Tran X. Phuoc, Mehrdad Massoudi and Ping Wang
Since a photon carries both energy and momentum, when it interacts with a particle, photon-particle energy and momentum transfer occur, resulting in mechanical forces acting on the particle. In this paper we report our theoretical study on the use of a laser beam to manipulate and control the flow of nanofluids in a micro-channel. We calculate the velocity induced by a laser beam for TiO2, Fe2O3, Al2O3 MgO, and SiO2 nanoparticles with water as the base fluid. The particle diameter is 50 nm and the laser beam is a 4 W continuous beam of 6 mm diameter and 532 nm wavelength. The results indicate that, as the particle moves, a significant volume of the surrounding water (up to about 8 particle diameters away from the particle surface) is disturbed and dragged along with the moving particle. The results also show the effect of the particle refractive index on the particle velocity and the induced volume flow rate. The velocity and the volume flowrate induced by the TiO2 nanoparticle (refractive index n = 2.82) are about 0.552 mm/s and 9.86 fL, respectively, while those induced by SiO2 (n = 1.46) are only about 7.569 μm/s and 0.135, respectively.
DOI
Tuesday, November 8, 2016
Monday, November 7, 2016
Alternative modes of client binding enable functional plasticity of Hsp70
Alireza Mashaghi, Sergey Bezrukavnikov, David P. Minde, Anne S. Wentink, Roman Kityk, Beate Zachmann-Brand, Matthias P. Mayer, Günter Kramer, Bernd Bukau & Sander J. Tans
The Hsp70 system is a central hub of chaperone activity in all domains of life. Hsp70 performs a plethora of tasks, including folding assistance, protection against aggregation, protein trafficking, and enzyme activity regulation1, 2, 3, 4, 5, and interacts with non-folded chains, as well as near-native, misfolded, and aggregated proteins6, 7, 8, 9, 10. Hsp70 is thought to achieve its many physiological roles by binding peptide segments that extend from these different protein conformers within a groove that can be covered by an ATP-driven helical lid11, 12, 13, 14, 15. However, it has been difficult to test directly how Hsp70 interacts with protein substrates in different stages of folding and how it affects their structure. Moreover, recent indications of diverse lid conformations in Hsp70–substrate complexes raise the possibility of additional interaction mechanisms15, 16, 17, 18. Addressing these issues is technically challenging, given the conformational dynamics of both chaperone and client, the transient nature of their interaction, and the involvement of co-chaperones and the ATP hydrolysis cycle19. Here, using optical tweezers, we show that the bacterial Hsp70 homologue (DnaK) binds and stabilizes not only extended peptide segments, but also partially folded and near-native protein structures. The Hsp70 lid and groove act synergistically when stabilizing folded structures: stabilization is abolished when the lid is truncated and less efficient when the groove is mutated. The diversity of binding modes has important consequences: Hsp70 can both stabilize and destabilize folded structures, in a nucleotide-regulated manner; like Hsp90 and GroEL, Hsp70 can affect the late stages of protein folding; and Hsp70 can suppress aggregation by protecting partially folded structures as well as unfolded protein chains. Overall, these findings in the DnaK system indicate an extension of the Hsp70 canonical model that potentially affects a wide range of physiological roles of the Hsp70 system.
DOI
The Hsp70 system is a central hub of chaperone activity in all domains of life. Hsp70 performs a plethora of tasks, including folding assistance, protection against aggregation, protein trafficking, and enzyme activity regulation1, 2, 3, 4, 5, and interacts with non-folded chains, as well as near-native, misfolded, and aggregated proteins6, 7, 8, 9, 10. Hsp70 is thought to achieve its many physiological roles by binding peptide segments that extend from these different protein conformers within a groove that can be covered by an ATP-driven helical lid11, 12, 13, 14, 15. However, it has been difficult to test directly how Hsp70 interacts with protein substrates in different stages of folding and how it affects their structure. Moreover, recent indications of diverse lid conformations in Hsp70–substrate complexes raise the possibility of additional interaction mechanisms15, 16, 17, 18. Addressing these issues is technically challenging, given the conformational dynamics of both chaperone and client, the transient nature of their interaction, and the involvement of co-chaperones and the ATP hydrolysis cycle19. Here, using optical tweezers, we show that the bacterial Hsp70 homologue (DnaK) binds and stabilizes not only extended peptide segments, but also partially folded and near-native protein structures. The Hsp70 lid and groove act synergistically when stabilizing folded structures: stabilization is abolished when the lid is truncated and less efficient when the groove is mutated. The diversity of binding modes has important consequences: Hsp70 can both stabilize and destabilize folded structures, in a nucleotide-regulated manner; like Hsp90 and GroEL, Hsp70 can affect the late stages of protein folding; and Hsp70 can suppress aggregation by protecting partially folded structures as well as unfolded protein chains. Overall, these findings in the DnaK system indicate an extension of the Hsp70 canonical model that potentially affects a wide range of physiological roles of the Hsp70 system.
DOI
Dynamic viscosity mapping of the oxidation of squalene aerosol particles
Athanasios Athanasiadis, Clare Fitzgerald, Nicholas M. Davidson, Chiara Giorio, Stanley W. Botchway, Andrew D. Ward, Markus Kalberer, Francis D. Pope and Marina K. Kuimova
Organic aerosols (OAs) play important roles in multiple atmospheric processes, including climate change, and can impact human health. The physico-chemical properties of OAs are important for all these processes and can evolve through reactions with various atmospheric components, including oxidants. The dynamic nature of these reactions makes it challenging to obtain a true representation of their composition and surface chemistry. Here we investigate the microscopic viscosity of the model OA composed of squalene, undergoing chemical aging. We employ Fluorescent Lifetime Imaging Microscopy (FLIM) in conjunction with viscosity sensitive probes termed molecular rotors, in order to image the changes in microviscosity in real time during oxidation with ozone and hydroxyl radicals, which are two key oxidising species in the troposphere. We also recorded the Raman spectra of the levitated particles to follow the reactivity during particle ozonolysis. The levitation of droplets was achieved via optical trapping that enabled simultaneous levitation and measurement via FLIM or Raman spectroscopy and allowed the true aerosol phase to be probed. Our data revealed a very significant increase in viscosity of the levitated squalene droplets upon ozonolysis, following their transformation from the liquid to solid phase that was not observable when the oxidation was carried out on coverslip mounted droplets. FLIM imaging with sub-micron spatial resolution also revealed spatial heterogeneity in the viscosity distribution of oxidised droplets. Overall, a combination of molecular rotors, FLIM and optical trapping is able to provide powerful insights into OA chemistry and the microscopic structure that enables the dynamic monitoring of microscopic viscosity in aerosol particles in their true phase.
DOI
Organic aerosols (OAs) play important roles in multiple atmospheric processes, including climate change, and can impact human health. The physico-chemical properties of OAs are important for all these processes and can evolve through reactions with various atmospheric components, including oxidants. The dynamic nature of these reactions makes it challenging to obtain a true representation of their composition and surface chemistry. Here we investigate the microscopic viscosity of the model OA composed of squalene, undergoing chemical aging. We employ Fluorescent Lifetime Imaging Microscopy (FLIM) in conjunction with viscosity sensitive probes termed molecular rotors, in order to image the changes in microviscosity in real time during oxidation with ozone and hydroxyl radicals, which are two key oxidising species in the troposphere. We also recorded the Raman spectra of the levitated particles to follow the reactivity during particle ozonolysis. The levitation of droplets was achieved via optical trapping that enabled simultaneous levitation and measurement via FLIM or Raman spectroscopy and allowed the true aerosol phase to be probed. Our data revealed a very significant increase in viscosity of the levitated squalene droplets upon ozonolysis, following their transformation from the liquid to solid phase that was not observable when the oxidation was carried out on coverslip mounted droplets. FLIM imaging with sub-micron spatial resolution also revealed spatial heterogeneity in the viscosity distribution of oxidised droplets. Overall, a combination of molecular rotors, FLIM and optical trapping is able to provide powerful insights into OA chemistry and the microscopic structure that enables the dynamic monitoring of microscopic viscosity in aerosol particles in their true phase.
DOI
Single-Molecule Optical-Trapping Techniques to Study Molecular Mechanisms of a Replisome
B. Sun, M.D. Wang
The replisome is a multiprotein molecular machinery responsible for the replication of DNA. It is composed of several specialized proteins each with dedicated enzymatic activities, and in particular, helicase unwinds double-stranded DNA and DNA polymerase catalyzes the synthesis of DNA. Understanding how a replisome functions in the process of DNA replication requires methods to dissect the mechanisms of individual proteins and of multiproteins acting in concert. Single-molecule optical-trapping techniques have proved to be a powerful approach, offering the unique ability to observe and manipulate biomolecules at the single-molecule level and providing insights into the mechanisms of molecular motors and their interactions and coordination in a complex. Here, we describe a practical guide to applying these techniques to study the dynamics of individual proteins in the bacteriophage T7 replisome, as well as the coordination among them. We also summarize major findings from these studies, including nucleotide-specific helicase slippage and new lesion bypass pathway in T7 replication.
DOI
The replisome is a multiprotein molecular machinery responsible for the replication of DNA. It is composed of several specialized proteins each with dedicated enzymatic activities, and in particular, helicase unwinds double-stranded DNA and DNA polymerase catalyzes the synthesis of DNA. Understanding how a replisome functions in the process of DNA replication requires methods to dissect the mechanisms of individual proteins and of multiproteins acting in concert. Single-molecule optical-trapping techniques have proved to be a powerful approach, offering the unique ability to observe and manipulate biomolecules at the single-molecule level and providing insights into the mechanisms of molecular motors and their interactions and coordination in a complex. Here, we describe a practical guide to applying these techniques to study the dynamics of individual proteins in the bacteriophage T7 replisome, as well as the coordination among them. We also summarize major findings from these studies, including nucleotide-specific helicase slippage and new lesion bypass pathway in T7 replication.
DOI
Direct Visualization of Helicase Dynamics Using Fluorescence Localization and Optical Trapping
C.-T. Lin, T. Ha
Helicases control the accessibility of single-stranded (ss) nucleic acid (NA) generated as a transient intermediate during almost every step in cells related to nucleic acid metabolisms. For subsequent processing, however, helicases need to adjust the pace of unwinding adequately to avoid ssNA exposure to nucleases. Therefore, understanding how the unwinding process of helicases is regulated is crucial to address genome integrity and repair mechanisms. Using single-molecule fluorescence-force spectroscopy with fluorescence localization, we recently observed the stoichiometry of UvrD helicase, which determines the functions of UvrD: translocation and unwinding. For the first time, we provide direct evidence that a UvrD dimer is required to initiate the unwinding pathway. Moreover, with subpixel precision of fluorescence localization, the dynamic parameters of helicases can be obtained directly. Here, we present detailed single-molecule assays for observing the biochemical activities of helicases in real time and revealing how mechanical forces are involved in protein–nucleic acid interactions. These single-molecule approaches are generally applicable to many other protein–nucleic acid systems.
DOI
Helicases control the accessibility of single-stranded (ss) nucleic acid (NA) generated as a transient intermediate during almost every step in cells related to nucleic acid metabolisms. For subsequent processing, however, helicases need to adjust the pace of unwinding adequately to avoid ssNA exposure to nucleases. Therefore, understanding how the unwinding process of helicases is regulated is crucial to address genome integrity and repair mechanisms. Using single-molecule fluorescence-force spectroscopy with fluorescence localization, we recently observed the stoichiometry of UvrD helicase, which determines the functions of UvrD: translocation and unwinding. For the first time, we provide direct evidence that a UvrD dimer is required to initiate the unwinding pathway. Moreover, with subpixel precision of fluorescence localization, the dynamic parameters of helicases can be obtained directly. Here, we present detailed single-molecule assays for observing the biochemical activities of helicases in real time and revealing how mechanical forces are involved in protein–nucleic acid interactions. These single-molecule approaches are generally applicable to many other protein–nucleic acid systems.
DOI
Numerical analysis of an optical nanoscale particles trapping device based on a slotted nanobeam cavity
Senlin Zhang, Zhengdong Yong, Yaocheng Shi & Sailing He
A slotted nanobeam cavity (SNC) is utilized to trap a polystyrene (PS) particle with a radius of only 2 nm. The carefully designed SNC shows an ultrahigh Q factor of 4.5 × 107 while maintaining a small mode volume of 0.067(λ/nwater)3. Strongly enhanced optical trapping force is numerically demonstrated when the 2 nm PS particle is introduced into the central, slotted part of the SNC. In the vertical direction, the numerical calculation results show that a trapping stiffness of 0.4 pN/(nm · mW) around the equilibrium position and a trapping potential barrier of ~2000 kBT/mW can be reached. To our best knowledge, the trapping capability (trapping stiffness and trapping potential barrier) of the proposed structure significantly outperforms the theoretical results of those in previously reported work. In addition, the SNC system does not suffer from the metal induced heat issue that restricts the performance of state-of-the-art optical trapping systems involving plasmonic enhancement. Based on the proposed cavity, applications such as lab-on-a-chip platforms for nanoscale particle trapping and analysis can be expected in future.
DOI
A slotted nanobeam cavity (SNC) is utilized to trap a polystyrene (PS) particle with a radius of only 2 nm. The carefully designed SNC shows an ultrahigh Q factor of 4.5 × 107 while maintaining a small mode volume of 0.067(λ/nwater)3. Strongly enhanced optical trapping force is numerically demonstrated when the 2 nm PS particle is introduced into the central, slotted part of the SNC. In the vertical direction, the numerical calculation results show that a trapping stiffness of 0.4 pN/(nm · mW) around the equilibrium position and a trapping potential barrier of ~2000 kBT/mW can be reached. To our best knowledge, the trapping capability (trapping stiffness and trapping potential barrier) of the proposed structure significantly outperforms the theoretical results of those in previously reported work. In addition, the SNC system does not suffer from the metal induced heat issue that restricts the performance of state-of-the-art optical trapping systems involving plasmonic enhancement. Based on the proposed cavity, applications such as lab-on-a-chip platforms for nanoscale particle trapping and analysis can be expected in future.
DOI
Tuesday, November 1, 2016
Characterization of drug effect on leukemia cells through single cell assay with optical tweezers and dielectrophoresis
Jundi Hou ; Tao Luo ; Ka Lam Ng ; Raymond Liang ; Anskar Y. H. Leung ; Dong Sun
One of the greatest challenges in acute myeloid leukemia (AML) treatment is preventing relapse. Leukemia cells can hide in bone marrow niche or vascular niche. Hence, many chemical drugs cannot kill these cells. To characterize migration and adhesion properties of leukemia cells in specific niches, CXCR4/SDF-1α signal pathway has been widely used for investigation. AMD3100 is treated as one of the most common chemical drugs that can inhibit this signal. In the current study, we particularly investigate the effect of AMD3100 on the adhesion property of leukemia cells on stromal cells by using engineering tools, namely, optical tweezers (OT) and dielectrophoresis (DEP), to probe single cell property. AMD3100 not only inhibits the CXCR4/SDF-1α signal pathway but also reduces gene expression of CXCR4 and VLA4 on leukemia cells. The drug also softens leukemia cells. This work provides a new way to investigate cell behavior under drug treatment. The use of combined engineering tools will benefit drug discovery and assessment for leukemia treatment.
DOI
One of the greatest challenges in acute myeloid leukemia (AML) treatment is preventing relapse. Leukemia cells can hide in bone marrow niche or vascular niche. Hence, many chemical drugs cannot kill these cells. To characterize migration and adhesion properties of leukemia cells in specific niches, CXCR4/SDF-1α signal pathway has been widely used for investigation. AMD3100 is treated as one of the most common chemical drugs that can inhibit this signal. In the current study, we particularly investigate the effect of AMD3100 on the adhesion property of leukemia cells on stromal cells by using engineering tools, namely, optical tweezers (OT) and dielectrophoresis (DEP), to probe single cell property. AMD3100 not only inhibits the CXCR4/SDF-1α signal pathway but also reduces gene expression of CXCR4 and VLA4 on leukemia cells. The drug also softens leukemia cells. This work provides a new way to investigate cell behavior under drug treatment. The use of combined engineering tools will benefit drug discovery and assessment for leukemia treatment.
DOI
Cooperative Optical Trapping and Manipulation of Multiple Cells With Robot Tweezers
Xiang Li; Chien Chern Cheah; Quang Minh Ta
While several control schemes and automation techniques have been proposed for coordination and manipulation of multiple cells using optical tweezers, open-loop control methods are always utilized by treating the positions of lasers as the control inputs instead of feedback variables. As the positions of laser beams are not utilized in feedback control, it is, therefore, assumed in the literature that the multiple cells are always trapped by the laser beams throughout the manipulation task. However, the control techniques fail when the cells are not initially trapped by the laser beams, or when the laser beams move too fast, such that the cells escape from the traps during manipulation. This paper presents a closed-loop control formulation and strategy for optical manipulation of multiple cells, based on the cooperative movement of the motorized stage and the beam steering system. The closed-loop control method enables the trapping operation to be automatically activated whenever the cells are not inside the optical traps. The proposed controller does not require exact knowledge on the dynamic parameters and the varying trapping stiffness. Lyapunov methods are employed in the stability analysis of the optical tweezers system. Experimental results are presented to illustrate the performance of the proposed controller.
DOI
While several control schemes and automation techniques have been proposed for coordination and manipulation of multiple cells using optical tweezers, open-loop control methods are always utilized by treating the positions of lasers as the control inputs instead of feedback variables. As the positions of laser beams are not utilized in feedback control, it is, therefore, assumed in the literature that the multiple cells are always trapped by the laser beams throughout the manipulation task. However, the control techniques fail when the cells are not initially trapped by the laser beams, or when the laser beams move too fast, such that the cells escape from the traps during manipulation. This paper presents a closed-loop control formulation and strategy for optical manipulation of multiple cells, based on the cooperative movement of the motorized stage and the beam steering system. The closed-loop control method enables the trapping operation to be automatically activated whenever the cells are not inside the optical traps. The proposed controller does not require exact knowledge on the dynamic parameters and the varying trapping stiffness. Lyapunov methods are employed in the stability analysis of the optical tweezers system. Experimental results are presented to illustrate the performance of the proposed controller.
DOI
Potentialities of laser trapping and manipulation of blood cells in hemorheologic research
Priezzhev, A.; Lee, K.
Laser trapping and manipulation of blood cells without mechanical contact have become feasible with implication of laser tweezers. They open up new horizons for the hemorheologic researches, offer new possibilities for studying live cells interactions on individual cell level under the influence of different endogenous and exogenous factors. The operation principle of laser tweezers is based on the property of strongly focused laser beam to act on a dielectric microparticle located in the vicinity of the beam waist with a force that drives the particle to the equilibrium location and holds it there. If the beam waist position is manipulated, so is the position of the particle. The displacement of the particle from the equilibrium position by external forces can be calibrated so that these forces can be precisely measured in the range ca. 0.1–100 pN. This is the range of forces of elastic deformation of blood cells and of their interaction with each other and with vessel walls. Being able to measure these forces without mechanical contact allows for studying on single cell level the mechanisms of interactions that was impossible earlier. Here we discuss the basic features of these techniques and give some examples of challenging hemorheologic studies.
DOI
Laser trapping and manipulation of blood cells without mechanical contact have become feasible with implication of laser tweezers. They open up new horizons for the hemorheologic researches, offer new possibilities for studying live cells interactions on individual cell level under the influence of different endogenous and exogenous factors. The operation principle of laser tweezers is based on the property of strongly focused laser beam to act on a dielectric microparticle located in the vicinity of the beam waist with a force that drives the particle to the equilibrium location and holds it there. If the beam waist position is manipulated, so is the position of the particle. The displacement of the particle from the equilibrium position by external forces can be calibrated so that these forces can be precisely measured in the range ca. 0.1–100 pN. This is the range of forces of elastic deformation of blood cells and of their interaction with each other and with vessel walls. Being able to measure these forces without mechanical contact allows for studying on single cell level the mechanisms of interactions that was impossible earlier. Here we discuss the basic features of these techniques and give some examples of challenging hemorheologic studies.
DOI
Nucleosomal arrangement affects single-molecule transcription dynamics
Veronika Fitz, Jaeoh Shin, Christoph Ehrlich, Lucas Farnung, Patrick Cramer, Vasily Zaburdaev, and Stephan W. Grill
In eukaryotes, gene expression depends on chromatin organization. However, how chromatin affects the transcription dynamics of individual RNA polymerases has remained elusive. Here, we use dual trap optical tweezers to study single yeast RNA polymerase II (Pol II) molecules transcribing along a DNA template with two nucleosomes. The slowdown and the changes in pausing behavior within the nucleosomal region allow us to determine a drift coefficient, χ, which characterizes the ability of the enzyme to recover from a nucleosomal backtrack. Notably, χ can be used to predict the probability to pass the first nucleosome. Importantly, the presence of a second nucleosome changes χ in a manner that depends on the spacing between the two nucleosomes, as well as on their rotational arrangement on the helical DNA molecule. Our results indicate that the ability of Pol II to pass the first nucleosome is increased when the next nucleosome is turned away from the first one to face the opposite side of the DNA template. These findings help to rationalize how chromatin arrangement affects Pol II transcription dynamics.
DOI
In eukaryotes, gene expression depends on chromatin organization. However, how chromatin affects the transcription dynamics of individual RNA polymerases has remained elusive. Here, we use dual trap optical tweezers to study single yeast RNA polymerase II (Pol II) molecules transcribing along a DNA template with two nucleosomes. The slowdown and the changes in pausing behavior within the nucleosomal region allow us to determine a drift coefficient, χ, which characterizes the ability of the enzyme to recover from a nucleosomal backtrack. Notably, χ can be used to predict the probability to pass the first nucleosome. Importantly, the presence of a second nucleosome changes χ in a manner that depends on the spacing between the two nucleosomes, as well as on their rotational arrangement on the helical DNA molecule. Our results indicate that the ability of Pol II to pass the first nucleosome is increased when the next nucleosome is turned away from the first one to face the opposite side of the DNA template. These findings help to rationalize how chromatin arrangement affects Pol II transcription dynamics.
DOI
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