Ihab Sraj, Joshua Francois, David W M Marr and Charles D Eggleton
In this paper, we seek to numerically study the deformation of nucleated cells by single diode-laser bar optical stretchers. We employ a recently developed computational model, the dynamic ray-tracing method, to determine the force distribution induced by optical stretchers on a cell encapsulating a nucleus of different optical properties. These optical forces are shape dependent and can deform real non-rigid objects; thus resulting in dynamically changing distributions with cell and nucleus deformation. A Chinese hamster ovary (CHO) cell is a common biological cell that is of interest to the biomedical community because of its use in recombinant protein therapeutics and is an example of a nucleated cell. To this end, we model CHO cells as two concentric three-dimensional elastic capsules immersed in a fluid where the hydrodynamic forces are calculated using the immersed boundary method. We vary the inner capsule size to simulate different nucleus sizes. Our results show that the presence of a nucleus has a major effect on the force distribution on the cell surface and consequently on its net deformation. Scattering and gradient forces are reported for different nucleus sizes and the effect of nucleus size on the cell deformation is discussed quantitatively.
DOI
Friday, July 31, 2015
Excitation of multipolar surface plasmon resonance in plasmonic nanoparticles by complex accelerating beams
Yang Yang, Jiafang Li, Zhi-Yuan Li and Yue-Gang Chen
In this paper, through a vector-spherical harmonics approach, we investigate the optical spectra of plasmonic Au nanoparticles excited by two special accelerating beams: a non-paraxial Airy beam and a Bessel beam. We systematically analyze the impacts of the beam profile, phase, and helical wave front of the electromagnetic fields on the optical spectrum and the excitation of the surface plasmon resonance (SPR). We find that the high-order phase in the Airy beam would result in strong plasmonic oscillations in the optical spectra, while the cone angle and orbital angular momentum carried by the Bessel beam could be employed to engineer the plasmon modes excited in Au nanoparticles. Furthermore, the optical spectrum excited by a combined Airy–Bessel–Gauss beam is discussed. The study could help to deeply explore new ways to manipulate SPR in metal nanoparticles via the wave front engineering of optical beams for enhancing light–matter interaction and optical sensing performance.
DOI
In this paper, through a vector-spherical harmonics approach, we investigate the optical spectra of plasmonic Au nanoparticles excited by two special accelerating beams: a non-paraxial Airy beam and a Bessel beam. We systematically analyze the impacts of the beam profile, phase, and helical wave front of the electromagnetic fields on the optical spectrum and the excitation of the surface plasmon resonance (SPR). We find that the high-order phase in the Airy beam would result in strong plasmonic oscillations in the optical spectra, while the cone angle and orbital angular momentum carried by the Bessel beam could be employed to engineer the plasmon modes excited in Au nanoparticles. Furthermore, the optical spectrum excited by a combined Airy–Bessel–Gauss beam is discussed. The study could help to deeply explore new ways to manipulate SPR in metal nanoparticles via the wave front engineering of optical beams for enhancing light–matter interaction and optical sensing performance.
DOI
Methodological challenges of optical tweezers-based X-ray fluorescence imaging of biological model organisms at synchrotron facilities
E. Vergucht, T. Brans, F. Beunis, J. Garrevoet, S. Bauters, M. De Rijcke, D. Deruytter, C. Janssen, C. Riekel, M. Burghammer and L. Vincze
Recently, a radically new synchrotron radiation-based elemental imaging approach for the analysis of biological model organisms and single cells in their natural in vivo state was introduced. The methodology combines optical tweezers (OT) technology for non-contact laser-based sample manipulation with synchrotron radiation confocal X-ray fluorescence (XRF) microimaging for the first time at ESRF-ID13. The optical manipulation possibilities and limitations of biological model organisms, the OT setup developments for XRF imaging and the confocal XRF-related challenges are reported. In general, the applicability of the OT-based setup is extended with the aim of introducing the OT XRF methodology in all research fields where highly sensitive in vivo multi-elemental analysis is of relevance at the (sub)micrometre spatial resolution level.
DOI
Recently, a radically new synchrotron radiation-based elemental imaging approach for the analysis of biological model organisms and single cells in their natural in vivo state was introduced. The methodology combines optical tweezers (OT) technology for non-contact laser-based sample manipulation with synchrotron radiation confocal X-ray fluorescence (XRF) microimaging for the first time at ESRF-ID13. The optical manipulation possibilities and limitations of biological model organisms, the OT setup developments for XRF imaging and the confocal XRF-related challenges are reported. In general, the applicability of the OT-based setup is extended with the aim of introducing the OT XRF methodology in all research fields where highly sensitive in vivo multi-elemental analysis is of relevance at the (sub)micrometre spatial resolution level.
DOI
Biomechanical and Structural Features of CS2 Fimbriae of Enterotoxigenic Escherichia coli
Narges Mortezaei, Bhupender Singh, Johan Zakrisson, Esther Bullitt, Magnus Andersson
Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrhea worldwide, and infection of children in under-developed countries often leads to high mortality rates. Isolated ETEC expresses a plethora of colonization factors (fimbriae/pili), of which CFA/I and CFA/II, which are assembled via the alternate chaperone pathway (ACP), are among the most common. Fimbriae are filamentous structures whose shafts are primarily composed of helically arranged single pilin-protein subunits, with a unique biomechanical ability to unwind and rewind. A sustained ETEC infection, under adverse conditions of dynamic shear forces, is primarily attributed to this biomechanical feature of ETEC fimbriae. Recent understanding about the role of fimbriae as virulence factors points to an evolutionary adaptation of their structural and biomechanical features. In this work, we investigated the biophysical properties of CS2 fimbriae from the CFA/II group. Homology modeling of its major structural subunit, CotA, reveals structural clues related to the niche in which they are expressed. Using optical-tweezers force spectroscopy, we found that CS2 fimbriae unwind at a constant force of 10 pN and have a corner velocity (i.e., the velocity at which the force required for unwinding rises exponentially with increased speed) of 1300 nm/s. The biophysical properties of CS2 fimbriae assessed in this work classify them into a low-force unwinding group of fimbriae together with the CFA/I and CS20 fimbriae expressed by ETEC strains. The three fimbriae are expressed by ETEC, colonize in similar gut environments, and exhibit similar biophysical features, but differ in their biogenesis. Our observation suggests that the environment has a strong impact on the biophysical characteristics of fimbriae expressed by ETEC.
DOI
Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrhea worldwide, and infection of children in under-developed countries often leads to high mortality rates. Isolated ETEC expresses a plethora of colonization factors (fimbriae/pili), of which CFA/I and CFA/II, which are assembled via the alternate chaperone pathway (ACP), are among the most common. Fimbriae are filamentous structures whose shafts are primarily composed of helically arranged single pilin-protein subunits, with a unique biomechanical ability to unwind and rewind. A sustained ETEC infection, under adverse conditions of dynamic shear forces, is primarily attributed to this biomechanical feature of ETEC fimbriae. Recent understanding about the role of fimbriae as virulence factors points to an evolutionary adaptation of their structural and biomechanical features. In this work, we investigated the biophysical properties of CS2 fimbriae from the CFA/II group. Homology modeling of its major structural subunit, CotA, reveals structural clues related to the niche in which they are expressed. Using optical-tweezers force spectroscopy, we found that CS2 fimbriae unwind at a constant force of 10 pN and have a corner velocity (i.e., the velocity at which the force required for unwinding rises exponentially with increased speed) of 1300 nm/s. The biophysical properties of CS2 fimbriae assessed in this work classify them into a low-force unwinding group of fimbriae together with the CFA/I and CS20 fimbriae expressed by ETEC strains. The three fimbriae are expressed by ETEC, colonize in similar gut environments, and exhibit similar biophysical features, but differ in their biogenesis. Our observation suggests that the environment has a strong impact on the biophysical characteristics of fimbriae expressed by ETEC.
DOI
Protein misfolding occurs by slow diffusion across multiple barriers in a rough energy landscape
Hao Yu, Derek R. Dee, Xia Liu, Angela M. Brigley, Iveta Sosova, and Michael T. Woodside
The timescale for the microscopic dynamics of proteins during conformational transitions is set by the intrachain diffusion coefficient, D. Despite the central role of protein misfolding and aggregation in many diseases, it has proven challenging to measure D for these processes because of their heterogeneity. We used single-molecule force spectroscopy to overcome these challenges and determine D for misfolding of the prion protein PrP. Observing directly the misfolding of individual dimers into minimal aggregates, we reconstructed the energy landscape governing nonnative structure formation. Remarkably, rather than displaying multiple pathways, as typically expected for aggregation, PrP dimers were funneled into a thermodynamically stable misfolded state along a single pathway containing several intermediates, one of which blocked native folding. Using Kramers’ rate theory, D was found to be 1,000-fold slower for misfolding than for native folding, reflecting local roughening of the misfolding landscape, likely due to increased internal friction. The slow diffusion also led to much longer transit times for barrier crossing, allowing transition paths to be observed directly for the first time to our knowledge. These results open a new window onto the microscopic mechanisms governing protein misfolding.
DOI
The timescale for the microscopic dynamics of proteins during conformational transitions is set by the intrachain diffusion coefficient, D. Despite the central role of protein misfolding and aggregation in many diseases, it has proven challenging to measure D for these processes because of their heterogeneity. We used single-molecule force spectroscopy to overcome these challenges and determine D for misfolding of the prion protein PrP. Observing directly the misfolding of individual dimers into minimal aggregates, we reconstructed the energy landscape governing nonnative structure formation. Remarkably, rather than displaying multiple pathways, as typically expected for aggregation, PrP dimers were funneled into a thermodynamically stable misfolded state along a single pathway containing several intermediates, one of which blocked native folding. Using Kramers’ rate theory, D was found to be 1,000-fold slower for misfolding than for native folding, reflecting local roughening of the misfolding landscape, likely due to increased internal friction. The slow diffusion also led to much longer transit times for barrier crossing, allowing transition paths to be observed directly for the first time to our knowledge. These results open a new window onto the microscopic mechanisms governing protein misfolding.
DOI
Wednesday, July 29, 2015
Ligand-Induced Changes of the Apparent Transition-State Position in Mechanical Protein Unfolding
Johannes Stigler, Matthias Rief
Force-spectroscopic measurements of ligand-receptor systems and the unfolding/folding of nucleic acids or proteins reveal information on the underlying energy landscape along the pulling coordinate. The slope Δx‡ of the force-dependent unfolding/unbinding rates is interpreted as the distance from the folded/bound state to the transition state for unfolding/unbinding and, hence, often related to the mechanical compliance of the sample molecule. Here we show that in ligand-binding proteins, the experimentally inferred Δx‡ can depend on the ligand concentration, unrelated to changes in mechanical compliance. We describe the effect in single-molecule, force-spectroscopy experiments of the calcium-binding protein calmodulin and explain it in a simple model where mechanical unfolding and ligand binding occur on orthogonal reaction coordinates. This model predicts changes in the experimentally inferred Δx‡, depending on ligand concentration and the associated shift of the dominant barrier between the two reaction coordinates. We demonstrate quantitative agreement between experiments and simulations using a realistic six-state kinetic scheme using literature values for calcium-binding kinetics and affinities. Our results have important consequences for the interpretation of force-spectroscopic data of ligand-binding proteins.
DOI
Force-spectroscopic measurements of ligand-receptor systems and the unfolding/folding of nucleic acids or proteins reveal information on the underlying energy landscape along the pulling coordinate. The slope Δx‡ of the force-dependent unfolding/unbinding rates is interpreted as the distance from the folded/bound state to the transition state for unfolding/unbinding and, hence, often related to the mechanical compliance of the sample molecule. Here we show that in ligand-binding proteins, the experimentally inferred Δx‡ can depend on the ligand concentration, unrelated to changes in mechanical compliance. We describe the effect in single-molecule, force-spectroscopy experiments of the calcium-binding protein calmodulin and explain it in a simple model where mechanical unfolding and ligand binding occur on orthogonal reaction coordinates. This model predicts changes in the experimentally inferred Δx‡, depending on ligand concentration and the associated shift of the dominant barrier between the two reaction coordinates. We demonstrate quantitative agreement between experiments and simulations using a realistic six-state kinetic scheme using literature values for calcium-binding kinetics and affinities. Our results have important consequences for the interpretation of force-spectroscopic data of ligand-binding proteins.
DOI
Stabilizing the Central Part of Tropomyosin Increases the Bending Stiffness of the Thin Filament
Salavat R. Nabiev, Denis A. Ovsyannikov, Galina V. Kopylova, Daniil V. Shchepkin, Alexander M. Matyushenko, Natalia A. Koubassova, Dmitrii I. Levitsky, Andrey K. Tsaturyan, Sergey Y. Bershitsky
A two-beam optical trap was used to measure the bending stiffness of F-actin and reconstructed thin filaments. A dumbbell was formed by a filament segment attached to two beads that were held in the two optical traps. One trap was static and held a bead used as a force transducer, whereas an acoustooptical deflector moved the beam holding the second bead, causing stretch of the dumbbell. The distance between the beads was measured using image analysis of micrographs. An exact solution to the problem of bending of an elastic filament attached to two beads and subjected to a stretch was used for data analysis. Substitution of noncanonical residues in the central part of tropomyosin with canonical ones, G126R and D137L, and especially their combination, caused an increase in the bending stiffness of the thin filaments. The data confirm that the effect of these mutations on the regulation of actin-myosin interactions may be caused by an increase in tropomyosin stiffness.
DOI
A two-beam optical trap was used to measure the bending stiffness of F-actin and reconstructed thin filaments. A dumbbell was formed by a filament segment attached to two beads that were held in the two optical traps. One trap was static and held a bead used as a force transducer, whereas an acoustooptical deflector moved the beam holding the second bead, causing stretch of the dumbbell. The distance between the beads was measured using image analysis of micrographs. An exact solution to the problem of bending of an elastic filament attached to two beads and subjected to a stretch was used for data analysis. Substitution of noncanonical residues in the central part of tropomyosin with canonical ones, G126R and D137L, and especially their combination, caused an increase in the bending stiffness of the thin filaments. The data confirm that the effect of these mutations on the regulation of actin-myosin interactions may be caused by an increase in tropomyosin stiffness.
DOI
Titin Domains Progressively Unfolded by Force Are Homogenously Distributed along the Molecule
Pasquale Bianco, Zsolt Mártonfalvi, Katalin Naftz, Dorina Kőszegi, Miklós Kellermayer
Titin is a giant filamentous protein of the muscle sarcomere in which stretch induces the unfolding of its globular domains. However, the mechanisms of how domains are progressively selected for unfolding and which domains eventually unfold have for long been elusive. Based on force-clamp optical tweezers experiments we report here that, in a paradoxical violation of mechanically driven activation kinetics, neither the global domain unfolding rate, nor the folded-state lifetime distributions of full-length titin are sensitive to force. This paradox is reconciled by a gradient of mechanical stability so that domains are gradually selected for unfolding as the magnitude of the force field increases. Atomic force microscopic screening of extended titin molecules revealed that the unfolded domains are distributed homogenously along the entire length of titin, and this homogeneity is maintained with increasing overstretch. Although the unfolding of domains with progressively increasing mechanical stability makes titin a variable viscosity damper, the spatially randomized variation of domain stability ensures that the induced structural changes are not localized but are distributed along the molecule's length. Titin may thereby provide complex safety mechanims for protecting the sarcomere against structural disintegration under excessive mechanical conditions.
DOI
Titin is a giant filamentous protein of the muscle sarcomere in which stretch induces the unfolding of its globular domains. However, the mechanisms of how domains are progressively selected for unfolding and which domains eventually unfold have for long been elusive. Based on force-clamp optical tweezers experiments we report here that, in a paradoxical violation of mechanically driven activation kinetics, neither the global domain unfolding rate, nor the folded-state lifetime distributions of full-length titin are sensitive to force. This paradox is reconciled by a gradient of mechanical stability so that domains are gradually selected for unfolding as the magnitude of the force field increases. Atomic force microscopic screening of extended titin molecules revealed that the unfolded domains are distributed homogenously along the entire length of titin, and this homogeneity is maintained with increasing overstretch. Although the unfolding of domains with progressively increasing mechanical stability makes titin a variable viscosity damper, the spatially randomized variation of domain stability ensures that the induced structural changes are not localized but are distributed along the molecule's length. Titin may thereby provide complex safety mechanims for protecting the sarcomere against structural disintegration under excessive mechanical conditions.
DOI
Monday, July 27, 2015
Controlled translocation of DNA through nanopores in carbon nano-, silicon-nitride- and lipid-coated membranes
Andy Sischka, Lukas Galla, Andreas J. Meyer, Andre Spiering, Sebastian Knust, Michael Mayer, Adam R. Hall, André Beyer, Peter Reimann, Armin Gölzhäuser and Dario Anselmetti
We investigated experimentally and theoretically the translocation forces when a charged polymer is threaded through a solid-state nanopore and found distinct dependencies on the nanopore diameter as well as on the nano membrane material chemistry. For this purpose we utilized dedicated optical tweezers force mechanics capable of probing the insertion of negatively charged double-stranded DNA inside a helium-ion drilled nanopore. We found that both the diameter of the nanopore and the membrane material itself have significant influences on the electroosmotic flow through the nanopore and thus on the threading force. Compared to a bare silicon-nitride membrane, the threading of DNA through only 3 nm thin carbon nano membranes as well as lipid bilayer-coated nanopores increased the threading force by 15% or 85%, respectively. This finding was quantitatively described by our recently developed theoretical model that also incorporates hydrodynamic slip effects on the translocating DNA molecule and the force dependence on the membrane thickness. The additional measurements presented in this paper further support our model.
DOI
We investigated experimentally and theoretically the translocation forces when a charged polymer is threaded through a solid-state nanopore and found distinct dependencies on the nanopore diameter as well as on the nano membrane material chemistry. For this purpose we utilized dedicated optical tweezers force mechanics capable of probing the insertion of negatively charged double-stranded DNA inside a helium-ion drilled nanopore. We found that both the diameter of the nanopore and the membrane material itself have significant influences on the electroosmotic flow through the nanopore and thus on the threading force. Compared to a bare silicon-nitride membrane, the threading of DNA through only 3 nm thin carbon nano membranes as well as lipid bilayer-coated nanopores increased the threading force by 15% or 85%, respectively. This finding was quantitatively described by our recently developed theoretical model that also incorporates hydrodynamic slip effects on the translocating DNA molecule and the force dependence on the membrane thickness. The additional measurements presented in this paper further support our model.
DOI
The impact of DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics
Andreas S. Biebricher, Iddo Heller, Roel F. H. Roijmans, Tjalle P. Hoekstra, Erwin J. G. Peterman & Gijs J. L. Wuite
DNA intercalators are widely used as fluorescent probes to visualize DNA and DNA transactions in vivo and in vitro. It is well known that they perturb DNA structure and stability, which can in turn influence DNA-processing by proteins. Here we elucidate this perturbation by combining single-dye fluorescence microscopy with force spectroscopy and measuring the kinetics of DNA intercalation by the mono- and bis-intercalating cyanine dyes SYTOX Orange, SYTOX Green, SYBR Gold, YO-PRO-1, YOYO-1 and POPO-3. We show that their DNA-binding affinity is mainly governed by a strongly tension-dependent dissociation rate. These rates can be tuned over a range of seven orders of magnitude by changing DNA tension, intercalating species and ionic strength. We show that optimizing these rates minimizes the impact of intercalators on strand separation and enzymatic activity. These new insights provide handles for the improved use of intercalators as DNA probes with minimal perturbation and maximal efficacy.
DOI
DNA intercalators are widely used as fluorescent probes to visualize DNA and DNA transactions in vivo and in vitro. It is well known that they perturb DNA structure and stability, which can in turn influence DNA-processing by proteins. Here we elucidate this perturbation by combining single-dye fluorescence microscopy with force spectroscopy and measuring the kinetics of DNA intercalation by the mono- and bis-intercalating cyanine dyes SYTOX Orange, SYTOX Green, SYBR Gold, YO-PRO-1, YOYO-1 and POPO-3. We show that their DNA-binding affinity is mainly governed by a strongly tension-dependent dissociation rate. These rates can be tuned over a range of seven orders of magnitude by changing DNA tension, intercalating species and ionic strength. We show that optimizing these rates minimizes the impact of intercalators on strand separation and enzymatic activity. These new insights provide handles for the improved use of intercalators as DNA probes with minimal perturbation and maximal efficacy.
DOI
Information and thermodynamics: experimental verification of Landauer's Erasure principle
Antoine Bérut, Artyom Petrosyan and Sergio Ciliberto
We present an experiment in which a one-bit memory is constructed, using a system of a single colloidal particle trapped in a modulated double-well potential. We measure the amount of heat dissipated to erase a bit and we establish that in the limit of long erasure cycles the mean dissipated heat saturates at the Landauer bound, i.e. the minimal quantity of heat necessarily produced to delete a classical bit of information. This result demonstrates the intimate link between information theory and thermodynamics. To stress this connection we also show that a detailed Jarzynski equality is verified, retrieving the Landauer's bound independently of the work done on the system. The experimental details are presented and the experimental errors carefully discussed.
We present an experiment in which a one-bit memory is constructed, using a system of a single colloidal particle trapped in a modulated double-well potential. We measure the amount of heat dissipated to erase a bit and we establish that in the limit of long erasure cycles the mean dissipated heat saturates at the Landauer bound, i.e. the minimal quantity of heat necessarily produced to delete a classical bit of information. This result demonstrates the intimate link between information theory and thermodynamics. To stress this connection we also show that a detailed Jarzynski equality is verified, retrieving the Landauer's bound independently of the work done on the system. The experimental details are presented and the experimental errors carefully discussed.
Wednesday, July 22, 2015
Nanochannel Electroporation as a Platform for Living Cell Interrogation in Acute Myeloid Leukemia
Xi Zhao, Xiaomeng Huang, Xinmei Wang, Yun Wu, Ann-Kathrin Eisfeld, Sebastian Schwind, Daniel Gallego-Perez, Pouyan E. Boukany, Guido I. Marcucci and Ly James Lee
A comprehensive micro/nanofluidics platform for single-cell analysis based on nanochannel electroporation (NEP) and molecular beacon (MB) is presented in this study. The platform can quantitatively analyze multiple RNA species in individual cells with minimal cell damage. Furthermore, it is capable of delivering nucleic acids into target cells and subsequently detecting their responses at RNA level, e.g., microRNA (miRNA). It is known that as the downstream targets of miR-29b, DNMT3A/B can be downregulated by miR-29b overexpression. To demonstrate the activity of delivered miR-29b by NEP and the analytical function of the platform, the decreased expression of DNMT3A/B in acute myeloid leukemia (AML) cells was verified at single-cell level by simultaneous detection of multiple genes in the same cell. The potential of such platform on intracellular pathway studies has also been explored by investigating the upregulation efficiencies of miR-181a through different pathways in AML cells. The results showed that an indirect approach by C/EBPα-p30 peptide expression would have a stronger effect than direct transfection of the miR-181a gene. The platform has also shown its advantages over established technologies in the analysis of cells that are hard to transfect.
DOI
A comprehensive micro/nanofluidics platform for single-cell analysis based on nanochannel electroporation (NEP) and molecular beacon (MB) is presented in this study. The platform can quantitatively analyze multiple RNA species in individual cells with minimal cell damage. Furthermore, it is capable of delivering nucleic acids into target cells and subsequently detecting their responses at RNA level, e.g., microRNA (miRNA). It is known that as the downstream targets of miR-29b, DNMT3A/B can be downregulated by miR-29b overexpression. To demonstrate the activity of delivered miR-29b by NEP and the analytical function of the platform, the decreased expression of DNMT3A/B in acute myeloid leukemia (AML) cells was verified at single-cell level by simultaneous detection of multiple genes in the same cell. The potential of such platform on intracellular pathway studies has also been explored by investigating the upregulation efficiencies of miR-181a through different pathways in AML cells. The results showed that an indirect approach by C/EBPα-p30 peptide expression would have a stronger effect than direct transfection of the miR-181a gene. The platform has also shown its advantages over established technologies in the analysis of cells that are hard to transfect.
DOI
Advances in the measurement of red blood cell deformability: A brief review
Kim, Jeongho | Lee, HoYoon | Shin, Sehyun
Red blood cells (RBCs) exhibit a unique deformability, which enables them to change shape reversibly in response to an external force. The deformability of RBCs allows them to flow in microvessels while transporting oxygen and carbon dioxide. In this review, we discussed the major determinants of RBC deformability, which include cell geometry, internal viscosity, rheological properties of the membrane, osmotic pressure, calcium, nitric oxide, temperature, ageing, and depletion of adenosine triphosphate. Additionally, we highlighted the various methods and techniques used to measure RBC deformability. Individual cell analyses (pipette aspiration and optical tweezers) and bulk cell analyses (ektacytometry, multiple channels) were described and compared. Finally, we reviewed the correlation between RBC deformability and clinical outcomes such as diabetic microangiopathy.
DOI
Red blood cells (RBCs) exhibit a unique deformability, which enables them to change shape reversibly in response to an external force. The deformability of RBCs allows them to flow in microvessels while transporting oxygen and carbon dioxide. In this review, we discussed the major determinants of RBC deformability, which include cell geometry, internal viscosity, rheological properties of the membrane, osmotic pressure, calcium, nitric oxide, temperature, ageing, and depletion of adenosine triphosphate. Additionally, we highlighted the various methods and techniques used to measure RBC deformability. Individual cell analyses (pipette aspiration and optical tweezers) and bulk cell analyses (ektacytometry, multiple channels) were described and compared. Finally, we reviewed the correlation between RBC deformability and clinical outcomes such as diabetic microangiopathy.
DOI
Probing the structural dynamics of proteins and nucleic acids with optical tweezers
Dustin B Ritchie, Michael T Woodside
Conformational changes are an essential feature of most molecular processes in biology. Optical tweezers have emerged as a powerful tool for probing conformational dynamics at the single-molecule level because of their high resolution and sensitivity, opening new windows on phenomena ranging from folding and ligand binding to enzyme function, molecular machines, and protein aggregation. By measuring conformational changes induced in a molecule by forces applied by optical tweezers, new insight has been gained into the relationship between dynamics and function. We discuss recent advances from studies of how structure forms in proteins and RNA, including non-native structures, fluctuations in disordered proteins, and interactions with chaperones assisting native folding. We also review the development of assays probing the dynamics of complex protein–nucleic acid and protein–protein assemblies that reveal the dynamic interactions between biomolecular machines and their substrates.
DOI
Conformational changes are an essential feature of most molecular processes in biology. Optical tweezers have emerged as a powerful tool for probing conformational dynamics at the single-molecule level because of their high resolution and sensitivity, opening new windows on phenomena ranging from folding and ligand binding to enzyme function, molecular machines, and protein aggregation. By measuring conformational changes induced in a molecule by forces applied by optical tweezers, new insight has been gained into the relationship between dynamics and function. We discuss recent advances from studies of how structure forms in proteins and RNA, including non-native structures, fluctuations in disordered proteins, and interactions with chaperones assisting native folding. We also review the development of assays probing the dynamics of complex protein–nucleic acid and protein–protein assemblies that reveal the dynamic interactions between biomolecular machines and their substrates.
DOI
Monday, July 20, 2015
Curved singular beams for three-dimensional particle manipulation
Juanying Zhao, Ioannis D. Chremmos, Daohong Song, Demetrios N. Christodoulides, Nikolaos K. Efremidis & Zhigang Chen
For decades, singular beams carrying angular momentum have been a topic of considerable interest. Their intriguing applications are ubiquitous in a variety of fields, ranging from optical manipulation to photon entanglement, and from microscopy and coronagraphy to free-space communications, detection of rotating black holes, and even relativistic electrons and strong-field physics. In most applications, however, singular beams travel naturally along a straight line, expanding during linear propagation or breaking up in nonlinear media. Here, we design and demonstrate diffraction-resisting singular beams that travel along arbitrary trajectories in space. These curved beams not only maintain an invariant dark “hole” in the center but also preserve their angular momentum, exhibiting combined features of optical vortex, Bessel, and Airy beams. Furthermore, we observe three-dimensional spiraling of microparticles driven by such fine-shaped dynamical beams. Our findings may open up new avenues for shaped light in various applications.
DOI
For decades, singular beams carrying angular momentum have been a topic of considerable interest. Their intriguing applications are ubiquitous in a variety of fields, ranging from optical manipulation to photon entanglement, and from microscopy and coronagraphy to free-space communications, detection of rotating black holes, and even relativistic electrons and strong-field physics. In most applications, however, singular beams travel naturally along a straight line, expanding during linear propagation or breaking up in nonlinear media. Here, we design and demonstrate diffraction-resisting singular beams that travel along arbitrary trajectories in space. These curved beams not only maintain an invariant dark “hole” in the center but also preserve their angular momentum, exhibiting combined features of optical vortex, Bessel, and Airy beams. Furthermore, we observe three-dimensional spiraling of microparticles driven by such fine-shaped dynamical beams. Our findings may open up new avenues for shaped light in various applications.
DOI
Push-Pull Phenomenon of a Dielectric Particle in a Rectangular Waveguide
N. K. Paul and B. Kemp
The electromagnetic force acting on a Rayleigh particle placed in a rectangular waveguide is studied. The particle is excited using the lowest order TE10 mode. It is determined that the particle is laterally trapped at the high intensity region of the electric field and either pushed away from or pulled toward the light source. This push-pull phenomenon depends on whether the frequency of the light wave is above or below the cutoff frequency (i.e. the particle can be pushed or pulled by tuning the frequency). While conventional optical tweezers rely on a balance of scattering and gradient force in the propagation direction, the phenomenon predicted here switches between the two forces near the lowest cutoff in a waveguide.
DOI
The electromagnetic force acting on a Rayleigh particle placed in a rectangular waveguide is studied. The particle is excited using the lowest order TE10 mode. It is determined that the particle is laterally trapped at the high intensity region of the electric field and either pushed away from or pulled toward the light source. This push-pull phenomenon depends on whether the frequency of the light wave is above or below the cutoff frequency (i.e. the particle can be pushed or pulled by tuning the frequency). While conventional optical tweezers rely on a balance of scattering and gradient force in the propagation direction, the phenomenon predicted here switches between the two forces near the lowest cutoff in a waveguide.
DOI
Perturbative theory for Brownian vortexes
Henrique W. Moyses, Ross O. Bauer, Alexander Y. Grosberg, and David G. Grier
Brownian vortexes are stochastic machines that use static nonconservative force fields to bias random thermal fluctuations into steadily circulating currents. The archetype for this class of systems is a colloidal sphere in an optical tweezer. Trapped near the focus of a strongly converging beam of light, the particle is displaced by random thermal kicks into the nonconservative part of the optical force field arising from radiation pressure, which then biases its diffusion. Assuming the particle remains localized within the trap, its time-averaged trajectory traces out a toroidal vortex. Unlike trivial Brownian vortexes, such as the biased Brownian pendulum, which circulate preferentially in the direction of the bias, the general Brownian vortex can change direction and even topology in response to temperature changes. Here we introduce a theory based on a perturbative expansion of the Fokker-Planck equation for weak nonconservative driving. The first-order solution takes the form of a modified Boltzmann relation and accounts for the rich phenomenology observed in experiments on micrometer-scale colloidal spheres in optical tweezers.
DOI
Brownian vortexes are stochastic machines that use static nonconservative force fields to bias random thermal fluctuations into steadily circulating currents. The archetype for this class of systems is a colloidal sphere in an optical tweezer. Trapped near the focus of a strongly converging beam of light, the particle is displaced by random thermal kicks into the nonconservative part of the optical force field arising from radiation pressure, which then biases its diffusion. Assuming the particle remains localized within the trap, its time-averaged trajectory traces out a toroidal vortex. Unlike trivial Brownian vortexes, such as the biased Brownian pendulum, which circulate preferentially in the direction of the bias, the general Brownian vortex can change direction and even topology in response to temperature changes. Here we introduce a theory based on a perturbative expansion of the Fokker-Planck equation for weak nonconservative driving. The first-order solution takes the form of a modified Boltzmann relation and accounts for the rich phenomenology observed in experiments on micrometer-scale colloidal spheres in optical tweezers.
DOI
Friday, July 17, 2015
Nanophotonic detection of freely interacting molecules on a single influenza virus
Pilgyu Kang, Perry Schein, Xavier Serey, Dakota O’Dell & David Erickson
Biomolecular interactions, such as antibody-antigen binding, are fundamental to many biological processes. At present, most techniques for analyzing these interactions require immobilizing one or both of the interacting molecules on an assay plate or a sensor surface. This is convenient experimentally but can constrain the natural binding affinity and capacity of the molecules, resulting in data that can deviate from the natural free-solution behavior. Here we demonstrate a label-free method for analyzing free-solution interactions between a single influenza virus and specific antibodies at the single particle level using near-field optical trapping and light-scattering techniques. We determine the number of specific antibodies binding to an optically trapped influenza virus by analyzing the change of the Brownian fluctuations of the virus. We develop an analytical model that determines the increased size of the virus resulting from antibodies binding to the virus membrane with uncertainty of ±1–2 nm. We present stoichiometric results of 26 ± 4 (6.8 ± 1.1 attogram) anti-influenza antibodies binding to an H1N1 influenza virus. Our technique can be applied to a wide range of molecular interactions because the nanophotonic tweezer can handle molecules from tens to thousands of nanometers in diameter.
DOI
Biomolecular interactions, such as antibody-antigen binding, are fundamental to many biological processes. At present, most techniques for analyzing these interactions require immobilizing one or both of the interacting molecules on an assay plate or a sensor surface. This is convenient experimentally but can constrain the natural binding affinity and capacity of the molecules, resulting in data that can deviate from the natural free-solution behavior. Here we demonstrate a label-free method for analyzing free-solution interactions between a single influenza virus and specific antibodies at the single particle level using near-field optical trapping and light-scattering techniques. We determine the number of specific antibodies binding to an optically trapped influenza virus by analyzing the change of the Brownian fluctuations of the virus. We develop an analytical model that determines the increased size of the virus resulting from antibodies binding to the virus membrane with uncertainty of ±1–2 nm. We present stoichiometric results of 26 ± 4 (6.8 ± 1.1 attogram) anti-influenza antibodies binding to an H1N1 influenza virus. Our technique can be applied to a wide range of molecular interactions because the nanophotonic tweezer can handle molecules from tens to thousands of nanometers in diameter.
DOI
Three-dimensional motion detection of a 20-nm gold nanoparticle using twilight-field digital holography with coherence regulation
Kazufumi Goto and Yoshio Hayasaki
In the twilight-field method for obtaining interference fringes with high contrast in in-line digital holography, only the intensity of the reference light is regulated to be close to the intensity of the object light, which is the ultra-weak scattered light from a nanoparticle, by using a low-frequency attenuation filter. Coherence of the light also strongly affects the contrast of the interference fringes. High coherence causes a lot of undesired coherent noise, which masks the fringes derived from the nanoparticles. Too-low coherence results in fringes with low contrast and a correspondingly low signal-to-noise ratio. Consequently, proper regulation of the coherence of the light source, in this study the spectral width, improves the minimum detectable size in holographic three-dimensional position measurement of nanoparticles. By using these methods, we were able to measure the position of a gold nanoparticle with a minimum diameter of 20 nm.
DOI
In the twilight-field method for obtaining interference fringes with high contrast in in-line digital holography, only the intensity of the reference light is regulated to be close to the intensity of the object light, which is the ultra-weak scattered light from a nanoparticle, by using a low-frequency attenuation filter. Coherence of the light also strongly affects the contrast of the interference fringes. High coherence causes a lot of undesired coherent noise, which masks the fringes derived from the nanoparticles. Too-low coherence results in fringes with low contrast and a correspondingly low signal-to-noise ratio. Consequently, proper regulation of the coherence of the light source, in this study the spectral width, improves the minimum detectable size in holographic three-dimensional position measurement of nanoparticles. By using these methods, we were able to measure the position of a gold nanoparticle with a minimum diameter of 20 nm.
DOI
Actin Filament Turnover Drives Leading Edge Growth during Myelin Sheath Formation in the Central Nervous System
Schanila Nawaz, Paula Sánchez, Sebastian Schmitt, Nicolas Snaidero, Mišo Mitkovski, Caroline Velte, Bastian R. Brückner, Ioannis Alexopoulos, Tim Czopka, Sang Y. Jung, Jeong S. Rhee, Andreas Janshoff, Walter Witke, Iwan A.T. Schaap, David A. Lyons, Mikael Simons
During CNS development, oligodendrocytes wrap their plasma membrane around axons to generate multilamellar myelin sheaths. To drive growth at the leading edge of myelin at the interface with the axon, mechanical forces are necessary, but the underlying mechanisms are not known. Using an interdisciplinary approach that combines morphological, genetic, and biophysical analyses, we identified a key role for actin filament network turnover in myelin growth. At the onset of myelin biogenesis, F-actin is redistributed to the leading edge, where its polymerization-based forces push out non-adhesive and motile protrusions. F-actin disassembly converts protrusions into sheets by reducing surface tension and in turn inducing membrane spreading and adhesion. We identified the actin depolymerizing factor ADF/cofilin1, which mediates high F-actin turnover rates, as an essential factor in this process. We propose that F-actin turnover is the driving force in myelin wrapping by regulating repetitive cycles of leading edge protrusion and spreading.
DOI
During CNS development, oligodendrocytes wrap their plasma membrane around axons to generate multilamellar myelin sheaths. To drive growth at the leading edge of myelin at the interface with the axon, mechanical forces are necessary, but the underlying mechanisms are not known. Using an interdisciplinary approach that combines morphological, genetic, and biophysical analyses, we identified a key role for actin filament network turnover in myelin growth. At the onset of myelin biogenesis, F-actin is redistributed to the leading edge, where its polymerization-based forces push out non-adhesive and motile protrusions. F-actin disassembly converts protrusions into sheets by reducing surface tension and in turn inducing membrane spreading and adhesion. We identified the actin depolymerizing factor ADF/cofilin1, which mediates high F-actin turnover rates, as an essential factor in this process. We propose that F-actin turnover is the driving force in myelin wrapping by regulating repetitive cycles of leading edge protrusion and spreading.
DOI
From free energy measurements to thermodynamic inference in nonequilibrium small systems
A Alemany, M Ribezzi-Crivellari and F Ritort
Fluctuation theorems (FTs), such as the Crooks or Jarzynski equalities (JEs), have become an important tool in single-molecule biophysics where they allow experimentalists to exploit thermal fluctuations and measure free-energy differences from non-equilibrium pulling experiments. The rich phenomenology of biomolecular systems has stimulated the development of extensions to the standard FTs, to encompass different experimental situations. Here we discuss an extension of the Crooks fluctuation relation that allows the thermodynamic characterization of kinetic molecular states. This extension can be connected to the generalized JE under feedback. Finally we address the recently introduced concept of thermodynamic inference or how FTs can be used to extract the total entropy production distribution in nonequilibrium systems from partial entropy production measurements. We discuss the significance of the concept of effective temperature in this context and show how thermodynamic inference provides a unifying comprehensive picture in several nonequilibrium problems.
DOI
Fluctuation theorems (FTs), such as the Crooks or Jarzynski equalities (JEs), have become an important tool in single-molecule biophysics where they allow experimentalists to exploit thermal fluctuations and measure free-energy differences from non-equilibrium pulling experiments. The rich phenomenology of biomolecular systems has stimulated the development of extensions to the standard FTs, to encompass different experimental situations. Here we discuss an extension of the Crooks fluctuation relation that allows the thermodynamic characterization of kinetic molecular states. This extension can be connected to the generalized JE under feedback. Finally we address the recently introduced concept of thermodynamic inference or how FTs can be used to extract the total entropy production distribution in nonequilibrium systems from partial entropy production measurements. We discuss the significance of the concept of effective temperature in this context and show how thermodynamic inference provides a unifying comprehensive picture in several nonequilibrium problems.
DOI
Tuesday, July 14, 2015
Single-Molecule Folding Mechanisms of the apo- and Mg2+-Bound States of Human Neuronal Calcium Sensor-1
Mohsin M. Naqvi, Pétur O. Heidarsson, Mariela R. Otazo, Alessandro Mossa, Birthe B. Kragelund, Ciro Cecconi
Neuronal calcium sensor-1 (NCS-1) is the primordial member of a family of proteins responsible primarily for sensing changes in neuronal Ca2+ concentration. NCS-1 is a multispecific protein interacting with a number of binding partners in both calcium-dependent and independent manners, and acting in a variety of cellular processes in which it has been linked to a number of disorders such as schizophrenia and autism. Despite extensive studies on the Ca2+-activated state of NCS proteins, little is known about the conformational dynamics of the Mg2+-bound and apo states, both of which are populated, at least transiently, at resting Ca2+ conditions. Here, we used optical tweezers to study the folding behavior of individual NCS-1 molecules in the presence of Mg2+ and in the absence of divalent ions. Under tension, the Mg2+-bound state of NCS-1 unfolds and refolds in a three-state process by populating one intermediate state consisting of a folded C-domain and an unfolded N-domain. The interconversion at equilibrium between the different molecular states populated by NCS-1 was monitored in real time through constant-force measurements and the energy landscapes underlying the observed transitions were reconstructed through hidden Markov model analysis. Unlike what has been observed with the Ca2+-bound state, the presence of Mg2+ allows both the N- and C-domain to fold through all-or-none transitions with similar refolding rates. In the absence of divalent ions, NCS-1 unfolds and refolds reversibly in a two-state reaction involving only the C-domain, whereas the N-domain has no detectable transitions. Overall, the results allowed us to trace the progression of NCS-1 folding along its energy landscapes and provided a solid platform for understanding the conformational dynamics of similar EF-hand proteins.
DOI
Neuronal calcium sensor-1 (NCS-1) is the primordial member of a family of proteins responsible primarily for sensing changes in neuronal Ca2+ concentration. NCS-1 is a multispecific protein interacting with a number of binding partners in both calcium-dependent and independent manners, and acting in a variety of cellular processes in which it has been linked to a number of disorders such as schizophrenia and autism. Despite extensive studies on the Ca2+-activated state of NCS proteins, little is known about the conformational dynamics of the Mg2+-bound and apo states, both of which are populated, at least transiently, at resting Ca2+ conditions. Here, we used optical tweezers to study the folding behavior of individual NCS-1 molecules in the presence of Mg2+ and in the absence of divalent ions. Under tension, the Mg2+-bound state of NCS-1 unfolds and refolds in a three-state process by populating one intermediate state consisting of a folded C-domain and an unfolded N-domain. The interconversion at equilibrium between the different molecular states populated by NCS-1 was monitored in real time through constant-force measurements and the energy landscapes underlying the observed transitions were reconstructed through hidden Markov model analysis. Unlike what has been observed with the Ca2+-bound state, the presence of Mg2+ allows both the N- and C-domain to fold through all-or-none transitions with similar refolding rates. In the absence of divalent ions, NCS-1 unfolds and refolds reversibly in a two-state reaction involving only the C-domain, whereas the N-domain has no detectable transitions. Overall, the results allowed us to trace the progression of NCS-1 folding along its energy landscapes and provided a solid platform for understanding the conformational dynamics of similar EF-hand proteins.
DOI
Mechanical Properties of a Primary Cilium As Measured by Resonant Oscillation
Andrew Resnick
Primary cilia are ubiquitous mammalian cellular substructures implicated in an ever-increasing number of regulatory pathways. The well-established ciliary hypothesis states that physical bending of the cilium (for example, due to fluid flow) initiates signaling cascades, yet the mechanical properties of the cilium remain incompletely measured, resulting in confusion regarding the biological significance of flow-induced ciliary mechanotransduction. In this work we measure the mechanical properties of a primary cilium by using an optical trap to induce resonant oscillation of the structure. Our data indicate 1) the primary cilium is not a simple cantilevered beam; 2) the base of the cilium may be modeled as a nonlinear rotatory spring, with the linear spring constant k of the cilium base calculated to be (4.6 ± 0.62) × 10−12 N/rad and nonlinear spring constant α to be (−1 ± 0.34) × 10−10 N/rad2; and 3) the ciliary base may be an essential regulator of mechanotransduction signaling. Our method is also particularly suited to measure mechanical properties of nodal cilia, stereocilia, and motile cilia—anatomically similar structures with very different physiological functions.
DOI
Primary cilia are ubiquitous mammalian cellular substructures implicated in an ever-increasing number of regulatory pathways. The well-established ciliary hypothesis states that physical bending of the cilium (for example, due to fluid flow) initiates signaling cascades, yet the mechanical properties of the cilium remain incompletely measured, resulting in confusion regarding the biological significance of flow-induced ciliary mechanotransduction. In this work we measure the mechanical properties of a primary cilium by using an optical trap to induce resonant oscillation of the structure. Our data indicate 1) the primary cilium is not a simple cantilevered beam; 2) the base of the cilium may be modeled as a nonlinear rotatory spring, with the linear spring constant k of the cilium base calculated to be (4.6 ± 0.62) × 10−12 N/rad and nonlinear spring constant α to be (−1 ± 0.34) × 10−10 N/rad2; and 3) the ciliary base may be an essential regulator of mechanotransduction signaling. Our method is also particularly suited to measure mechanical properties of nodal cilia, stereocilia, and motile cilia—anatomically similar structures with very different physiological functions.
DOI
Fano resonance of the ultrasensitve optical force excited by Gaussian evanescent field
Yang Yang, Jiafang Li and Zhi-Yuan Li
In this paper, we study the angle-dependent Fano-like optical force spectra of plasmonic Ag nanoparticles, which exhibit extraordinary transformation from Lorentzian resonance to Fano resonance when excited by a Gaussian evanescent wave. We systematically analyze the behavior of this asymmetric scattering induced optical force under different conditions and find that this Fano interference-induced force is ultrasensitive to the excitation wavelength, incident angle and particle size, as well as the core–shell configuration, which could be useful for wavelength- and angle-dependent size-selective optical manipulation. The origin of this Fano resonance is further identified as the interference between the two adjacent-order multipolar plasmonic modes excited in the Ag particle under the excitation of an inhomogeneously distributed evanescent field.
DOI
In this paper, we study the angle-dependent Fano-like optical force spectra of plasmonic Ag nanoparticles, which exhibit extraordinary transformation from Lorentzian resonance to Fano resonance when excited by a Gaussian evanescent wave. We systematically analyze the behavior of this asymmetric scattering induced optical force under different conditions and find that this Fano interference-induced force is ultrasensitive to the excitation wavelength, incident angle and particle size, as well as the core–shell configuration, which could be useful for wavelength- and angle-dependent size-selective optical manipulation. The origin of this Fano resonance is further identified as the interference between the two adjacent-order multipolar plasmonic modes excited in the Ag particle under the excitation of an inhomogeneously distributed evanescent field.
DOI
Single Molecules Trapped by Dynamic Inhomogeneous Temperature Fields
Marco Braun, Andreas P. Bregulla, Katrin Günther, Michael Mertig, and Frank Cichos
We demonstrate a single molecule trapping concept that modulates the actual driving force of Brownian motion—the temperature. By spatially and temporally varying the temperature at a plasmonic nanostructure, thermodiffusive drifts are induced that are used to trap single nano-objects. A feedback controlled switching of local temperature fields allows us to confine the motion of a single DNA molecule for minutes and tailoring complex effective trapping potentials. This new type of thermophoretic microbeaker even provides control over a well-defined number of single molecules and is scalable to large arrays of trapping structures.
DOI
We demonstrate a single molecule trapping concept that modulates the actual driving force of Brownian motion—the temperature. By spatially and temporally varying the temperature at a plasmonic nanostructure, thermodiffusive drifts are induced that are used to trap single nano-objects. A feedback controlled switching of local temperature fields allows us to confine the motion of a single DNA molecule for minutes and tailoring complex effective trapping potentials. This new type of thermophoretic microbeaker even provides control over a well-defined number of single molecules and is scalable to large arrays of trapping structures.
DOI
Monday, July 13, 2015
Biased Brownian motion as a mechanism to facilitate nanometer-scale exploration of the microtubule plus end by a kinesin-8
Yongdae Shin, Yaqing Du, Scott E. Collier, Melanie D. Ohi, Matthew J. Lang, and Ryoma Ohi
Kinesin-8s are plus-end–directed motors that negatively regulate microtubule (MT) length. Well-characterized members of this subfamily (Kip3, Kif18A) exhibit two important properties: (i) They are “ultraprocessive,” a feature enabled by a second MT-binding site that tethers the motors to a MT track, and (ii) they dissociate infrequently from the plus end. Together, these characteristics combined with their plus-end motility cause Kip3 and Kif18A to enrich preferentially at the plus ends of long MTs, promoting MT catastrophes or pausing. Kif18B, an understudied human kinesin-8, also limits MT growth during mitosis. In contrast to Kif18A and Kip3, localization of Kif18B to plus ends relies on binding to the plus-end tracking protein EB1, making the relationship between its potential plus-end–directed motility and plus-end accumulation unclear. Using single-molecule assays, we show that Kif18B is only modestly processive and that the motor switches frequently between directed and diffusive modes of motility. Diffusion is promoted by the tail domain, which also contains a second MT-binding site that decreases the off rate of the motor from the MT lattice. In cells, Kif18B concentrates at the extreme tip of a subset of MTs, superseding EB1. Our data demonstrate that kinesin-8 motors use diverse design principles to target MT plus ends, which likely target them to the plus ends of distinct MT subpopulations in the mitotic spindle.
DOI
Kinesin-8s are plus-end–directed motors that negatively regulate microtubule (MT) length. Well-characterized members of this subfamily (Kip3, Kif18A) exhibit two important properties: (i) They are “ultraprocessive,” a feature enabled by a second MT-binding site that tethers the motors to a MT track, and (ii) they dissociate infrequently from the plus end. Together, these characteristics combined with their plus-end motility cause Kip3 and Kif18A to enrich preferentially at the plus ends of long MTs, promoting MT catastrophes or pausing. Kif18B, an understudied human kinesin-8, also limits MT growth during mitosis. In contrast to Kif18A and Kip3, localization of Kif18B to plus ends relies on binding to the plus-end tracking protein EB1, making the relationship between its potential plus-end–directed motility and plus-end accumulation unclear. Using single-molecule assays, we show that Kif18B is only modestly processive and that the motor switches frequently between directed and diffusive modes of motility. Diffusion is promoted by the tail domain, which also contains a second MT-binding site that decreases the off rate of the motor from the MT lattice. In cells, Kif18B concentrates at the extreme tip of a subset of MTs, superseding EB1. Our data demonstrate that kinesin-8 motors use diverse design principles to target MT plus ends, which likely target them to the plus ends of distinct MT subpopulations in the mitotic spindle.
DOI
Vascular disease-causing mutation R258C in ACTA2 disrupts actin dynamics and interaction with myosin
Hailong Lu, Patricia M. Fagnant, Carol S. Bookwalter, Peteranne Joel, and Kathleen M. Trybus
Point mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2, are the most prevalent cause of familial thoracic aortic aneurysms and dissections (TAAD). Here, we provide the first molecular characterization, to our knowledge, of the effect of the R258C mutation in SM α-actin, expressed with the baculovirus system. Smooth muscles are unique in that force generation requires both interaction of stable actin filaments with myosin and polymerization of actin in the subcortical region. Both aspects of R258C function therefore need investigation. Total internal reflection fluorescence (TIRF) microscopy was used to quantify the growth of single actin filaments as a function of time. R258C filaments are less stable than WT and more susceptible to severing by cofilin. Smooth muscle tropomyosin offers little protection from cofilin cleavage, unlike its effect on WT actin. Unexpectedly, profilin binds tighter to the R258C monomer, which will increase the pool of globular actin (G-actin). In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly than WT, and the slowing is exacerbated by smooth muscle tropomyosin. Under loaded conditions, small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting that tropomyosin occupies an inhibitory position on actin. Many of the observed defects cannot be explained by a direct interaction with the mutated residue, and thus the mutation allosterically affects multiple regions of the monomer. Our results align with the hypothesis that defective contractile function contributes to the pathogenesis of TAAD.
DOI
Point mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2, are the most prevalent cause of familial thoracic aortic aneurysms and dissections (TAAD). Here, we provide the first molecular characterization, to our knowledge, of the effect of the R258C mutation in SM α-actin, expressed with the baculovirus system. Smooth muscles are unique in that force generation requires both interaction of stable actin filaments with myosin and polymerization of actin in the subcortical region. Both aspects of R258C function therefore need investigation. Total internal reflection fluorescence (TIRF) microscopy was used to quantify the growth of single actin filaments as a function of time. R258C filaments are less stable than WT and more susceptible to severing by cofilin. Smooth muscle tropomyosin offers little protection from cofilin cleavage, unlike its effect on WT actin. Unexpectedly, profilin binds tighter to the R258C monomer, which will increase the pool of globular actin (G-actin). In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly than WT, and the slowing is exacerbated by smooth muscle tropomyosin. Under loaded conditions, small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting that tropomyosin occupies an inhibitory position on actin. Many of the observed defects cannot be explained by a direct interaction with the mutated residue, and thus the mutation allosterically affects multiple regions of the monomer. Our results align with the hypothesis that defective contractile function contributes to the pathogenesis of TAAD.
DOI
Label-Free DNA Sequencing Using Millikan Detection
Roger Dettloff, Danielle Leiske, Andrea Chow, Javier Farinas
A label-free method for DNA sequencing based on the principle of the Millikan oil drop experiment was developed. This sequencing-by-synthesis approach sensed increases in bead charge as nucleotides were added by a polymerase to DNA templates attached to beads. The balance between an electrical force, which was dependent on the number of nucleotide charges on a bead, and opposing hydrodynamic drag and restoring tether forces resulted in a bead velocity that was a function of the number of nucleotides attached to the bead. The velocity of beads tethered via a polymer to a microfluidic channel and subjected to an oscillating electric field was measured using dark-field microscopy and used to determine how many nucleotides were incorporated during each sequencing-by-synthesis cycle. Increases in bead velocity of ∼ 1% were reliably detected during DNA polymerization allowing for sequencing of short DNA templates. The method could lead to a low-cost, high-throughput sequencing platform that could enable routine sequencing in medical applications.
DOI
A label-free method for DNA sequencing based on the principle of the Millikan oil drop experiment was developed. This sequencing-by-synthesis approach sensed increases in bead charge as nucleotides were added by a polymerase to DNA templates attached to beads. The balance between an electrical force, which was dependent on the number of nucleotide charges on a bead, and opposing hydrodynamic drag and restoring tether forces resulted in a bead velocity that was a function of the number of nucleotides attached to the bead. The velocity of beads tethered via a polymer to a microfluidic channel and subjected to an oscillating electric field was measured using dark-field microscopy and used to determine how many nucleotides were incorporated during each sequencing-by-synthesis cycle. Increases in bead velocity of ∼ 1% were reliably detected during DNA polymerization allowing for sequencing of short DNA templates. The method could lead to a low-cost, high-throughput sequencing platform that could enable routine sequencing in medical applications.
DOI
Nonlinear irreversible thermodynamics of single-molecule experiments
I. Santamaría-Holek, N. J. López-Alamilla, M. Hidalgo-Soria, and A. Pérez-Madrid
Irreversible thermodynamics of single-molecule experiments subject to external constraining forces of a mechanical nature is presented. Extending Onsager's formalism to the nonlinear case of systems under nonequilibrium external constraints, we are able to calculate the entropy production and the general nonlinear kinetic equations for the variables involved. In particular, we analyze the case of RNA stretching protocols obtaining critical oscillations between different configurational states when forced by external means to remain in the unstable region of its free-energy landscape, as observed in experiments. We also calculate the entropy produced during these hopping events and show how resonant phenomena in stretching experiments of single RNA macromolecules may arise. We also calculate the hopping rates using Kramer's approach obtaining a good comparison with experiments.
DOI
Irreversible thermodynamics of single-molecule experiments subject to external constraining forces of a mechanical nature is presented. Extending Onsager's formalism to the nonlinear case of systems under nonequilibrium external constraints, we are able to calculate the entropy production and the general nonlinear kinetic equations for the variables involved. In particular, we analyze the case of RNA stretching protocols obtaining critical oscillations between different configurational states when forced by external means to remain in the unstable region of its free-energy landscape, as observed in experiments. We also calculate the entropy produced during these hopping events and show how resonant phenomena in stretching experiments of single RNA macromolecules may arise. We also calculate the hopping rates using Kramer's approach obtaining a good comparison with experiments.
DOI
Imaging Nanoscale Electromagnetic Near-Field Distributions Using Optical Forces
Fei Huang, Venkata Ananth Tamma, Zahra Mardy, Jonathan Burdett & H. Kumar Wickramasinghe
We demonstrate the application of Atomic Force Microscopy (AFM) for mapping optical near-fields with nanometer resolution, limited only by the AFM probe geometry. By detecting the optical force between a gold coated AFM probe and its image dipole on a glass substrate, we profile the electric field distributions of tightly focused laser beams with different polarizations. The experimentally recorded focal force maps agree well with theoretical predictions based on a dipole-dipole interaction model. We experimentally estimate the aspect ratio of the apex of gold coated AFM probe using only optical forces. We also show that the optical force between a sharp gold coated AFM probe and a spherical gold nanoparticle of radius 15 nm, is indicative of the electric field distribution between the two interacting particles. Photo Induced Force Microscopy (PIFM) allows for background free, thermal noise limited mechanical imaging of optical phenomenon over wide range of wavelengths from Visible to RF with detection sensitivity limited only by AFM performance.
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Saturday, July 11, 2015
Macro-optical trapping for sample confinement in light sheet microscopy
Zhengyi Yang, Peeter Piksarv, David E.K. Ferrier, Frank J. Gunn-Moore, and Kishan Dholakia
Light sheet microscopy is a powerful approach to construct three-dimensional images of large specimens with minimal photo-damage and photo-bleaching. To date, the specimens are usually mounted in agents such as agarose, potentially restricting the development of live samples, and also highly mobile specimens need to be anaesthetized before imaging. To overcome these problems, here we demonstrate an integrated light sheet microscope which solely uses optical forces to trap and hold the sample using a counter-propagating laser beam geometry. Specifically, tobacco plant cells and living Spirobranchus lamarcki larvae were successfully trapped and sectional images acquired. This novel approach has the potential to significantly expand the range of applications for light sheet imaging.
DOI
Light sheet microscopy is a powerful approach to construct three-dimensional images of large specimens with minimal photo-damage and photo-bleaching. To date, the specimens are usually mounted in agents such as agarose, potentially restricting the development of live samples, and also highly mobile specimens need to be anaesthetized before imaging. To overcome these problems, here we demonstrate an integrated light sheet microscope which solely uses optical forces to trap and hold the sample using a counter-propagating laser beam geometry. Specifically, tobacco plant cells and living Spirobranchus lamarcki larvae were successfully trapped and sectional images acquired. This novel approach has the potential to significantly expand the range of applications for light sheet imaging.
DOI
Raman spectroscopy for physiological investigations of tissues and cells
Thomas Huser, James Chan
Raman micro-spectroscopy provides a convenient non-destructive and location-specific means of probing cellular physiology and tissue physiology at sub-micron length scales. By probing the vibrational signature of molecules and molecular groups, the distribution and metabolic products of small molecules that cannot be labeled with fluorescent dyes can be analyzed. This method works well for molecular concentrations in the micro-molar range and has been demonstrated as a valuable tool for monitoring drug–cell interactions. If the small molecule of interest does not contain groups that would allow for a discrimination against cytoplasmic background signals, “labeling” of the molecule by isotope substitution or by incorporating other unique small groups, e.g. alkynes provides a stable signal even for time-lapse imaging such compounds in living cells. In this review we highlight recent progress in assessing the physiology of cells and tissue by Raman spectroscopy and imaging.
DOI
Raman micro-spectroscopy provides a convenient non-destructive and location-specific means of probing cellular physiology and tissue physiology at sub-micron length scales. By probing the vibrational signature of molecules and molecular groups, the distribution and metabolic products of small molecules that cannot be labeled with fluorescent dyes can be analyzed. This method works well for molecular concentrations in the micro-molar range and has been demonstrated as a valuable tool for monitoring drug–cell interactions. If the small molecule of interest does not contain groups that would allow for a discrimination against cytoplasmic background signals, “labeling” of the molecule by isotope substitution or by incorporating other unique small groups, e.g. alkynes provides a stable signal even for time-lapse imaging such compounds in living cells. In this review we highlight recent progress in assessing the physiology of cells and tissue by Raman spectroscopy and imaging.
DOI
Confocal Raman Microscopy for pH-Gradient Preconcentration and Quantitative Analyte Detection in Optically-trapped Phospholipid Vesicles
Chris D. Hardcastle and Joel M. Harris
The ability of a vesicle membrane to preserve a pH gradient, while allowing for diffusion of neutral molecules across the phospholipid bilayer, can provide the isolation and preconcentration of ionizable compounds within the vesicle interior. In this work, confocal-Raman microscopy is used to observe (in situ) the pH-gradient preconcentration of compounds into individual optically-trapped vesicles that provide sub-femtoliter collectors for small-volume samples. The concentration of analyte accumulated in the vesicle interior is determined relative to a perchlorate-ion internal standard, preloaded into the vesicle along with a high-concentration buffer. As a guide to the experiments, a model for the transfer of analyte into the vesicle based on acid-base equilibria is developed to predict the concentration enrichment as a function of source-phase pH and analyte concentration. To test the concept, the accumulation of benzyldimethylamine (BDMA) was measured within individual 1-µm phospholipid vesicles having a stable initial pH that is 7 units lower than the source phase. For low analyte concentrations in the source phase (100 nM), a concentration enrichment into the vesicle interior of (5.2 ± 0.4) x 105 was observed, in agreement with the model predictions. Detection of BDMA from a 25-nM source-phase sample was demonstrated, a noteworthy result for an unenhanced Raman scattering measurement. The developed model accurately predicts the fall-off of enrichment (and measurement sensitivity) at higher analyte concentrations, where the transfer of greater amounts of BDMA into the vesicle titrates the internal buffer and decreases the pH gradient. The predictable calibration response over 4-orders-of-magnitude in source-phase concentration makes it suitable for quantitative analysis of ionizable compounds from small volume samples. The kinetics of analyte accumulation are relatively fast (~15 min) and are consistent with the rate of transfer of a polar aromatic molecule across a gel-phase phospholipid membrane.
DOI
The ability of a vesicle membrane to preserve a pH gradient, while allowing for diffusion of neutral molecules across the phospholipid bilayer, can provide the isolation and preconcentration of ionizable compounds within the vesicle interior. In this work, confocal-Raman microscopy is used to observe (in situ) the pH-gradient preconcentration of compounds into individual optically-trapped vesicles that provide sub-femtoliter collectors for small-volume samples. The concentration of analyte accumulated in the vesicle interior is determined relative to a perchlorate-ion internal standard, preloaded into the vesicle along with a high-concentration buffer. As a guide to the experiments, a model for the transfer of analyte into the vesicle based on acid-base equilibria is developed to predict the concentration enrichment as a function of source-phase pH and analyte concentration. To test the concept, the accumulation of benzyldimethylamine (BDMA) was measured within individual 1-µm phospholipid vesicles having a stable initial pH that is 7 units lower than the source phase. For low analyte concentrations in the source phase (100 nM), a concentration enrichment into the vesicle interior of (5.2 ± 0.4) x 105 was observed, in agreement with the model predictions. Detection of BDMA from a 25-nM source-phase sample was demonstrated, a noteworthy result for an unenhanced Raman scattering measurement. The developed model accurately predicts the fall-off of enrichment (and measurement sensitivity) at higher analyte concentrations, where the transfer of greater amounts of BDMA into the vesicle titrates the internal buffer and decreases the pH gradient. The predictable calibration response over 4-orders-of-magnitude in source-phase concentration makes it suitable for quantitative analysis of ionizable compounds from small volume samples. The kinetics of analyte accumulation are relatively fast (~15 min) and are consistent with the rate of transfer of a polar aromatic molecule across a gel-phase phospholipid membrane.
DOI
Plasmofluidics: Merging Light and Fluids at the Micro-/Nanoscale
Mingsong Wang, Chenglong Zhao, Xiaoyu Miao, Yanhui Zhao, Joseph Rufo, Yan Jun Liu, Tony Jun Huang and Yuebing Zheng
Plasmofluidics is the synergistic integration of plasmonics and micro/nanofluidics in devices and applications in order to enhance performance. There has been significant progress in the emerging field of plasmofluidics in recent years. By utilizing the capability of plasmonics to manipulate light at the nanoscale, combined with the unique optical properties of fluids and precise manipulation via micro/nanofluidics, plasmofluidic technologies enable innovations in lab-on-a-chip systems, reconfigurable photonic devices, optical sensing, imaging, and spectroscopy. In this review article, the most recent advances in plasmofluidics are examined and categorized into plasmon-enhanced functionalities in microfluidics and microfluidics-enhanced plasmonic devices. The former focuses on plasmonic manipulations of fluids, bubbles, particles, biological cells, and molecules at the micro/nanoscale. The latter includes technological advances that apply microfluidic principles to enable reconfigurable plasmonic devices and performance-enhanced plasmonic sensors. The article is concluded with perspectives on the upcoming challenges, opportunities, and possible future directions of the emerging field of plasmofluidics.
DOI
Plasmofluidics is the synergistic integration of plasmonics and micro/nanofluidics in devices and applications in order to enhance performance. There has been significant progress in the emerging field of plasmofluidics in recent years. By utilizing the capability of plasmonics to manipulate light at the nanoscale, combined with the unique optical properties of fluids and precise manipulation via micro/nanofluidics, plasmofluidic technologies enable innovations in lab-on-a-chip systems, reconfigurable photonic devices, optical sensing, imaging, and spectroscopy. In this review article, the most recent advances in plasmofluidics are examined and categorized into plasmon-enhanced functionalities in microfluidics and microfluidics-enhanced plasmonic devices. The former focuses on plasmonic manipulations of fluids, bubbles, particles, biological cells, and molecules at the micro/nanoscale. The latter includes technological advances that apply microfluidic principles to enable reconfigurable plasmonic devices and performance-enhanced plasmonic sensors. The article is concluded with perspectives on the upcoming challenges, opportunities, and possible future directions of the emerging field of plasmofluidics.
DOI
Monday, July 6, 2015
Single-nanoparticle detection with slot-mode photonic crystal cavities
Cheng Wang, Qimin Quan, Shota Kita, Yihang Li and Marko Lončar
Optical cavities that are capable for detecting single nanoparticles could lead to great progress in early stage disease diagnostics and the study of biological interactions on the single-molecule level. In particular, photonic crystal (PhC) cavities are excellent platforms for label-free single-nanoparticle detection, owing to their high quality (Q) factors and wavelength-scale modal volumes. Here, we demonstrate the design and fabrication of a high-Q (>104) slot-mode PhC nanobeam cavity, which is able to strongly confine light in the slotted regions. The enhanced light-matter interaction results in an order of magnitude improvement in both refractive index sensitivity (439 nm/RIU) and single-nanoparticle sensitivity compared with conventional dielectric-mode PhC cavities. Detection of single polystyrene nanoparticles with radii of 20 nm and 30 nm is demonstrated in aqueous environments (D2O), without additional laser and temperature stabilization techniques.
DOI
Optical cavities that are capable for detecting single nanoparticles could lead to great progress in early stage disease diagnostics and the study of biological interactions on the single-molecule level. In particular, photonic crystal (PhC) cavities are excellent platforms for label-free single-nanoparticle detection, owing to their high quality (Q) factors and wavelength-scale modal volumes. Here, we demonstrate the design and fabrication of a high-Q (>104) slot-mode PhC nanobeam cavity, which is able to strongly confine light in the slotted regions. The enhanced light-matter interaction results in an order of magnitude improvement in both refractive index sensitivity (439 nm/RIU) and single-nanoparticle sensitivity compared with conventional dielectric-mode PhC cavities. Detection of single polystyrene nanoparticles with radii of 20 nm and 30 nm is demonstrated in aqueous environments (D2O), without additional laser and temperature stabilization techniques.
DOI
Microemulsion droplets in optical traps
Alex L. Hargreaves, Florence Gregson, Andrew K. Kirby, Sandra Engelskirchen, Colin D. Bain
While the optical manipulation of solid particles is increasingly familiar, complex liquids represent a relatively new area of investigation. In this study, microscopic liquid–liquid interfaces are deformed at ultralow interfacial tension using near-infrared optical tweezers. The chosen emulsions, of hydrocarbon oil (heptane and decane) in aqueous solutions of surfactant (AOT, C12E4, C12E5, Brij-L4) and salt, are capable of forming Winsor microemulsions, although the macroscopic phase volumes are unaffected by changes in ambient temperature. The amphiphilic ratio of nonionic (C12E4,5) and anionic (AOT) surfactants is chosen to minimise the temperature dependence of the monolayer curvature. These phases and their dispersions are examined, both with and without equilibration of the compositions. Under laser action at powers > 0.1 W, a plethora of metastable phases, multiple emulsions and vesicles are produced.
The rates of phase separation of microemulsion increase near the coverglass, which absorbs the laser more strongly. Conversely, substitution with heavy water reduces the absorptive heating, whilst maintaining a similar refractive index contrast. Localised laser heating is found to be the major driving force behind phase separation. The observed changes are rationalised in terms of the phase diagram of the microemulsion and the value of the amphiphilic ratio. The relative importance and origin of the thermal and optical effects on phase separation are discussed.
DOI
While the optical manipulation of solid particles is increasingly familiar, complex liquids represent a relatively new area of investigation. In this study, microscopic liquid–liquid interfaces are deformed at ultralow interfacial tension using near-infrared optical tweezers. The chosen emulsions, of hydrocarbon oil (heptane and decane) in aqueous solutions of surfactant (AOT, C12E4, C12E5, Brij-L4) and salt, are capable of forming Winsor microemulsions, although the macroscopic phase volumes are unaffected by changes in ambient temperature. The amphiphilic ratio of nonionic (C12E4,5) and anionic (AOT) surfactants is chosen to minimise the temperature dependence of the monolayer curvature. These phases and their dispersions are examined, both with and without equilibration of the compositions. Under laser action at powers > 0.1 W, a plethora of metastable phases, multiple emulsions and vesicles are produced.
The rates of phase separation of microemulsion increase near the coverglass, which absorbs the laser more strongly. Conversely, substitution with heavy water reduces the absorptive heating, whilst maintaining a similar refractive index contrast. Localised laser heating is found to be the major driving force behind phase separation. The observed changes are rationalised in terms of the phase diagram of the microemulsion and the value of the amphiphilic ratio. The relative importance and origin of the thermal and optical effects on phase separation are discussed.
DOI
Optical trapping Rayleigh dielectric particles with focused partially coherent dark hollow beams
Hua-Feng Xu, Wei-Jun Zhang, Jun Qu & Wei Huang
The focusing properties of coherent and partially coherent dark hollow beams (DHBs) through a paraxial ABCD optical system are theoretically investigated. It is found that the evolution behavior of the intensity distribution of focused partially coherent DHBs is closely related to their spatial coherence. The radiation forces (RFs) of focused coherent and partially coherent DHBs acting on a Rayleigh dielectric particle are also theoretically investigated. Numerical results show that the coherent and partially coherent DHBs can be focused into a tight focal spot, which can be used to stably trap a Rayleigh dielectric particle with high refractive index at the focus point. The influences of different beam parameters, including the spatial coherence, beam waist width, beam order, and hollow parameter of partially coherent DHBs, on the RFs and the trap stiffness are analyzed in detail. Finally, the stability conditions for effective trapping particles are also discussed.
DOI
The focusing properties of coherent and partially coherent dark hollow beams (DHBs) through a paraxial ABCD optical system are theoretically investigated. It is found that the evolution behavior of the intensity distribution of focused partially coherent DHBs is closely related to their spatial coherence. The radiation forces (RFs) of focused coherent and partially coherent DHBs acting on a Rayleigh dielectric particle are also theoretically investigated. Numerical results show that the coherent and partially coherent DHBs can be focused into a tight focal spot, which can be used to stably trap a Rayleigh dielectric particle with high refractive index at the focus point. The influences of different beam parameters, including the spatial coherence, beam waist width, beam order, and hollow parameter of partially coherent DHBs, on the RFs and the trap stiffness are analyzed in detail. Finally, the stability conditions for effective trapping particles are also discussed.
DOI
Thursday, July 2, 2015
Rigid multibody simulation of a helix-like structure: the dynamics of bacterial adhesion pili
Johan Zakrisson, Krister Wiklund, Martin Servin, Ove Axner, Claude Lacoursière, Magnus Andersson
We present a coarse-grained rigid multibody model of a subunit assembled helix-like polymer, e.g., adhesion pili expressed by bacteria, that is capable of describing the polymer’s force-extension response. With building blocks representing individual subunits, the model appropriately describes the complex behavior of pili expressed by the gram-negative uropathogenic Escherichia coli bacteria under the action of an external force. Numerical simulations show that the dynamics of the model, which include the effects of both unwinding and rewinding, are in good quantitative agreement with the characteristic force-extension response as observed experimentally for type 1 and P pili. By tuning the model, it is also possible to reproduce the force-extension response in the presence of anti-shaft antibodies, which dramatically changes the mechanical properties. Thus, the model and results in this work give enhanced understanding of how a pilus unwinds under the action of external forces and provide a new perspective of the complex bacterial adhesion processes.
DOI
We present a coarse-grained rigid multibody model of a subunit assembled helix-like polymer, e.g., adhesion pili expressed by bacteria, that is capable of describing the polymer’s force-extension response. With building blocks representing individual subunits, the model appropriately describes the complex behavior of pili expressed by the gram-negative uropathogenic Escherichia coli bacteria under the action of an external force. Numerical simulations show that the dynamics of the model, which include the effects of both unwinding and rewinding, are in good quantitative agreement with the characteristic force-extension response as observed experimentally for type 1 and P pili. By tuning the model, it is also possible to reproduce the force-extension response in the presence of anti-shaft antibodies, which dramatically changes the mechanical properties. Thus, the model and results in this work give enhanced understanding of how a pilus unwinds under the action of external forces and provide a new perspective of the complex bacterial adhesion processes.
DOI
Recent Progress on Man-Made Inorganic Nanomachines
Kwanoh Kim, Jianhe Guo, Xiaobin Xu and D. L. Fan
The successful development of nanoscale machinery, which can operate with high controllability, high precision, long lifetimes, and tunable driving powers, is pivotal for the realization of future intelligent nanorobots, nanofactories, and advanced biomedical devices. However, the development of nanomachines remains one of the most difficult research areas, largely due to the grand challenges in fabrication of devices with complex components and actuation with desired efficiency, precision, lifetime, and/or environmental friendliness. In this work, the cutting-edge efforts toward fabricating and actuating various types of nanomachines and their applications are reviewed, with a special focus on nanomotors made from inorganic nanoscale building blocks, which are introduced according to the employed actuation mechanism. The unique characteristics and obstacles for each type of nanomachine are discussed, and perspectives and challenges of this exciting field are presented.
DOI
The successful development of nanoscale machinery, which can operate with high controllability, high precision, long lifetimes, and tunable driving powers, is pivotal for the realization of future intelligent nanorobots, nanofactories, and advanced biomedical devices. However, the development of nanomachines remains one of the most difficult research areas, largely due to the grand challenges in fabrication of devices with complex components and actuation with desired efficiency, precision, lifetime, and/or environmental friendliness. In this work, the cutting-edge efforts toward fabricating and actuating various types of nanomachines and their applications are reviewed, with a special focus on nanomotors made from inorganic nanoscale building blocks, which are introduced according to the employed actuation mechanism. The unique characteristics and obstacles for each type of nanomachine are discussed, and perspectives and challenges of this exciting field are presented.
DOI
Optical Nanofiber Integrated into Optical Tweezers for In Situ Fiber Probing and Optical Binding Studies
Ivan Gusachenko, Viet Giang Truong, Mary C. Frawley and Síle Nic Chormaic
Precise control of particle positioning is desirable in many optical propulsion and sorting applications. Here, we develop an integrated platform for particle manipulation consisting of a combined optical nanofiber and optical tweezers system. We show that consistent and reversible transmission modulations arise when individual silica microspheres are introduced to the nanofiber surface using the optical tweezers. The observed transmission changes depend on both particle and fiber diameter and can be used as a reference point for in situ nanofiber or particle size measurement. Thence, we combine scanning electron microscope (SEM) size measurements with nanofiber transmission data to provide calibration for particle-based fiber assessment. This integrated optical platform provides a method for selective evanescent field manipulation of micron-sized particles and facilitates studies of optical binding and light-particle interaction dynamics.
DOI
Precise control of particle positioning is desirable in many optical propulsion and sorting applications. Here, we develop an integrated platform for particle manipulation consisting of a combined optical nanofiber and optical tweezers system. We show that consistent and reversible transmission modulations arise when individual silica microspheres are introduced to the nanofiber surface using the optical tweezers. The observed transmission changes depend on both particle and fiber diameter and can be used as a reference point for in situ nanofiber or particle size measurement. Thence, we combine scanning electron microscope (SEM) size measurements with nanofiber transmission data to provide calibration for particle-based fiber assessment. This integrated optical platform provides a method for selective evanescent field manipulation of micron-sized particles and facilitates studies of optical binding and light-particle interaction dynamics.
DOI
Wednesday, July 1, 2015
Factor-dependent processivity in human eIF4A DEAD-box helicase
Cuauhtémoc García-García, Kirsten L. Frieda, Kateryna Feoktistova, Christopher S. Fraser, Steven M. Block
During eukaryotic translation initiation, the small ribosomal subunit, assisted by initiation factors, locates the messenger RNA start codon by scanning from the 5′ cap. This process is powered by the eukaryotic initiation factor 4A (eIF4A), a DEAD-box helicase. eIF4A has been thought to unwind structures formed in the untranslated 5′ region via a nonprocessive mechanism. Using a single-molecule assay, we found that eIF4A functions instead as an adenosine triphosphate–dependent processive helicase when complexed with two accessory proteins, eIF4G and eIF4B. Translocation occurred in discrete steps of 11 ± 2 base pairs, irrespective of the accessory factor combination. Our findings support a memory-less stepwise mechanism for translation initiation and suggest that similar factor-dependent processivity may be shared by other members of the DEAD-box helicase family.
DOI
During eukaryotic translation initiation, the small ribosomal subunit, assisted by initiation factors, locates the messenger RNA start codon by scanning from the 5′ cap. This process is powered by the eukaryotic initiation factor 4A (eIF4A), a DEAD-box helicase. eIF4A has been thought to unwind structures formed in the untranslated 5′ region via a nonprocessive mechanism. Using a single-molecule assay, we found that eIF4A functions instead as an adenosine triphosphate–dependent processive helicase when complexed with two accessory proteins, eIF4G and eIF4B. Translocation occurred in discrete steps of 11 ± 2 base pairs, irrespective of the accessory factor combination. Our findings support a memory-less stepwise mechanism for translation initiation and suggest that similar factor-dependent processivity may be shared by other members of the DEAD-box helicase family.
DOI
Assessing single upconverting nanoparticle luminescence by optical tweezers
Paloma Rodriguez-Sevilla , Hector Rodriguez-Rodriguez , Marco Pedroni , Adolfo Speghini , Marco Bettinelli , Jose Antonio Garcia Sole , Daniel Jaque , and Patricia Haro-Gonzalez
We report on stable, long-term immobilization and localization of a single colloidal Er3+/Yb3+ codoped upconverting fluorescent nanoparticle (UCNP) by optical trapping with a single infrared laser beam. Contrary to expectations, the single UCNP emission differs from that generated by an assembly of UCNPs. The experimental data reveal that the differences can be explained in terms of modulations caused by radiation-trapping, a phenomenon not considered before but this work reveals to be of great relevance.
DOI
We report on stable, long-term immobilization and localization of a single colloidal Er3+/Yb3+ codoped upconverting fluorescent nanoparticle (UCNP) by optical trapping with a single infrared laser beam. Contrary to expectations, the single UCNP emission differs from that generated by an assembly of UCNPs. The experimental data reveal that the differences can be explained in terms of modulations caused by radiation-trapping, a phenomenon not considered before but this work reveals to be of great relevance.
DOI
The Effect of Short Duration Ultrasound Pulses on the Interaction Between Individual Microbubbles and Fibrin Clots
Christopher Acconcia, Ben Y.C. Leung, Anoop Manjunath, David E. Goertz
In previous work, we examined microscale interactions between microbubbles and fibrin clots under exposure to 1 ms ultrasound pulses. This provided direct evidence that microbubbles were capable of deforming clot boundaries and penetrating into clots, while also affecting fluid uptake and inducing fibrin network damage. Here, we investigate the effect of short duration (15 μs) pulses on microscale bubble-clot interactions as function of bubble diameter (3–9 μm) and pressure. Individual microbubbles (n = 45) were placed at the clot boundary with optical tweezers and exposed to 1 MHz ultrasound. High-speed (10 kfps) imaging and 2-photon microscopy were performed during and after exposure, respectively. While broadly similar phenomena were observed as in the 1 ms pulse case (i.e., bubble penetration, network damage and fluid uptake), substantial quantitative differences were present. The pressure threshold for bubble penetration was increased from 0.39 MPa to 0.6 MPa, and those bubbles that did enter clots had reduced penetration depths and were associated with less fibrin network damage and nanobead uptake. This appeared to be due in large part to increased bubble shrinkage relative to the 1 ms pulse case. Stroboscopic imaging was performed on a subset of bubbles (n = 11) and indicated that complex bubble oscillations can occur during this process.
DOI
In previous work, we examined microscale interactions between microbubbles and fibrin clots under exposure to 1 ms ultrasound pulses. This provided direct evidence that microbubbles were capable of deforming clot boundaries and penetrating into clots, while also affecting fluid uptake and inducing fibrin network damage. Here, we investigate the effect of short duration (15 μs) pulses on microscale bubble-clot interactions as function of bubble diameter (3–9 μm) and pressure. Individual microbubbles (n = 45) were placed at the clot boundary with optical tweezers and exposed to 1 MHz ultrasound. High-speed (10 kfps) imaging and 2-photon microscopy were performed during and after exposure, respectively. While broadly similar phenomena were observed as in the 1 ms pulse case (i.e., bubble penetration, network damage and fluid uptake), substantial quantitative differences were present. The pressure threshold for bubble penetration was increased from 0.39 MPa to 0.6 MPa, and those bubbles that did enter clots had reduced penetration depths and were associated with less fibrin network damage and nanobead uptake. This appeared to be due in large part to increased bubble shrinkage relative to the 1 ms pulse case. Stroboscopic imaging was performed on a subset of bubbles (n = 11) and indicated that complex bubble oscillations can occur during this process.
DOI
Microtubule C-terminal tails can change characteristics of motor force production
Mitra Shojania Feizabadi, Babu Reddy J N, Omid Vadpey, Yonggun Jun, Dail Chapman, Steven Rosenfeld and Steven P. Gross
Control of intracellular transport is poorly understood, and functional ramifications of tubulin isoform differences between cell types are mostly unexplored. Motors’ force production and detachment kinetics are critical for their group function, but how microtubule details affect these properties—if at all—are unknown. We investigated these questions using both a vesicular transport human kinesin, Kinesin-1 and also a mitotic kinesin likely optimized for group function, Kinesin-5, moving along either bovine brain or MCF7(breast cancer) microtubules. We found that kinesin-1 functioned similarly on the two sets of microtubules—in particular, its mean force production was approximately the same, though due to its previously reported decreased processivity, the mean duration of Kinesin-1 force production was slightly decreased on MCF7 MTs.
In contrast, Kinesin-5's function changed dramatically on MCF7 microtubules: its average detachment force was reduced and its force-velocity curve was different. In spite of the reduced detachment force, the force-velocity alteration surprisingly improved high-load group function for Kinesin-5 on the cancer-cell microtubules, potentially contributing to functions such as spindle-mediated chromosome separation. Significant differences were previously reported for C-terminal tubulin tails in MCF7 vs bovine brain tubulin. Consistent with this difference being functionally important, elimination of the tails made transport along the two sets of microtubules similar.
DOI
Control of intracellular transport is poorly understood, and functional ramifications of tubulin isoform differences between cell types are mostly unexplored. Motors’ force production and detachment kinetics are critical for their group function, but how microtubule details affect these properties—if at all—are unknown. We investigated these questions using both a vesicular transport human kinesin, Kinesin-1 and also a mitotic kinesin likely optimized for group function, Kinesin-5, moving along either bovine brain or MCF7(breast cancer) microtubules. We found that kinesin-1 functioned similarly on the two sets of microtubules—in particular, its mean force production was approximately the same, though due to its previously reported decreased processivity, the mean duration of Kinesin-1 force production was slightly decreased on MCF7 MTs.
In contrast, Kinesin-5's function changed dramatically on MCF7 microtubules: its average detachment force was reduced and its force-velocity curve was different. In spite of the reduced detachment force, the force-velocity alteration surprisingly improved high-load group function for Kinesin-5 on the cancer-cell microtubules, potentially contributing to functions such as spindle-mediated chromosome separation. Significant differences were previously reported for C-terminal tubulin tails in MCF7 vs bovine brain tubulin. Consistent with this difference being functionally important, elimination of the tails made transport along the two sets of microtubules similar.
DOI
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