by the tryptophan pair stacking. We anticipate the method of population deconvolution described here can offer a unique capability to investigate fast transitions in small biological structures.
DOI
Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions
Carina Monico, Gionata Belcastro, Francesco Vanzi, Francesco S. Pavone, Marco Capitanio
The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.
DOI
The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.
DOI
Tuesday, September 23, 2014
Direct single-molecule observation of calcium-dependent misfolding in human neuronal calcium sensor-1
Pétur O. Heidarsson, Mohsin M. Naqvi, Mariela R. Otazo, Alessandro Mossa, Birthe B. Kragelund, and Ciro Cecconi
Neurodegenerative disorders are strongly linked to protein misfolding, and crucial to their explication is a detailed understanding of the underlying structural rearrangements and pathways that govern the formation of misfolded states. Here we use single-molecule optical tweezers to monitor misfolding reactions of the human neuronal calcium sensor-1, a multispecific EF-hand protein involved in neurotransmitter release and linked to severe neurological diseases. We directly observed two misfolding trajectories leading to distinct kinetically trapped misfolded conformations. Both trajectories originate from an on-pathway intermediate state and compete with native folding in a calcium-dependent manner. The relative probability of the different trajectories could be affected by modulating the relaxation rate of applied force, demonstrating an unprecedented real-time control over the free-energy landscape of a protein. Constant-force experiments in combination with hidden Markov analysis revealed the free-energy landscape of the misfolding transitions under both physiological and pathological calcium concentrations. Remarkably for a calcium sensor, we found that higher calcium concentrations increased the lifetimes of the misfolded conformations, slowing productive folding to the native state. We propose a rugged, multidimensional energy landscape for neuronal calcium sensor-1 and speculate on a direct link between protein misfolding and calcium dysregulation that could play a role in neurodegeneration.
Neurodegenerative disorders are strongly linked to protein misfolding, and crucial to their explication is a detailed understanding of the underlying structural rearrangements and pathways that govern the formation of misfolded states. Here we use single-molecule optical tweezers to monitor misfolding reactions of the human neuronal calcium sensor-1, a multispecific EF-hand protein involved in neurotransmitter release and linked to severe neurological diseases. We directly observed two misfolding trajectories leading to distinct kinetically trapped misfolded conformations. Both trajectories originate from an on-pathway intermediate state and compete with native folding in a calcium-dependent manner. The relative probability of the different trajectories could be affected by modulating the relaxation rate of applied force, demonstrating an unprecedented real-time control over the free-energy landscape of a protein. Constant-force experiments in combination with hidden Markov analysis revealed the free-energy landscape of the misfolding transitions under both physiological and pathological calcium concentrations. Remarkably for a calcium sensor, we found that higher calcium concentrations increased the lifetimes of the misfolded conformations, slowing productive folding to the native state. We propose a rugged, multidimensional energy landscape for neuronal calcium sensor-1 and speculate on a direct link between protein misfolding and calcium dysregulation that could play a role in neurodegeneration.
The interplay of cell–cell and cell–substrate adhesion in collective cell migration
Chenlu Wang, Sagar Chowdhury, Meghan Driscoll, Carole A. Parent, S. K. Gupta and Wolfgang Losert
Collective cell migration often involves notable cell–cell and cell–substrate adhesions and highly coordinated motion of touching cells. We focus on the interplay between cell–substrate adhesion and cell–cell adhesion. We show that the loss of cell-surface contact does not significantly alter the dynamic pattern of protrusions and retractions of fast migrating amoeboid cells (Dictyostelium discoideum), but significantly changes their ability to adhere to other cells. Analysis of the dynamics of cell shapes reveals that cells that are adherent to a surface may coordinate their motion with neighbouring cells through protrusion waves that travel across cell–cell contacts. However, while shape waves exist if cells are detached from surfaces, they do not couple cell to cell. In addition, our investigation of actin polymerization indicates that loss of cell-surface adhesion changes actin polymerization at cell–cell contacts. To further investigate cell–cell/cell–substrate interactions, we used optical micromanipulation to form cell–substrate contact at controlled locations. We find that both cell-shape dynamics and cytoskeletal activity respond rapidly to the formation of cell–substrate contact.
DOI
Collective cell migration often involves notable cell–cell and cell–substrate adhesions and highly coordinated motion of touching cells. We focus on the interplay between cell–substrate adhesion and cell–cell adhesion. We show that the loss of cell-surface contact does not significantly alter the dynamic pattern of protrusions and retractions of fast migrating amoeboid cells (Dictyostelium discoideum), but significantly changes their ability to adhere to other cells. Analysis of the dynamics of cell shapes reveals that cells that are adherent to a surface may coordinate their motion with neighbouring cells through protrusion waves that travel across cell–cell contacts. However, while shape waves exist if cells are detached from surfaces, they do not couple cell to cell. In addition, our investigation of actin polymerization indicates that loss of cell-surface adhesion changes actin polymerization at cell–cell contacts. To further investigate cell–cell/cell–substrate interactions, we used optical micromanipulation to form cell–substrate contact at controlled locations. We find that both cell-shape dynamics and cytoskeletal activity respond rapidly to the formation of cell–substrate contact.
DOI
Light-Assisted, Templated Self-Assembly of Gold Nanoparticle Chains
Eric Jaquay, Luis Javier Martínez, Ningfeng Huang, Camilo A. Mejia, Debarghya Sarkar, and Michelle L. Povinelli
We experimentally demonstrate the technique of light-assisted, templated self-assembly (LATS) to trap and assemble 200 nm diameter gold nanoparticles. We excite a guided-resonance mode of a photonic-crystal slab with 1.55 μm laser light to create an array of optical traps. Unlike our previous demonstration of LATS with polystyrene particles, we find that the interparticle interactions play a significant role in the resulting particle patterns. Despite a two-dimensionally periodic intensity profile in the slab, the particles form one-dimensional chains whose orientations can be controlled by the incident polarization of the light. The formation of chains can be understood in terms of a competition between the gradient force due to the excitation of the mode in the slab and optical binding between particles.
DOI
We experimentally demonstrate the technique of light-assisted, templated self-assembly (LATS) to trap and assemble 200 nm diameter gold nanoparticles. We excite a guided-resonance mode of a photonic-crystal slab with 1.55 μm laser light to create an array of optical traps. Unlike our previous demonstration of LATS with polystyrene particles, we find that the interparticle interactions play a significant role in the resulting particle patterns. Despite a two-dimensionally periodic intensity profile in the slab, the particles form one-dimensional chains whose orientations can be controlled by the incident polarization of the light. The formation of chains can be understood in terms of a competition between the gradient force due to the excitation of the mode in the slab and optical binding between particles.
DOI
Optical torque from enhanced scattering by multipolar plasmonic resonance
Yoonkyung E. Lee, Kin Hung Fung, Dafei Jin, Nicholas X. Fang
We present a theoretical study of the optical angular momentum transfer from a circularly polarized plane wave to thin metal nanoparticles of different rotational symmetries. While absorption has been regarded as the predominant mechanism of torque generation on the nanoscale, we demonstrate numerically how the contribution from scattering can be enhanced by using multipolar plasmon resonance. The multipolar modes in non-circular particles can convert the angular momentum carried by the scattered field and thereby produce scattering-dominant optical torque, while a circularly symmetric particle cannot. Our results show that the optical torque induced by resonant scattering can contribute to 80% of the total optical torque in gold particles. This scattering-dominant torque generation is extremely mode-specific, and deserves to be distinguished from the absorption-dominant mechanism. Our findings might have applications in optical manipulation on the nanoscale as well as new designs in plasmonics and metamaterials.
DOI
We present a theoretical study of the optical angular momentum transfer from a circularly polarized plane wave to thin metal nanoparticles of different rotational symmetries. While absorption has been regarded as the predominant mechanism of torque generation on the nanoscale, we demonstrate numerically how the contribution from scattering can be enhanced by using multipolar plasmon resonance. The multipolar modes in non-circular particles can convert the angular momentum carried by the scattered field and thereby produce scattering-dominant optical torque, while a circularly symmetric particle cannot. Our results show that the optical torque induced by resonant scattering can contribute to 80% of the total optical torque in gold particles. This scattering-dominant torque generation is extremely mode-specific, and deserves to be distinguished from the absorption-dominant mechanism. Our findings might have applications in optical manipulation on the nanoscale as well as new designs in plasmonics and metamaterials.
DOI
Trapping of resonant metallic nanoparticles with engineered vectorial optical field
Guanghao Rui, Qiwen Zhan
Optical trapping and manipulation using focused laser beams has emerged as a powerful tool in the biological and physical sciences. However, scaling this technique to metallic nanoparticles remains challenging due to the strong scattering force and optical heating effect. In this work, we propose a novel strategy to optically trap metallic nanoparticles even under the resonant condition using engineered optical field. The distribution of the optical forces can be tailored through optimizing the spatial distribution of a vectorial optical illumination to favour the stable trapping of a variety of metallic nanoparticles under various conditions. It is shown that this optical tweezers has the ability of generating negative scattering force and supporting stable three-dimensional trapping for gold nanoparticles at resonance while avoiding trap destabilization due to optical overheating. The technique presented in this work offers a versatile solution for trapping metallic nanoparticles and may open up new avenues for optical manipulation.
DOI
Optical trapping and manipulation using focused laser beams has emerged as a powerful tool in the biological and physical sciences. However, scaling this technique to metallic nanoparticles remains challenging due to the strong scattering force and optical heating effect. In this work, we propose a novel strategy to optically trap metallic nanoparticles even under the resonant condition using engineered optical field. The distribution of the optical forces can be tailored through optimizing the spatial distribution of a vectorial optical illumination to favour the stable trapping of a variety of metallic nanoparticles under various conditions. It is shown that this optical tweezers has the ability of generating negative scattering force and supporting stable three-dimensional trapping for gold nanoparticles at resonance while avoiding trap destabilization due to optical overheating. The technique presented in this work offers a versatile solution for trapping metallic nanoparticles and may open up new avenues for optical manipulation.
DOI
Evanescent wave optical trapping and transport of polystyrene microspheres on microfibers
N. Irawati, V. John, M. M. Aeinehvand, F. Ibrahim, H. Ahmad and S. W. Harun
We investigate the manipulation of polystyrene microspheres using the evanescent optical field surrounding a silica microfiber. The microfiber is produced using a flame brushing technique from a standard single-mode optical fiber. It is observed that the polystyrene microspheres can be attracted and trapped along the microfiber by means of evanescent field, which provides both scattering and gradient forces. The number of microspheres attached to a microfiber decreases in a quadratic manner with the increase in microfiber waist diameter. Scattering and gradient forces exerted on the microspheres also depend on the microsphere size where a larger number of polystyrene microspheres were seen attached to the microfiber if the microsphere size is smaller. It is also observed that the microspheres attached to the microfiber are propelled along the microfiber in the direction of the propagating light. These results show that the proposed optical trapping mechanism can be used for manipulating and identifying minute biological objects such as bacteria, yeast, and organelles.
DOI
We investigate the manipulation of polystyrene microspheres using the evanescent optical field surrounding a silica microfiber. The microfiber is produced using a flame brushing technique from a standard single-mode optical fiber. It is observed that the polystyrene microspheres can be attracted and trapped along the microfiber by means of evanescent field, which provides both scattering and gradient forces. The number of microspheres attached to a microfiber decreases in a quadratic manner with the increase in microfiber waist diameter. Scattering and gradient forces exerted on the microspheres also depend on the microsphere size where a larger number of polystyrene microspheres were seen attached to the microfiber if the microsphere size is smaller. It is also observed that the microspheres attached to the microfiber are propelled along the microfiber in the direction of the propagating light. These results show that the proposed optical trapping mechanism can be used for manipulating and identifying minute biological objects such as bacteria, yeast, and organelles.
DOI
RecA Protein Plays a Role in the Chemotactic Response and Chemoreceptor Clustering of Salmonella enterica
Albert Mayola, Oihane Irazoki, Ignacio A. Martínez, Dmitri Petrov, Filippo Menolascina, Roman Stocker, José A. Reyes-Darias, Tino Krell, Jordi Barbé, Susana Campoy
The RecA protein is the main bacterial recombinase and the activator of the SOS system. In Escherichia coli and Salmonella enterica sv. Typhimurium, RecA is also essential for swarming, a flagellar-driven surface translocation mechanism widespread among bacteria. In this work, the direct interaction between RecA and the CheW coupling protein was confirmed, and the motility and chemotactic phenotype of a S. Typhimurium ΔrecA mutant was characterized through microfluidics, optical trapping, and quantitative capillary assays. The results demonstrate the tight association of RecA with the chemotaxis pathway and also its involvement in polar chemoreceptor cluster formation. RecA is therefore necessary for standard flagellar rotation switching, implying its essential role not only in swarming motility but also in the normal chemotactic response of S. Typhimurium.
DOI
The RecA protein is the main bacterial recombinase and the activator of the SOS system. In Escherichia coli and Salmonella enterica sv. Typhimurium, RecA is also essential for swarming, a flagellar-driven surface translocation mechanism widespread among bacteria. In this work, the direct interaction between RecA and the CheW coupling protein was confirmed, and the motility and chemotactic phenotype of a S. Typhimurium ΔrecA mutant was characterized through microfluidics, optical trapping, and quantitative capillary assays. The results demonstrate the tight association of RecA with the chemotaxis pathway and also its involvement in polar chemoreceptor cluster formation. RecA is therefore necessary for standard flagellar rotation switching, implying its essential role not only in swarming motility but also in the normal chemotactic response of S. Typhimurium.
DOI
Friday, September 19, 2014
Direct Observation of Dynamic Mechanical Regulation of DNA Condensation by Environmental Stimuli
Amy Lee, Adam Karcz, Ryan Akman, Tai Zheng, Sara Kwon, Szu-Ting Chou, Sarah Sucayan, Dr. Lucas J. Tricoli, Jason M. Hustedt, Dr. Qixin Leng, Prof. Jason D. Kahn, Prof. A. James Mixson and Prof. Joonil Seog
Gene delivery is a promising way to treat hereditary diseases and cancer; however, there is little understanding of DNA:carrier complex mechanical properties, which may be critical for the protection and release of nucleic acids. We applied optical tweezers to directly measure single-molecule mechanical properties of DNA condensed using 19-mer poly-l-lysine (PLL) or branched histidine–lysine (HK) peptides. Force–extension profiles indicate that both carriers condense DNA actively, showing force plateaus during stretching and relaxation cycles. As the environment such as carrier concentration, pH, and the presence of zinc ions changes, DNA:HK complexes showed dynamically regulated mechanical properties at multiple force levels. The fundamental knowledge from this study can be applied to design a mechanically tailored complex which may enhance transfection efficiency by controlling the stability of the complex temporally and spatially.
DOI
Gene delivery is a promising way to treat hereditary diseases and cancer; however, there is little understanding of DNA:carrier complex mechanical properties, which may be critical for the protection and release of nucleic acids. We applied optical tweezers to directly measure single-molecule mechanical properties of DNA condensed using 19-mer poly-l-lysine (PLL) or branched histidine–lysine (HK) peptides. Force–extension profiles indicate that both carriers condense DNA actively, showing force plateaus during stretching and relaxation cycles. As the environment such as carrier concentration, pH, and the presence of zinc ions changes, DNA:HK complexes showed dynamically regulated mechanical properties at multiple force levels. The fundamental knowledge from this study can be applied to design a mechanically tailored complex which may enhance transfection efficiency by controlling the stability of the complex temporally and spatially.
DOI
Cell-cell proximity effects in multi-cell electroporation
Brian E. Henslee, Andrew Morss, Xin Hu, Gregory P. Lafyatis and L. James Lee
We report a fundamental study of how the electropermeabilization of a cell is affected by nearby cells. Previous researchers studying electroporation of dense suspensions of cells have observed, both theoretically and experimentally, that such samples cannot be treated simply as collections of independent cells. However, the complexity of those systems makes quantitative modeling difficult. We studied the change in the minimum applied electric field, the threshold field, required to affect electropermeabilization of a cell due to the presence of a second cell. Experimentally, we used optical tweezers to accurately position two cells in a custom fluidic electroporation device and measured the threshold field for electropermeabilization. We also captured video of the process. In parallel, finite element simulations of the electrostatic potential distributions in our systems were generated using the 3-layer model and the contact resistance methods. Reasonably good agreement with measurements was found assuming a model in which changes in a cell's threshold field were predicted from the calculated changes in the maximum voltage across the cell's membrane induced by the presence of a second cell. The threshold field required to electroporate a cell is changed ∼5%–10% by a nearby, nearly touching second cell. Cells aligned parallel to the porating field shield one another. Those oriented perpendicular to the field enhance the applied field's effect. In addition, we found that the dynamics of the electropermeabilization process are important in explaining observations for even our simple two-cell system.
DOI
We report a fundamental study of how the electropermeabilization of a cell is affected by nearby cells. Previous researchers studying electroporation of dense suspensions of cells have observed, both theoretically and experimentally, that such samples cannot be treated simply as collections of independent cells. However, the complexity of those systems makes quantitative modeling difficult. We studied the change in the minimum applied electric field, the threshold field, required to affect electropermeabilization of a cell due to the presence of a second cell. Experimentally, we used optical tweezers to accurately position two cells in a custom fluidic electroporation device and measured the threshold field for electropermeabilization. We also captured video of the process. In parallel, finite element simulations of the electrostatic potential distributions in our systems were generated using the 3-layer model and the contact resistance methods. Reasonably good agreement with measurements was found assuming a model in which changes in a cell's threshold field were predicted from the calculated changes in the maximum voltage across the cell's membrane induced by the presence of a second cell. The threshold field required to electroporate a cell is changed ∼5%–10% by a nearby, nearly touching second cell. Cells aligned parallel to the porating field shield one another. Those oriented perpendicular to the field enhance the applied field's effect. In addition, we found that the dynamics of the electropermeabilization process are important in explaining observations for even our simple two-cell system.
DOI
Probing the coupled adhesion and deformation characteristics of suspension cells
T. H. Hui, Q. Zhu, Z. L. Zhou, J. Qian and Y. Lin
By combining optical trapping with fluorescence imaging, the adhesion and deformation characteristics of suspension cells were probed on single cell level. We found that, after 24 h of co-culturing, stable attachment between non-adherent K562 cells and polystyrene beads coated with fibronectin, collagen I, or G-actin can all be formed with an adhesion energy density in the range of 1–3×10−2 mJ/m2, which is about one order of magnitude lower than the reported values for several adherent cells. In addition, it was observed that the formation of a stronger adhesion is accompanied with the appearance of a denser actin cell cortex, especially in the region close to the cell-bead interface, resulting in a significant increase in the apparent modulus of the cell. Findings here could be important for our understanding of why the aggregation of circulating cells, like that in leukostasis, takes place in vivo as well as how such clusters of non-adherent cells behave. The method proposed can also be useful in investigating adhesion and related phenomena for other cell types in the future.
DOI
By combining optical trapping with fluorescence imaging, the adhesion and deformation characteristics of suspension cells were probed on single cell level. We found that, after 24 h of co-culturing, stable attachment between non-adherent K562 cells and polystyrene beads coated with fibronectin, collagen I, or G-actin can all be formed with an adhesion energy density in the range of 1–3×10−2 mJ/m2, which is about one order of magnitude lower than the reported values for several adherent cells. In addition, it was observed that the formation of a stronger adhesion is accompanied with the appearance of a denser actin cell cortex, especially in the region close to the cell-bead interface, resulting in a significant increase in the apparent modulus of the cell. Findings here could be important for our understanding of why the aggregation of circulating cells, like that in leukostasis, takes place in vivo as well as how such clusters of non-adherent cells behave. The method proposed can also be useful in investigating adhesion and related phenomena for other cell types in the future.
DOI
A Quantitative Comparison of Single-Cell Whole Genome Amplification Methods
Charles F. A. de Bourcy, Iwijn De Vlaminck, Jad N. Kanbar, Jianbin Wang, Charles Gawad, Stephen R. Quake
Single-cell sequencing is emerging as an important tool for studies of genomic heterogeneity. Whole genome amplification (WGA) is a key step in single-cell sequencing workflows and a multitude of methods have been introduced. Here, we compare three state-of-the-art methods on both bulk and single-cell samples of E. coli DNA: Multiple Displacement Amplification (MDA), Multiple Annealing and Looping Based Amplification Cycles (MALBAC), and the PicoPLEX single-cell WGA kit (NEB-WGA). We considered the effects of reaction gain on coverage uniformity, error rates and the level of background contamination. We compared the suitability of the different WGA methods for the detection of copy-number variations, for the detection of single-nucleotide polymorphisms and for de-novo genome assembly. No single method performed best across all criteria and significant differences in characteristics were observed; the choice of which amplifier to use will depend strongly on the details of the type of question being asked in any given experiment.
DOI
Single-cell sequencing is emerging as an important tool for studies of genomic heterogeneity. Whole genome amplification (WGA) is a key step in single-cell sequencing workflows and a multitude of methods have been introduced. Here, we compare three state-of-the-art methods on both bulk and single-cell samples of E. coli DNA: Multiple Displacement Amplification (MDA), Multiple Annealing and Looping Based Amplification Cycles (MALBAC), and the PicoPLEX single-cell WGA kit (NEB-WGA). We considered the effects of reaction gain on coverage uniformity, error rates and the level of background contamination. We compared the suitability of the different WGA methods for the detection of copy-number variations, for the detection of single-nucleotide polymorphisms and for de-novo genome assembly. No single method performed best across all criteria and significant differences in characteristics were observed; the choice of which amplifier to use will depend strongly on the details of the type of question being asked in any given experiment.
DOI
Single-beam three-dimensional optical trapping at extremely low insertion angles via optical fiber optimization
Steven Ross; Mark F. Murphy; Francis Lilley; Michael J. Lalor; David R. Burton
Employing optical fiber to deliver the trapping laser to the sample chamber significantly reduces the size and costs of optical tweezers (OT). The utilization of fiber decouples the OT from the microscope, providing scope for system portability, and the potential for uncomplicated integration with other advanced microscopy systems. For use with an atomic force microscope, the fiber must be inserted at an angle of 10 deg to the plane of the sample chamber floor. However, the literature states that optical trapping with a single fiber inserted at an angle ≤20 deg is not possible. This paper investigates this limitation and proposes a hypothesis that explains it. Based on this explanation, a tapered-fiber optical tweezer system is developed. This system demonstrates that such traps can indeed be made to function in three-dimensions (3-D) at insertion angles of ≤10 deg using relatively low optical powers, provided the fiber taper is optimized. Three such optimized tapered fiber tips are presented, and their ability to optically trap both organic and inanimate material in 3-D is demonstrated. The near-horizontal insertion angle introduced a maximum trapping range (MTR). The MTR of the three tips is determined empirically, evaluated against simulated data, and found to be tunable through taper optimization.
DOI
Employing optical fiber to deliver the trapping laser to the sample chamber significantly reduces the size and costs of optical tweezers (OT). The utilization of fiber decouples the OT from the microscope, providing scope for system portability, and the potential for uncomplicated integration with other advanced microscopy systems. For use with an atomic force microscope, the fiber must be inserted at an angle of 10 deg to the plane of the sample chamber floor. However, the literature states that optical trapping with a single fiber inserted at an angle ≤20 deg is not possible. This paper investigates this limitation and proposes a hypothesis that explains it. Based on this explanation, a tapered-fiber optical tweezer system is developed. This system demonstrates that such traps can indeed be made to function in three-dimensions (3-D) at insertion angles of ≤10 deg using relatively low optical powers, provided the fiber taper is optimized. Three such optimized tapered fiber tips are presented, and their ability to optically trap both organic and inanimate material in 3-D is demonstrated. The near-horizontal insertion angle introduced a maximum trapping range (MTR). The MTR of the three tips is determined empirically, evaluated against simulated data, and found to be tunable through taper optimization.
DOI
Effects of Coating on the Optical Trapping Efficiency of Microspheres via Geometrical Optics Approximation
Bum Jun Park and Eric M. Furst
We present the optical trapping forces that are generated when a single laser beam strongly focuses on a coated dielectric microsphere. On the basis of geometrical optics approximation (GOA), in which a particle intercepts all of the rays that make up a single laser beam, we calculate the trapping forces with varying coating thickness and refractive index values. To increase the optical trapping efficiency, the refractive index (nb) of the coating is selected such that na < nb < nc, where na and nc are the refractive indices of the medium and the core material, respectively. The thickness of the coating also increases trapping efficiency. Importantly, we find that trapping forces for the coated particles are predominantly determined by two rays: the incident ray and the first refracted ray to the medium.
DOI
We present the optical trapping forces that are generated when a single laser beam strongly focuses on a coated dielectric microsphere. On the basis of geometrical optics approximation (GOA), in which a particle intercepts all of the rays that make up a single laser beam, we calculate the trapping forces with varying coating thickness and refractive index values. To increase the optical trapping efficiency, the refractive index (nb) of the coating is selected such that na < nb < nc, where na and nc are the refractive indices of the medium and the core material, respectively. The thickness of the coating also increases trapping efficiency. Importantly, we find that trapping forces for the coated particles are predominantly determined by two rays: the incident ray and the first refracted ray to the medium.
DOI
Mechanical Detection of a Long-Range Actin Network Emanating from a Biomimetic Cortex
Matthias Bussonnier, Kevin Carvalho, Joël Lemière, Jean-François Joanny, Cécile Sykes, Timo Betz
Actin is ubiquitous globular protein that polymerizes into filaments and forms networks that participate in the force generation of eukaryotic cells. Such forces are used for cell motility, cytokinesis, and tissue remodeling. Among those actin networks, we focus on the actin cortex, a dense branched network beneath the plasma membrane that is of particular importance for the mechanical properties of the cell. Here we reproduce the cellular cortex by activating actin filament growth on a solid surface. We unveil the existence of a sparse actin network that emanates from the surface and extends over a distance that is at least 10 times larger than the cortex itself. We call this sparse actin network the “actin cloud” and characterize its mechanical properties with optical tweezers. We show, both experimentally and theoretically, that the actin cloud is mechanically relevant and that it should be taken into account because it can sustain forces as high as several picoNewtons (pN). In particular, it is known that in plant cells, actin networks similar to the actin cloud have a role in positioning the nucleus; in large oocytes, they play a role in driving chromosome movement. Recent evidence shows that such networks even prevent granule condensation in large cells.
DOI
Actin is ubiquitous globular protein that polymerizes into filaments and forms networks that participate in the force generation of eukaryotic cells. Such forces are used for cell motility, cytokinesis, and tissue remodeling. Among those actin networks, we focus on the actin cortex, a dense branched network beneath the plasma membrane that is of particular importance for the mechanical properties of the cell. Here we reproduce the cellular cortex by activating actin filament growth on a solid surface. We unveil the existence of a sparse actin network that emanates from the surface and extends over a distance that is at least 10 times larger than the cortex itself. We call this sparse actin network the “actin cloud” and characterize its mechanical properties with optical tweezers. We show, both experimentally and theoretically, that the actin cloud is mechanically relevant and that it should be taken into account because it can sustain forces as high as several picoNewtons (pN). In particular, it is known that in plant cells, actin networks similar to the actin cloud have a role in positioning the nucleus; in large oocytes, they play a role in driving chromosome movement. Recent evidence shows that such networks even prevent granule condensation in large cells.
DOI
Numerical study of nanoparticle sensors based on the detection of the two-photon-induced luminescence of gold nanorod antennas
Zaoshan Huang, Qiaofeng Dai, Sheng Lan, Shaolong Tie
We investigate numerically the modification of the nonlinear optical properties of a nanoantenna in the trapping of nanoparticles (NPs) by using both the discrete dipole approximation method and the finite-difference time-domain technique. The nanoantenna, which is formed by two gold nanorods (GNRs) aligned end to end and separated by a small gap, can emit strong two-photon-induced luminescence (TPL) under the excitation of a femtosecond laser light which is resonant with its longitudinal surface plasmon resonance. In addition, the excited antenna can stably trap small NPs which in turn induce modifications in the emitted TPL. These two features make it a promising candidate for building highly sensitive detectors for NPs of different materials and sizes. It is demonstrated that sensors built with antennas possess higher sensitivities than those built with single GNRs and nanorod-based antennas are more sensitive than nanoprism-based antennas. In addition, it is found that the trapping probability for a second NP is significantly reduced for the antenna with a trapped NP, implying that trapping of NPs may occur sequentially. A relationship between the TPL of the system (antenna + NP) and the optical potential energy of the NP is established, enabling the extraction of the information on the optical potential energy and optical force by recording the TPL of the system. It is shown that the sequential trapping and releasing of NPs flowing in a microfluid channel can be realized by designing two different antennas arranged closely.
DOI
We investigate numerically the modification of the nonlinear optical properties of a nanoantenna in the trapping of nanoparticles (NPs) by using both the discrete dipole approximation method and the finite-difference time-domain technique. The nanoantenna, which is formed by two gold nanorods (GNRs) aligned end to end and separated by a small gap, can emit strong two-photon-induced luminescence (TPL) under the excitation of a femtosecond laser light which is resonant with its longitudinal surface plasmon resonance. In addition, the excited antenna can stably trap small NPs which in turn induce modifications in the emitted TPL. These two features make it a promising candidate for building highly sensitive detectors for NPs of different materials and sizes. It is demonstrated that sensors built with antennas possess higher sensitivities than those built with single GNRs and nanorod-based antennas are more sensitive than nanoprism-based antennas. In addition, it is found that the trapping probability for a second NP is significantly reduced for the antenna with a trapped NP, implying that trapping of NPs may occur sequentially. A relationship between the TPL of the system (antenna + NP) and the optical potential energy of the NP is established, enabling the extraction of the information on the optical potential energy and optical force by recording the TPL of the system. It is shown that the sequential trapping and releasing of NPs flowing in a microfluid channel can be realized by designing two different antennas arranged closely.
DOI
Wednesday, September 17, 2014
Nanomanipulation of Single RNA Molecules by Optical Tweezers
William Stephenson, Gorby Wan, Scott A. Tenenbaum, Pan T. X. Li
A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.
DOI
A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.
DOI
Quantitation of Malaria Parasite-Erythrocyte Cell-Cell Interactions Using Optical Tweezers
Alex J. Crick, Michel Theron, Teresa Tiffert, Virgilio L. Lew, Pietro Cicuta, Julian C. Rayner
Erythrocyte invasion by Plasmodium falciparum merozoites is an essential step for parasite survival and hence the pathogenesis of malaria. Invasion has been studied intensively, but our cellular understanding has been limited by the fact that it occurs very rapidly: invasion is generally complete within 1 min, and shortly thereafter the merozoites, at least in in vitro culture, lose their invasive capacity. The rapid nature of the process, and hence the narrow time window in which measurements can be taken, have limited the tools available to quantitate invasion. Here we employ optical tweezers to study individual invasion events for what we believe is the first time, showing that newly released P. falciparum merozoites, delivered via optical tweezers to a target erythrocyte, retain their ability to invade. Even spent merozoites, which had lost the ability to invade, retain the ability to adhere to erythrocytes, and furthermore can still induce transient local membrane deformations in the erythrocyte membrane. We use this technology to measure the strength of the adhesive force between merozoites and erythrocytes, and to probe the cellular mode of action of known invasion inhibitory treatments. These data add to our understanding of the erythrocyte-merozoite interactions that occur during invasion, and demonstrate the power of optical tweezers technologies in unraveling the blood-stage biology of malaria.
DOI
Erythrocyte invasion by Plasmodium falciparum merozoites is an essential step for parasite survival and hence the pathogenesis of malaria. Invasion has been studied intensively, but our cellular understanding has been limited by the fact that it occurs very rapidly: invasion is generally complete within 1 min, and shortly thereafter the merozoites, at least in in vitro culture, lose their invasive capacity. The rapid nature of the process, and hence the narrow time window in which measurements can be taken, have limited the tools available to quantitate invasion. Here we employ optical tweezers to study individual invasion events for what we believe is the first time, showing that newly released P. falciparum merozoites, delivered via optical tweezers to a target erythrocyte, retain their ability to invade. Even spent merozoites, which had lost the ability to invade, retain the ability to adhere to erythrocytes, and furthermore can still induce transient local membrane deformations in the erythrocyte membrane. We use this technology to measure the strength of the adhesive force between merozoites and erythrocytes, and to probe the cellular mode of action of known invasion inhibitory treatments. These data add to our understanding of the erythrocyte-merozoite interactions that occur during invasion, and demonstrate the power of optical tweezers technologies in unraveling the blood-stage biology of malaria.
DOI
Chemoenvironmental modulators of fluidity in the suspended biological cell
John M. Maloney and Krystyn J. Van Vliet
Biological cells can be characterized as “soft matter” with mechanical characteristics potentially modulated by external cues such as pharmaceutical dosage or fever temperature. Further, quantifying the effects of chemical and physical stimuli on a cell's mechanical response informs models of living cells as complex materials. Here, we investigate the mechanical behavior of single biological cells in terms of fluidity, or mechanical hysteresivity normalized to the extremes of an elastic solid or a viscous liquid. This parameter, which complements stiffness when describing whole-cell viscoelastic response, can be determined for a suspended cell within subsecond times. Questions remain, however, about the origin of fluidity as a conserved parameter across timescales, the physical interpretation of its magnitude, and its potential use for high-throughput sorting and separation of interesting cells by mechanical means. Therefore, we exposed suspended CH27 lymphoma cells to various chemoenvironmental conditions—temperature, pharmacological agents, pH, and osmolarity—and measured cell fluidity with a non-contact technique to extend familiarity with suspended-cell mechanics in the context of both soft-matter physics and mechanical flow cytometry development. The actin-cytoskeleton-disassembling drug latrunculin exacted a large effect on mechanical behavior, amenable to dose-dependence analysis of coupled changes in fluidity and stiffness. Fluidity was minimally affected by pH changes from 6.5 to 8.5, but strongly modulated by osmotic challenge to the cell, where the range spanned halfway from solid to liquid behavior. Together, these results support the interpretation of fluidity as a reciprocal friction within the actin cytoskeleton, with implications both for cytoskeletal models and for expectations when separating interesting cell subpopulations by mechanical means in the suspended state.
DOI
Biological cells can be characterized as “soft matter” with mechanical characteristics potentially modulated by external cues such as pharmaceutical dosage or fever temperature. Further, quantifying the effects of chemical and physical stimuli on a cell's mechanical response informs models of living cells as complex materials. Here, we investigate the mechanical behavior of single biological cells in terms of fluidity, or mechanical hysteresivity normalized to the extremes of an elastic solid or a viscous liquid. This parameter, which complements stiffness when describing whole-cell viscoelastic response, can be determined for a suspended cell within subsecond times. Questions remain, however, about the origin of fluidity as a conserved parameter across timescales, the physical interpretation of its magnitude, and its potential use for high-throughput sorting and separation of interesting cells by mechanical means. Therefore, we exposed suspended CH27 lymphoma cells to various chemoenvironmental conditions—temperature, pharmacological agents, pH, and osmolarity—and measured cell fluidity with a non-contact technique to extend familiarity with suspended-cell mechanics in the context of both soft-matter physics and mechanical flow cytometry development. The actin-cytoskeleton-disassembling drug latrunculin exacted a large effect on mechanical behavior, amenable to dose-dependence analysis of coupled changes in fluidity and stiffness. Fluidity was minimally affected by pH changes from 6.5 to 8.5, but strongly modulated by osmotic challenge to the cell, where the range spanned halfway from solid to liquid behavior. Together, these results support the interpretation of fluidity as a reciprocal friction within the actin cytoskeleton, with implications both for cytoskeletal models and for expectations when separating interesting cell subpopulations by mechanical means in the suspended state.
DOI
Monday, September 8, 2014
Probing the Stochastic, Motor-Driven Properties of the Cytoplasm Using Force Spectrum Microscopy
Ming Guo, Allen J. Ehrlicher, Mikkel H. Jensen, Malte Renz, Jeffrey R. Moore, Robert D. Goldman, Jennifer Lippincott-Schwartz, Frederick C. Mackintosh, David A. Weitz
Molecular motors in cells typically produce highly directed motion; however, the aggregate, incoherent effect of all active processes also creates randomly fluctuating forces, which drive diffusive-like, nonthermal motion. Here, we introduce force-spectrum-microscopy (FSM) to directly quantify random forces within the cytoplasm of cells and thereby probe stochastic motor activity. This technique combines measurements of the random motion of probe particles with independent micromechanical measurements of the cytoplasm to quantify the spectrum of force fluctuations. Using FSM, we show that force fluctuations substantially enhance intracellular movement of small and large components. The fluctuations are three times larger in malignant cells than in their benign counterparts. We further demonstrate that vimentin acts globally to anchor organelles against randomly fluctuating forces in the cytoplasm, with no effect on their magnitude. Thus, FSM has broad applications for understanding the cytoplasm and its intracellular processes in relation to cell physiology in healthy and diseased states.
DOI
Molecular motors in cells typically produce highly directed motion; however, the aggregate, incoherent effect of all active processes also creates randomly fluctuating forces, which drive diffusive-like, nonthermal motion. Here, we introduce force-spectrum-microscopy (FSM) to directly quantify random forces within the cytoplasm of cells and thereby probe stochastic motor activity. This technique combines measurements of the random motion of probe particles with independent micromechanical measurements of the cytoplasm to quantify the spectrum of force fluctuations. Using FSM, we show that force fluctuations substantially enhance intracellular movement of small and large components. The fluctuations are three times larger in malignant cells than in their benign counterparts. We further demonstrate that vimentin acts globally to anchor organelles against randomly fluctuating forces in the cytoplasm, with no effect on their magnitude. Thus, FSM has broad applications for understanding the cytoplasm and its intracellular processes in relation to cell physiology in healthy and diseased states.
DOI
The complex folding behavior of HIV-1-protease monomer revealed by optical-tweezer single-molecule experiments and molecular-dynamics simulations
M. Caldarini, P. Sonar, I. Valpapuram, D. Tavella, C. Volonte, V. Pandini, M.A. Vanoni, A. Aliverti, R.A. Broglia, G. Tiana, C. Cecconic
We have used optical tweezers and molecular dynamics simulations to investigate the unfolding and refolding process of a stable monomeric form of HIV-1-protease (PR). We have characterized the behavior under tension of the native state (N), and that of the ensemble of partially folded (PF) conformations the protein visits en route to N, which collectively act as a long lived state controlling the slow kinetic phase of the folding process. Our results reveal a rich network of unfolding events, where the native state unfolds either in a two-state manner or by populating an intermediate state I, while the PF state unravels through a multitude of pathways, underscoring its structural heterogeneity. Under our experimental conditions the PF state is quite compact, its extension being essentially indistinguishable from that of the N state. Refolding of mechanically-denatured HIV-1-PR monomers is a multiple-pathway process, where the protein reaches its native state through different trajectories. Molecular dynamics simulations allowed us to gain insight into possible conformations the protein adopts along the unfolding pathways, which differ for number and type of non-native contacts, and provide information regarding possible structural features of the PF state.
DOI
We have used optical tweezers and molecular dynamics simulations to investigate the unfolding and refolding process of a stable monomeric form of HIV-1-protease (PR). We have characterized the behavior under tension of the native state (N), and that of the ensemble of partially folded (PF) conformations the protein visits en route to N, which collectively act as a long lived state controlling the slow kinetic phase of the folding process. Our results reveal a rich network of unfolding events, where the native state unfolds either in a two-state manner or by populating an intermediate state I, while the PF state unravels through a multitude of pathways, underscoring its structural heterogeneity. Under our experimental conditions the PF state is quite compact, its extension being essentially indistinguishable from that of the N state. Refolding of mechanically-denatured HIV-1-PR monomers is a multiple-pathway process, where the protein reaches its native state through different trajectories. Molecular dynamics simulations allowed us to gain insight into possible conformations the protein adopts along the unfolding pathways, which differ for number and type of non-native contacts, and provide information regarding possible structural features of the PF state.
DOI
Bidirectional cargo transport: moving beyond tug of war
William O. Hancock
Vesicles, organelles and other intracellular cargo are transported by kinesin and dynein motors, which move in opposite directions along microtubules. This bidirectional cargo movement is frequently described as a 'tug of war' between oppositely directed molecular motors attached to the same cargo. However, although many experimental and modelling studies support the tug-of-war paradigm, numerous knockout and inhibition studies in various systems have found that inhibiting one motor leads to diminished motility in both directions, which is a 'paradox of co-dependence' that challenges the paradigm. In an effort to resolve this paradox, three classes of bidirectional transport models — microtubule tethering, mechanical activation and steric disinhibition — are proposed, and a general mathematical modelling framework for bidirectional cargo transport is put forward to guide future experiments.
DOI
Vesicles, organelles and other intracellular cargo are transported by kinesin and dynein motors, which move in opposite directions along microtubules. This bidirectional cargo movement is frequently described as a 'tug of war' between oppositely directed molecular motors attached to the same cargo. However, although many experimental and modelling studies support the tug-of-war paradigm, numerous knockout and inhibition studies in various systems have found that inhibiting one motor leads to diminished motility in both directions, which is a 'paradox of co-dependence' that challenges the paradigm. In an effort to resolve this paradox, three classes of bidirectional transport models — microtubule tethering, mechanical activation and steric disinhibition — are proposed, and a general mathematical modelling framework for bidirectional cargo transport is put forward to guide future experiments.
DOI
Myosin Light Chain Kinase (MLCK) Regulates Cell Migration in a Myosin Regulatory Light Chain Phosphorylation-Independent Mechanism
Chen Chen, Tao Tao, Cheng Wen, Wei-Qi He, Yan-Ning Qiao, Yun-Qian Gao, Xin Chen, Pei Wang, Cai-Ping Chen, Wei Zhao, Hua-Qun Chen, An-Pei Ye, Ya-Jing Peng and Min-Sheng Zhu
Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL-motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration.
DOI
Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL-motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration.
DOI
Polarized Raman spectroscopic investigations on hemoglobin ordering in red blood cells
Sunita Ahlawat; Aniket Chowdhury; Nitin Kumar; Abha Uppal; Ravi Shanker Verma; Pradeep Kumar Gupta
We have investigated the dependence of the Raman spectrum of an optically trapped red blood cell (RBC) on the orientation of the cell, relative to the polarization direction of the Raman excitation beam. The Raman scattered light polarized parallel to the polarization direction of the excitation beam was observed to depend upon the orientation of the cell. In particular, the heme bands at ∼754 cm−1 and in the 1500 to 1700 cm−1 region were observed to become maximum when the cells’ equatorial plane was parallel to the excitation beam polarization direction and minimum when the cells’ plane was normal to the polarization direction. In contrast, no significant orientational dependence was seen in the Raman scattered light polarized orthogonal to the polarization direction of the excitation beam. Theoretical simulations carried out to investigate these observations suggest that inside the RBCs, the hemoglobin molecules must be present in an ordered arrangement, such that heme-porphyrin planes become preferentially orientated parallel to the RBCs’ equatorial plane.
DOI
We have investigated the dependence of the Raman spectrum of an optically trapped red blood cell (RBC) on the orientation of the cell, relative to the polarization direction of the Raman excitation beam. The Raman scattered light polarized parallel to the polarization direction of the excitation beam was observed to depend upon the orientation of the cell. In particular, the heme bands at ∼754 cm−1 and in the 1500 to 1700 cm−1 region were observed to become maximum when the cells’ equatorial plane was parallel to the excitation beam polarization direction and minimum when the cells’ plane was normal to the polarization direction. In contrast, no significant orientational dependence was seen in the Raman scattered light polarized orthogonal to the polarization direction of the excitation beam. Theoretical simulations carried out to investigate these observations suggest that inside the RBCs, the hemoglobin molecules must be present in an ordered arrangement, such that heme-porphyrin planes become preferentially orientated parallel to the RBCs’ equatorial plane.
DOI
Polarization evolution characteristics of focused hybridly polarized vector fields
Bing Gu, Yang Pan, Guanghao Rui, Danfeng Xu, Qiwen Zhan, Yiping Cui
We investigate the focusing property and the polarization evolution characteristics of hybridly polarized vector fields in the focal region. Three types of hybridly polarized vector fields, namely azimuthal-variant hybridly polarized vector field, radial-variant hybridly polarized vector field, and spatial-variant hybridly polarized vector field, are experimentally generated. The intensity distributions and the polarization evolution of these hybridly polarized vector fields focused under low numerical aperture (NA) are experimentally studied and good agreements with the numerical simulations are obtained. The three-dimensional (3D) state of polarization and the field distribution within the focal volume of these hybridly polarized vector fields under high-NA focusing are studied numerically. The optical curl force on Rayleigh particles induced by tightly focused hybridly polarized vector fields, which results in the orbital motion of trapped particles, is analyzed. Simulation results demonstrate that polarization-only modulation provided by the hybridly polarized vector field allows one to control both the intensity distribution and 3D elliptical polarization in the focal region, which may find interesting applications in particle trapping, manipulation, and orientation analysis.
DOI
We investigate the focusing property and the polarization evolution characteristics of hybridly polarized vector fields in the focal region. Three types of hybridly polarized vector fields, namely azimuthal-variant hybridly polarized vector field, radial-variant hybridly polarized vector field, and spatial-variant hybridly polarized vector field, are experimentally generated. The intensity distributions and the polarization evolution of these hybridly polarized vector fields focused under low numerical aperture (NA) are experimentally studied and good agreements with the numerical simulations are obtained. The three-dimensional (3D) state of polarization and the field distribution within the focal volume of these hybridly polarized vector fields under high-NA focusing are studied numerically. The optical curl force on Rayleigh particles induced by tightly focused hybridly polarized vector fields, which results in the orbital motion of trapped particles, is analyzed. Simulation results demonstrate that polarization-only modulation provided by the hybridly polarized vector field allows one to control both the intensity distribution and 3D elliptical polarization in the focal region, which may find interesting applications in particle trapping, manipulation, and orientation analysis.
DOI
Misfolding of Luciferase at the Single-Molecule Level
Dr. Alireza Mashaghi, Samaneh Mashaghi and Prof. Dr. Ir. Sander J. Tans
The folding of complex proteins can be dramatically affected by misfolding transitions. Directly observing misfolding and distinguishing it from aggregation is challenging. Experiments with optical tweezers revealed transitions between the folded states of a single protein in the absence of mechanical tension. Nonfolded chains of the multidomain protein luciferase folded within seconds to different partially folded states, one of which was stable over several minutes and was more resistant to forced unfolding than other partially folded states. Luciferase monomers can thus adopt a stable misfolded state and can do so without interacting with aggregation partners. This result supports the notion that luciferase misfolding is the cause of the low refolding yields and aggregation observed with this protein. This approach could be used to study misfolding transitions in other large proteins, as well as the factors that affect misfolding.
DOI
The folding of complex proteins can be dramatically affected by misfolding transitions. Directly observing misfolding and distinguishing it from aggregation is challenging. Experiments with optical tweezers revealed transitions between the folded states of a single protein in the absence of mechanical tension. Nonfolded chains of the multidomain protein luciferase folded within seconds to different partially folded states, one of which was stable over several minutes and was more resistant to forced unfolding than other partially folded states. Luciferase monomers can thus adopt a stable misfolded state and can do so without interacting with aggregation partners. This result supports the notion that luciferase misfolding is the cause of the low refolding yields and aggregation observed with this protein. This approach could be used to study misfolding transitions in other large proteins, as well as the factors that affect misfolding.
DOI
Optically and elastically assembled plasmonic nanoantennae for spatially resolved characterization of chemical composition in soft matter systems using surface enhanced spontaneous and stimulated Raman scattering
Haridas Mundoor, Taewoo Lee, Derek G. Gann, Paul J. Ackerman, Bohdan Senyuk, Jao van de Lagemaat and Ivan I. Smalyukh
We present a method to locally probe spatially varying chemical composition of soft matter systems by use of optically controlled and elastically self-assembled plasmonic nanoantennae. Disc-shaped metal particles with sharp irregular edges are optically trapped, manipulated, and assembled into small clusters to provide a strong enhancement of the Raman scattering signal coming from the sample regions around and in-between these particles. As the particles are reassembled and spatially translated by computer-controlled laser tweezers, we probe chemical composition as a function of spatial coordinates. This allows us to reliably detect tiny quantities of organic molecules, such as capping ligands present on various nanoparticles, as well as to probe chemical composition of the interior of liquid crystal defect cores that can be filled with, for example, polymer chains. The strong electromagnetic field enhancement of optically manipulated nanoparticles' rough surfaces is demonstrated in different forms of spectroscopy and microscopy, including enhanced spontaneous Raman scattering, coherent anti-Stokes Raman scattering, and stimulated Raman scattering imaging modes.
DOI
We present a method to locally probe spatially varying chemical composition of soft matter systems by use of optically controlled and elastically self-assembled plasmonic nanoantennae. Disc-shaped metal particles with sharp irregular edges are optically trapped, manipulated, and assembled into small clusters to provide a strong enhancement of the Raman scattering signal coming from the sample regions around and in-between these particles. As the particles are reassembled and spatially translated by computer-controlled laser tweezers, we probe chemical composition as a function of spatial coordinates. This allows us to reliably detect tiny quantities of organic molecules, such as capping ligands present on various nanoparticles, as well as to probe chemical composition of the interior of liquid crystal defect cores that can be filled with, for example, polymer chains. The strong electromagnetic field enhancement of optically manipulated nanoparticles' rough surfaces is demonstrated in different forms of spectroscopy and microscopy, including enhanced spontaneous Raman scattering, coherent anti-Stokes Raman scattering, and stimulated Raman scattering imaging modes.
DOI
Friday, September 5, 2014
Optical forces from evanescent Bessel beams, multiple reflections, and Kerker conditions in magnetodielectric spheres and cylinders
Juan Miguel Auñón and Manuel Nieto-Vesperinas
In this work we address, first, the optical force on a magnetodielectric particle on a flat dielectric surface due to an evanescent Bessel beam and, second, the effects on the force of multiple scattering with the substrate. For the first question we find analytical solutions showing that due to the interference of the excited electric and magnetic particle dipoles, the vertical force unusually pushes the object out from the plane. The incident wavelength rules whether or not the illumination constitutes an optical trap. As for the second problem, we make a 2D study with a single evanescent plane wave, and we present the Kerker conditions (so far established for spheres) for magnetodielectric cylinders, showing that in p polarization these conditions are practically reproduced by the latter particles and are associated to minima of the horizontal and vertical forces.
DOI
In this work we address, first, the optical force on a magnetodielectric particle on a flat dielectric surface due to an evanescent Bessel beam and, second, the effects on the force of multiple scattering with the substrate. For the first question we find analytical solutions showing that due to the interference of the excited electric and magnetic particle dipoles, the vertical force unusually pushes the object out from the plane. The incident wavelength rules whether or not the illumination constitutes an optical trap. As for the second problem, we make a 2D study with a single evanescent plane wave, and we present the Kerker conditions (so far established for spheres) for magnetodielectric cylinders, showing that in p polarization these conditions are practically reproduced by the latter particles and are associated to minima of the horizontal and vertical forces.
DOI
Plasmonic Optical Tweezers toward Molecular Manipulation: Tailoring Plasmonic Nanostructure, Light Source, and Resonant Trapping
Tatsuya Shoji and Yasuyuki Tsuboi
This Perspective describes recent progress in optical trappings of nanoparticles based on localized surface plasmon. This plasmonic optical trapping has great advantages over the conventional optical tweezers, being potentially applicable for a molecular manipulation technique. We review this novel trapping technique from the viewpoints of (i) plasmonic nanostructure, (ii) the light source for plasmon excitation, and (iii) the polarizability of the trapping target. These findings give us future outlook for plasmonic optical trapping. In addition to a brief review, recent developments on plasmonic optical trapping of soft nanomaterials such as proteins, polymer chains, and DNA will be discussed to point out the important issue for further development on this trapping method. Finally, we explore new directions of plasmonic optical trapping.
DOI
DOI
Tension on the linker gates the ATP-dependent release of dynein from microtubules
Frank B. Cleary, Mark A. Dewitt, Thomas Bilyard, Zaw Min Htet, Vladislav Belyy, Danna D. Chan, Amy Y. Chang & Ahmet Yildiz
Cytoplasmic dynein is a dimeric motor that transports intracellular cargoes towards the minus end of microtubules (MTs). In contrast to other processive motors, stepping of the dynein motor domains (heads) is not precisely coordinated. Therefore, the mechanism of dynein processivity remains unclear. Here, by engineering the mechanical and catalytic properties of the motor, we show that dynein processivity minimally requires a single active head and a second inert MT-binding domain. Processivity arises from a high ratio of MT-bound to unbound time, and not from interhead communication. In addition, nucleotide-dependent microtubule release is gated by tension on the linker domain. Intramolecular tension sensing is observed in dynein’s stepping motion at high interhead separations. On the basis of these results, we propose a quantitative model for the stepping characteristics of dynein and its response to chemical and mechanical perturbation.
DOI
Cytoplasmic dynein is a dimeric motor that transports intracellular cargoes towards the minus end of microtubules (MTs). In contrast to other processive motors, stepping of the dynein motor domains (heads) is not precisely coordinated. Therefore, the mechanism of dynein processivity remains unclear. Here, by engineering the mechanical and catalytic properties of the motor, we show that dynein processivity minimally requires a single active head and a second inert MT-binding domain. Processivity arises from a high ratio of MT-bound to unbound time, and not from interhead communication. In addition, nucleotide-dependent microtubule release is gated by tension on the linker domain. Intramolecular tension sensing is observed in dynein’s stepping motion at high interhead separations. On the basis of these results, we propose a quantitative model for the stepping characteristics of dynein and its response to chemical and mechanical perturbation.
DOI
Chaperone-enhanced purification of unconventional myosin 15, a molecular motor specialized for stereocilia protein trafficking
Jonathan E. Bird, Yasuharu Takagi, Neil Billington, Marie-Paule Strub, James R. Sellers, and Thomas B. Friedman
Mutations in unconventional myosin 15 cause nonsyndromic autosomal recessive deafness, a common form of hereditary hearing loss in humans. Myosin 15 is required for the development of hair cell mechanosensory stereocilia that detect sounds within the inner ear. To our knowledge, our work offers the first insight into the biophysical properties of purified myosin 15. Using ensemble and single molecule techniques, we show that myosin 15 is a high-duty ratio motor, which is a characteristic of myosins that can move processively along actin filaments. We also introduce a new strategy for producing myosins by chaperone coexpression in Spodoptera frugiperda insect cells. This approach may help optimize expression of skeletal and cardiac muscle myosins, which are emerging as translational drug targets but are presently refractory to larger-scale purification.
DOI
Mutations in unconventional myosin 15 cause nonsyndromic autosomal recessive deafness, a common form of hereditary hearing loss in humans. Myosin 15 is required for the development of hair cell mechanosensory stereocilia that detect sounds within the inner ear. To our knowledge, our work offers the first insight into the biophysical properties of purified myosin 15. Using ensemble and single molecule techniques, we show that myosin 15 is a high-duty ratio motor, which is a characteristic of myosins that can move processively along actin filaments. We also introduce a new strategy for producing myosins by chaperone coexpression in Spodoptera frugiperda insect cells. This approach may help optimize expression of skeletal and cardiac muscle myosins, which are emerging as translational drug targets but are presently refractory to larger-scale purification.
DOI
Imaging and Analysis of Single Optically Trapped Gold Nanoparticles Using Spatial Modulation Spectroscopy
Mary Sajini Devadas, Zhongming Li, and Gregory V. Hartland
The extinction cross sections and spectra of single nanoparticles can be directly measured by moving the particle in and out of a tightly focused laser beam. This technique, known as spatial modulation spectroscopy, yields detailed information about the size, shape, and environment of the particles. These experiments are typically done on particles immobilized on a substrate. Here we demonstrate for the first time the use of spatial modulation spectroscopy to interrogate single, optically trapped nanoparticles in solution. Gold nanoparticles as small as 15 nm were trapped and imaged. The experiments were performed by modulating the position of the probe laser beam while scanning it over the trapped particle with a galvo-scanning mirror system. This technique opens up the possibility of precisely measuring the optical properties of single nanoparticles in liquid environments, free from the influence of a surface.
DOI
The extinction cross sections and spectra of single nanoparticles can be directly measured by moving the particle in and out of a tightly focused laser beam. This technique, known as spatial modulation spectroscopy, yields detailed information about the size, shape, and environment of the particles. These experiments are typically done on particles immobilized on a substrate. Here we demonstrate for the first time the use of spatial modulation spectroscopy to interrogate single, optically trapped nanoparticles in solution. Gold nanoparticles as small as 15 nm were trapped and imaged. The experiments were performed by modulating the position of the probe laser beam while scanning it over the trapped particle with a galvo-scanning mirror system. This technique opens up the possibility of precisely measuring the optical properties of single nanoparticles in liquid environments, free from the influence of a surface.
DOI
Optical manipulation of the nematic director field around microspheres covered with an azo-dendrimer monolayer
Pemika Hirankittiwong, Nattaporn Chattham, Jumras Limtrakul, Osamu Haba, Koichiro Yonetake, Alexey Eremin, Ralf Stannarius, and Hideo Takezoe
We report here the optical manipulation of the director and topological defect structures of nematic liquid crystals around a silica microparticle with azobenzene-containing dendrimers (azo-dendrimers) on its surface. We successfully demonstrate the successive switching processes from hedgehog, to boojum, and further to Saturn ring configurations by ultraviolet (UV) light irradiation and termination. The switching time between these defect structures depends on the UV light intensity and attains about 50 ms. Since the pretreatment of microparticles is not necessary and the surface modification is spontaneously performed just by dissolving the azo-dendrimers in liquid crystals, this dendrimer supplies us with a variety of possible applications.
DOI
We report here the optical manipulation of the director and topological defect structures of nematic liquid crystals around a silica microparticle with azobenzene-containing dendrimers (azo-dendrimers) on its surface. We successfully demonstrate the successive switching processes from hedgehog, to boojum, and further to Saturn ring configurations by ultraviolet (UV) light irradiation and termination. The switching time between these defect structures depends on the UV light intensity and attains about 50 ms. Since the pretreatment of microparticles is not necessary and the surface modification is spontaneously performed just by dissolving the azo-dendrimers in liquid crystals, this dendrimer supplies us with a variety of possible applications.
DOI
Monday, September 1, 2014
Multi-photon excited luminescence of magnetic FePt core-shell nanoparticles
K.M. Seemann and B. Kuhn
We present magnetic FePt nanoparticles with a hydrophilic, inert, and biocompatible silico-tungsten oxide shell. The particles can be functionalized, optically detected, and optically manipulated. To show the functionalization the fluorescent dye NOPS was bound to the FePt core-shell nanoparticles with propyl-triethoxy-silane linkers and fluorescence of the labeled particles were observed in ethanol (EtOH). In aqueous dispersion the NOPS fluorescence is quenched making them invisible using 1-photon excitation. However, we observe bright luminescence of labeled and even unlabeled magnetic core-shell nanoparticles with multi-photon excitation. Luminescence can be detected in the near ultraviolet and the full visible spectral range by near infrared multi-photon excitation. For optical manipulation, we were able to drag clusters of particles, and maybe also single particles, by a focused laser beam that acts as optical tweezers by inducing an electric dipole in the insulated metal nanoparticles. In a first application, we show that the luminescence of the core-shell nanoparticles is bright enough for in vivo multi-photon imaging in the mouse neocortex down to cortical layer 5.
DOI
We present magnetic FePt nanoparticles with a hydrophilic, inert, and biocompatible silico-tungsten oxide shell. The particles can be functionalized, optically detected, and optically manipulated. To show the functionalization the fluorescent dye NOPS was bound to the FePt core-shell nanoparticles with propyl-triethoxy-silane linkers and fluorescence of the labeled particles were observed in ethanol (EtOH). In aqueous dispersion the NOPS fluorescence is quenched making them invisible using 1-photon excitation. However, we observe bright luminescence of labeled and even unlabeled magnetic core-shell nanoparticles with multi-photon excitation. Luminescence can be detected in the near ultraviolet and the full visible spectral range by near infrared multi-photon excitation. For optical manipulation, we were able to drag clusters of particles, and maybe also single particles, by a focused laser beam that acts as optical tweezers by inducing an electric dipole in the insulated metal nanoparticles. In a first application, we show that the luminescence of the core-shell nanoparticles is bright enough for in vivo multi-photon imaging in the mouse neocortex down to cortical layer 5.
DOI
The influence of compliant boundary proximity on the fundamental and subharmonic emissions from individual microbubbles
Brandon L. Helfield, Ben Y. C. Leung and David E. Goertz
The proximity of a solid-liquid boundary has been theoretically predicted to affect nonlinear microbubble emissions, but to date there has been no experimental validation of this effect. In this study, individual microbubbles (n = 15) were insonicated at f = 11 MHz as a function of offset distance from a compliant (agarose) planar boundary by employing an optical trapping apparatus. It was found that fundamental scattering increases while subharmonic scattering decreases as the microbubble approaches the boundary. Although a microbubble-boundary model can predict the qualitative trends observed for a subset of encapsulation properties, further modeling efforts are required to completely model compliant boundary-microbubble interactions.
DOI
The proximity of a solid-liquid boundary has been theoretically predicted to affect nonlinear microbubble emissions, but to date there has been no experimental validation of this effect. In this study, individual microbubbles (n = 15) were insonicated at f = 11 MHz as a function of offset distance from a compliant (agarose) planar boundary by employing an optical trapping apparatus. It was found that fundamental scattering increases while subharmonic scattering decreases as the microbubble approaches the boundary. Although a microbubble-boundary model can predict the qualitative trends observed for a subset of encapsulation properties, further modeling efforts are required to completely model compliant boundary-microbubble interactions.
DOI
Routes to DNA Accessibility: Alternative Pathways for Nucleosome Unwinding
Daniel J. Schlingman, Andrew H. Mack, Masha Kamenetska, Simon G.J. Mochrie, Lynne Regan
The dynamic packaging of DNA into chromatin is a key determinant of eukaryotic gene regulation and epigenetic inheritance. Nucleosomes are the basic unit of chromatin, and therefore the accessible states of the nucleosome must be the starting point for mechanistic models regarding these essential processes. Although the existence of different unwound nucleosome states has been hypothesized, there have been few studies of these states. The consequences of multiple states are far reaching. These states will behave differently in all aspects, including their interactions with chromatin remodelers, histone variant exchange, and kinetic properties. Here, we demonstrate the existence of two distinct states of the unwound nucleosome, which are accessible at physiological forces and ionic strengths. Using optical tweezers, we measure the rates of unwinding and rewinding for these two states and show that the rewinding rates from each state are different. In addition, we show that the probability of unwinding into each state is dependent on the applied force and ionic strength. Our results demonstrate not only that multiple unwound states exist but that their accessibility can be differentially perturbed, suggesting possible roles for these states in gene regulation. For example, different histone variants or modifications may facilitate or suppress access to DNA by promoting unwinding into one state or the other. We anticipate that the two unwound states reported here will be the basis for future models of eukaryotic transcriptional control.
DOI
The dynamic packaging of DNA into chromatin is a key determinant of eukaryotic gene regulation and epigenetic inheritance. Nucleosomes are the basic unit of chromatin, and therefore the accessible states of the nucleosome must be the starting point for mechanistic models regarding these essential processes. Although the existence of different unwound nucleosome states has been hypothesized, there have been few studies of these states. The consequences of multiple states are far reaching. These states will behave differently in all aspects, including their interactions with chromatin remodelers, histone variant exchange, and kinetic properties. Here, we demonstrate the existence of two distinct states of the unwound nucleosome, which are accessible at physiological forces and ionic strengths. Using optical tweezers, we measure the rates of unwinding and rewinding for these two states and show that the rewinding rates from each state are different. In addition, we show that the probability of unwinding into each state is dependent on the applied force and ionic strength. Our results demonstrate not only that multiple unwound states exist but that their accessibility can be differentially perturbed, suggesting possible roles for these states in gene regulation. For example, different histone variants or modifications may facilitate or suppress access to DNA by promoting unwinding into one state or the other. We anticipate that the two unwound states reported here will be the basis for future models of eukaryotic transcriptional control.
DOI
High-Throughput Phenotyping of Chlamydomonas Swimming Mutants Based on Nanoscale Video Analysis
Shohei Fujita, Takuya Matsuo, Masahiro Ishiura, Masahide Kikkawa
Studies on biflagellated algae Chlamydomonas reinhardtii mutants have resulted in significant contributions to our understanding of the functions of cilia/flagella components. However, visual inspection conducted under a microscope to screen and classify Chlamydomonas swimming requires considerable time, effort, and experience. In addition, it is likely that identification of mutants by this screening is biased toward individual cells with severe swimming defects, and mutants that swim slightly more slowly than wild-type cells may be missed by these screening methods. To systematically screen Chlamydomonas swimming mutants, we have here developed the cell-locating-with-nanoscale-accuracy (CLONA) method to identify the cell position to within 10-nm precision through the analysis of high-speed video images. Instead of analyzing the shape of the flagella, which is not always visible in images, we determine the position of Chlamydomonas cell bodies by determining the cross-correlation between a reference image and the image of the cell. From these positions, various parameters related to swimming, such as velocity and beat frequency, can be accurately estimated for each beat cycle. In the examination of wild-type and seven dynein arm mutants of Chlamydomonas, we found characteristic clustering on scatter plots of beat frequency versus swimming velocity. Using the CLONA method, we have screened 38 Chlamydomonas strains and detected believed-novel motility-deficient mutants that would be missed by visual screening. This CLONA method can automate the screening for mutants of Chlamydomonas and contribute to the elucidation of the functions of motility-associated proteins.
DOI
Studies on biflagellated algae Chlamydomonas reinhardtii mutants have resulted in significant contributions to our understanding of the functions of cilia/flagella components. However, visual inspection conducted under a microscope to screen and classify Chlamydomonas swimming requires considerable time, effort, and experience. In addition, it is likely that identification of mutants by this screening is biased toward individual cells with severe swimming defects, and mutants that swim slightly more slowly than wild-type cells may be missed by these screening methods. To systematically screen Chlamydomonas swimming mutants, we have here developed the cell-locating-with-nanoscale-accuracy (CLONA) method to identify the cell position to within 10-nm precision through the analysis of high-speed video images. Instead of analyzing the shape of the flagella, which is not always visible in images, we determine the position of Chlamydomonas cell bodies by determining the cross-correlation between a reference image and the image of the cell. From these positions, various parameters related to swimming, such as velocity and beat frequency, can be accurately estimated for each beat cycle. In the examination of wild-type and seven dynein arm mutants of Chlamydomonas, we found characteristic clustering on scatter plots of beat frequency versus swimming velocity. Using the CLONA method, we have screened 38 Chlamydomonas strains and detected believed-novel motility-deficient mutants that would be missed by visual screening. This CLONA method can automate the screening for mutants of Chlamydomonas and contribute to the elucidation of the functions of motility-associated proteins.
DOI
Mechanical Checkpoint For Persistent Cell Polarization In Adhesion-Naive Fibroblasts
Philippe Bun, JunJun Liu, Hervé Turlier, ZengZhen Liu, Karen Uriot, Jean-François Joanny, Maïté Coppey-Moisan
Cell polarization is a fundamental biological process implicated in nearly every aspect of multicellular development. The role of cell-extracellular matrix contacts in the establishment and the orientation of cell polarity have been extensively studied. However, the respective contributions of substrate mechanics and biochemistry remain unclear. Here we propose a believed novel single-cell approach to assess the minimal polarization trigger. Using nonadhered round fibroblast cells, we show that stiffness sensing through single localized integrin-mediated cues are necessary and sufficient to trigger and direct a shape polarization. In addition, the traction force developed by cells has to reach a minimal threshold of 56 ± 1.6 pN for persistent polarization. The polarization kinetics increases with the stiffness of the cue. The polarized state is characterized by cortical actomyosin redistribution together with cell shape change. We develop a physical model supporting the idea that a local and persistent inhibition of actin polymerization and/or myosin activity is sufficient to trigger and sustain the polarized state. Finally, the cortical polarity propagates to an intracellular polarity, evidenced by the reorientation of the centrosome. Our results define the minimal adhesive requirements and quantify the mechanical checkpoint for persistent cell shape and organelle polarization, which are critical regulators of tissue and cell development.
DOI
Cell polarization is a fundamental biological process implicated in nearly every aspect of multicellular development. The role of cell-extracellular matrix contacts in the establishment and the orientation of cell polarity have been extensively studied. However, the respective contributions of substrate mechanics and biochemistry remain unclear. Here we propose a believed novel single-cell approach to assess the minimal polarization trigger. Using nonadhered round fibroblast cells, we show that stiffness sensing through single localized integrin-mediated cues are necessary and sufficient to trigger and direct a shape polarization. In addition, the traction force developed by cells has to reach a minimal threshold of 56 ± 1.6 pN for persistent polarization. The polarization kinetics increases with the stiffness of the cue. The polarized state is characterized by cortical actomyosin redistribution together with cell shape change. We develop a physical model supporting the idea that a local and persistent inhibition of actin polymerization and/or myosin activity is sufficient to trigger and sustain the polarized state. Finally, the cortical polarity propagates to an intracellular polarity, evidenced by the reorientation of the centrosome. Our results define the minimal adhesive requirements and quantify the mechanical checkpoint for persistent cell shape and organelle polarization, which are critical regulators of tissue and cell development.
DOI
Comparative Analysis of the Trapping Force Using Laguerre-Gaussian Beam and Gaussian Beam
ZHOU Ye-peng, REN Hong-liang, WANG Juan, CHEN Ling
The axial trapping effect of hollow beam such as Laguerre-Gaussian beam is better than that of Gaussian beam. The intensity distributions of Gaussian beam and Laguerre-Gaussian beam were simulated according to their cross-section intensity expression. The trapping Q-factors of Gaussian beam and Laguerre-Gaussian beam were calculated in the ray-optics model. The trapping efficiencies of Gaussian beam and Laguerre-Gaussian beam were compared with different trappped bead size and refractive index, numerical aperture of microscope objective, and the distance from the bottom of the sample cell to the trapped bead. The results show that the maxmium backward trapping Q-factor and trap stiffness of Laguerre-Gaussian beam are higher than those of the Gaussian beam. Laguerre-Gaussian beam is more sensitive to spherical aberration due to its special intensity distribution, especially when the numerical aperture of microscope objective is high.
DOI
The axial trapping effect of hollow beam such as Laguerre-Gaussian beam is better than that of Gaussian beam. The intensity distributions of Gaussian beam and Laguerre-Gaussian beam were simulated according to their cross-section intensity expression. The trapping Q-factors of Gaussian beam and Laguerre-Gaussian beam were calculated in the ray-optics model. The trapping efficiencies of Gaussian beam and Laguerre-Gaussian beam were compared with different trappped bead size and refractive index, numerical aperture of microscope objective, and the distance from the bottom of the sample cell to the trapped bead. The results show that the maxmium backward trapping Q-factor and trap stiffness of Laguerre-Gaussian beam are higher than those of the Gaussian beam. Laguerre-Gaussian beam is more sensitive to spherical aberration due to its special intensity distribution, especially when the numerical aperture of microscope objective is high.
DOI
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