Nima Khatibzadeh , Sharad Gupta , Brenda Farrell , William E. Brownell and Bahman Anvari
In this study, we investigated the effects of membrane cholesterol content on the mechanical properties of cell membranes by using optical tweezers. We pulled membrane tethers from human embryonic kidney cells using single and multi-speed protocols, and obtained time-resolved tether forces. We quantified various mechanical characteristics including the tether equilibrium force, bending modulus, effective membrane viscosity, and plasma membrane–cytoskeleton adhesion energy, and correlated them to the membrane cholesterol level. Decreases in cholesterol concentration were associated with increases in the tether equilibrium force, tether stiffness, and adhesion energy. Tether diameter and effective viscosity increased with increasing cholesterol levels. Disruption of cytoskeletal F-actin significantly changed the tether diameters in both non-cholesterol and cholesterol-manipulated cells, while the effective membrane viscosity was unaffected by F-actin disruption. The findings are relevant to inner ear function where cochlear amplification is altered by changes in the membrane cholesterol content.
DOI
Tuesday, July 10, 2012
Calcium-dependent folding of single calmodulin molecules
Johannes Stigler and Matthias Rief
Calmodulin is the primary calcium binding protein in living cells. Its function and structure depend strongly on calcium concentration. We used single molecule force spectroscopy by optical tweezers to study the folding of calmodulin in the physiologically relevant range. We find that full-length calmodulin switches from a rich and complex folding behavior at high calcium to a simple folding pathway at apo conditions. Using truncation mutants, we studied the individual domains separately. Folding and stability of the individual domains differ significantly at low calcium concentrations. With increasing calcium, the folding rate constants increase while unfolding rate constants decrease. The complete kinetic as well as energetic behavior of both domains could be modeled using a calcium-dependent three-pathway model. We find that the dominant folding pathway at high calcium concentrations proceeds via a transition state capable of binding one calcium ion. The folding of calmodulin seems to be designed to occur fast robustly over a large range of calcium concentrations and hence energetic stabilities.
Calmodulin is the primary calcium binding protein in living cells. Its function and structure depend strongly on calcium concentration. We used single molecule force spectroscopy by optical tweezers to study the folding of calmodulin in the physiologically relevant range. We find that full-length calmodulin switches from a rich and complex folding behavior at high calcium to a simple folding pathway at apo conditions. Using truncation mutants, we studied the individual domains separately. Folding and stability of the individual domains differ significantly at low calcium concentrations. With increasing calcium, the folding rate constants increase while unfolding rate constants decrease. The complete kinetic as well as energetic behavior of both domains could be modeled using a calcium-dependent three-pathway model. We find that the dominant folding pathway at high calcium concentrations proceeds via a transition state capable of binding one calcium ion. The folding of calmodulin seems to be designed to occur fast robustly over a large range of calcium concentrations and hence energetic stabilities.
Monday, July 9, 2012
Comparing the mechanism of water condensation and evaporation in glassy aerosol
David L. Bones, Jonathan P. Reid, Daniel M. Lienhard, and Ulrich K. Krieger
Atmospheric models generally assume that aerosol particles are in equilibrium with the surrounding gas phase. However, recent observations that secondary organic aerosols can exist in a glassy state have highlighted the need to more fully understand the kinetic limitations that may control water partitioning in ambient particles. Here, we explore the influence of slow water diffusion in the condensed aerosol phase on the rates of both condensation and evaporation, demonstrating that significant inhibition in mass transfer occurs for ultraviscous aerosol, not just for glassy aerosol. Using coarse mode (3–4 um radius) ternary sucrose/sodium chloride/aqueous droplets as a proxy for multicomponent ambient aerosol, we demonstrate that the timescale for particle equilibration correlates with bulk viscosity and can be ≫103 s. Extrapolation of these timescales to particle sizes in the accumulation mode (e.g., approximately 100 nm) by applying the Stokes-Einstein equation suggests that the kinetic limitations imposed on mass transfer of water by slow bulk phase diffusion must be more fully investigated for atmospheric aerosol. Measurements have been made on particles covering a range in dynamic viscosity from < 0.1 to > 1013 Pa s. We also retrieve the radial inhomogeneities apparent in particle composition during condensation and evaporation and contrast the dynamics of slow dissolution of a viscous core into a labile shell during condensation with the slow percolation of water during evaporation through a more homogeneous viscous particle bulk.
DOI
Atmospheric models generally assume that aerosol particles are in equilibrium with the surrounding gas phase. However, recent observations that secondary organic aerosols can exist in a glassy state have highlighted the need to more fully understand the kinetic limitations that may control water partitioning in ambient particles. Here, we explore the influence of slow water diffusion in the condensed aerosol phase on the rates of both condensation and evaporation, demonstrating that significant inhibition in mass transfer occurs for ultraviscous aerosol, not just for glassy aerosol. Using coarse mode (3–4 um radius) ternary sucrose/sodium chloride/aqueous droplets as a proxy for multicomponent ambient aerosol, we demonstrate that the timescale for particle equilibration correlates with bulk viscosity and can be ≫103 s. Extrapolation of these timescales to particle sizes in the accumulation mode (e.g., approximately 100 nm) by applying the Stokes-Einstein equation suggests that the kinetic limitations imposed on mass transfer of water by slow bulk phase diffusion must be more fully investigated for atmospheric aerosol. Measurements have been made on particles covering a range in dynamic viscosity from < 0.1 to > 1013 Pa s. We also retrieve the radial inhomogeneities apparent in particle composition during condensation and evaporation and contrast the dynamics of slow dissolution of a viscous core into a labile shell during condensation with the slow percolation of water during evaporation through a more homogeneous viscous particle bulk.
DOI
Force-Dependent Detachment of Kinesin-2 Biases Track Switching at Cytoskeletal Filament Intersections
Harry W. Schroeder III, Adam G. Hendricks, Kazuho Ikeda, Henry Shuman, Vladimir Rodionov, Mitsuo Ikebe, Yale E. Goldman, Erika L.F. Holzbaur
Intracellular trafficking of organelles often involves cytoskeletal track switching. Organelles such as melanosomes are transported by multiple motors including kinesin-2, dynein, and myosin-V, which drive switching between microtubules and actin filaments during dispersion and aggregation. Here, we used optical trapping to determine the unitary and ensemble forces of kinesin-2, and to reconstitute cargo switching at cytoskeletal intersections in a minimal system with kinesin-2 and myosin-V motors bound to beads. Single kinesin-2 motors exerted forces up to ∼5 pN, similar to kinesin-1. However, kinesin-2 motors were more likely to detach at submaximal forces, and the duration of force maintenance was short as compared to kinesin-1. In multimotor assays, force increased with kinesin-2 density but was not affected by the presence of myosin-V. In crossed filament assays, switching frequencies of motor-bound beads were dependent on the starting track. At equal average forces, beads tended to switch from microtubules onto overlying actin filaments consistent with the relatively faster detachment of kinesin-2 at near-maximal forces. Thus, in addition to relative force, switching probability at filament intersections is determined by the dynamics of motor-filament interaction, such as the quick detachment of kinesin-2 under load. This may enable fine-tuning of filament switching in the cell.
DOI
Intracellular trafficking of organelles often involves cytoskeletal track switching. Organelles such as melanosomes are transported by multiple motors including kinesin-2, dynein, and myosin-V, which drive switching between microtubules and actin filaments during dispersion and aggregation. Here, we used optical trapping to determine the unitary and ensemble forces of kinesin-2, and to reconstitute cargo switching at cytoskeletal intersections in a minimal system with kinesin-2 and myosin-V motors bound to beads. Single kinesin-2 motors exerted forces up to ∼5 pN, similar to kinesin-1. However, kinesin-2 motors were more likely to detach at submaximal forces, and the duration of force maintenance was short as compared to kinesin-1. In multimotor assays, force increased with kinesin-2 density but was not affected by the presence of myosin-V. In crossed filament assays, switching frequencies of motor-bound beads were dependent on the starting track. At equal average forces, beads tended to switch from microtubules onto overlying actin filaments consistent with the relatively faster detachment of kinesin-2 at near-maximal forces. Thus, in addition to relative force, switching probability at filament intersections is determined by the dynamics of motor-filament interaction, such as the quick detachment of kinesin-2 under load. This may enable fine-tuning of filament switching in the cell.
DOI
How Malaria Parasites Reduce the Deformability of Infected Red Blood Cells
S. Majid Hosseini, James J. Feng
The pathogenesis of malaria is largely due to stiffening of the infected red blood cells (RBCs). Contemporary understanding ascribes the loss of RBC deformability to a 10-fold increase in membrane stiffness caused by extra cross-linking in the spectrin network. Local measurements by micropipette aspiration, however, have reported only an increase of ∼3-fold in the shear modulus. We believe the discrepancy stems from the rigid parasite particles inside infected cells, and have carried out numerical simulations to demonstrate this mechanism. The cell membrane is represented by a set of discrete particles connected by linearly elastic springs. The cytosol is modeled as a homogeneous Newtonian fluid, and discretized by particles as in standard smoothed particle hydrodynamics. The malaria parasite is modeled as an aggregate of particles constrained to rigid-body motion. We simulate RBC stretching tests by optical tweezers in three dimensions. The results demonstrate that the presence of a sizeable parasite greatly reduces the ability of RBCs to deform under stretching. With the solid inclusion, the observed loss of deformability can be predicted quantitatively using the local membrane elasticity measured by micropipettes.
DOI
The pathogenesis of malaria is largely due to stiffening of the infected red blood cells (RBCs). Contemporary understanding ascribes the loss of RBC deformability to a 10-fold increase in membrane stiffness caused by extra cross-linking in the spectrin network. Local measurements by micropipette aspiration, however, have reported only an increase of ∼3-fold in the shear modulus. We believe the discrepancy stems from the rigid parasite particles inside infected cells, and have carried out numerical simulations to demonstrate this mechanism. The cell membrane is represented by a set of discrete particles connected by linearly elastic springs. The cytosol is modeled as a homogeneous Newtonian fluid, and discretized by particles as in standard smoothed particle hydrodynamics. The malaria parasite is modeled as an aggregate of particles constrained to rigid-body motion. We simulate RBC stretching tests by optical tweezers in three dimensions. The results demonstrate that the presence of a sizeable parasite greatly reduces the ability of RBCs to deform under stretching. With the solid inclusion, the observed loss of deformability can be predicted quantitatively using the local membrane elasticity measured by micropipettes.
DOI
A non-contact single optical fiber multi-optical tweezers probe: Design and fabrication
Yu Zhang, Zhihai Liu, Jun Yang, Libo Yuan
Optical tweezers have developed into a versatile and widely used tool; compared with the traditional optical tweezers, optical traps built with a single optical fiber provide a more flexible solution towards compact, integrated multiple traps. But the single optical tweezers have their own short comings—the manipulation working on the micro-particles is not non-contact, because of the fabrication method of the optical fiber probe. However we propose new single fiber multi-optical tweezers which are simple, low-cost and can manipulate micro-particles in a non-contact manner and non-invasively.
DOI
Optical tweezers have developed into a versatile and widely used tool; compared with the traditional optical tweezers, optical traps built with a single optical fiber provide a more flexible solution towards compact, integrated multiple traps. But the single optical tweezers have their own short comings—the manipulation working on the micro-particles is not non-contact, because of the fabrication method of the optical fiber probe. However we propose new single fiber multi-optical tweezers which are simple, low-cost and can manipulate micro-particles in a non-contact manner and non-invasively.
DOI
Saturday, July 7, 2012
Electromagnetic plane-wave forces on homogeneous material
Kevin J. Webb and Shivanand
The electromagnetic plane-wave force on homogeneous materials having the possible set of constitutive parameter signs is evaluated. The force remains positive in all cases with loss, but a negative force results with gain. A negative force is shown to occur for an evanescent field when there is no component of the electric field in that direction. Both forces have significant magnitude and hence should be observable in experiments.
DOI
The electromagnetic plane-wave force on homogeneous materials having the possible set of constitutive parameter signs is evaluated. The force remains positive in all cases with loss, but a negative force results with gain. A negative force is shown to occur for an evanescent field when there is no component of the electric field in that direction. Both forces have significant magnitude and hence should be observable in experiments.
DOI
Calculation of radiation force and torque exerted on a uniaxial anisotropic sphere by an incident Gaussian beam with arbitrary propagation and polarization directions
Zheng-Jun Li, Zhen-Sen Wu, Qing-Chao Shang, Lu Bai, and Chun-Hui Cao
On the basis of spherical vector wave functions and coordinate rotation theory, the expansion of the fields of an incident Gaussian beam with arbitrary propagation and polarization directions in terms of spherical vector wave functions is investigated, and beam shape coefficients are derived. Using the results of electromagnetic scattering by a uniaxial anisotropic sphere, the analytical expressions of the radiation force and torque exerted on a homogeneous absorbing uniaxial anisotropic sphere by the arbitrary incident Gaussian beam. We numerically analyze and discuss the following: the effects of an anisotropic absorbing dielectric on the axial and transverse radiation forces exerted by an off-axis Gaussian beam on a uniaxial anisotropic sphere; the variations in the axial, transverse, and resultant radiation forces (with incident angle β and polarization angle α) imposed by an obliquely Gaussian beam on a uniaxial anisotropic sphere; and the results on the characteristics of the three components of the radiation forces versus the center-to-center distance between the sphere and beam. Selected numerically results on the radiation torque exerted on a stationary uniaxial anisotropic transparent or absorbing sphere by a linearly polarized Gaussian beam are shown, and the results are compared with those exerted an isotropic sphere. The accuracy of the theory and code is confirmed by comparing the axial radiation forces with the results obtained from references.
DOI
On the basis of spherical vector wave functions and coordinate rotation theory, the expansion of the fields of an incident Gaussian beam with arbitrary propagation and polarization directions in terms of spherical vector wave functions is investigated, and beam shape coefficients are derived. Using the results of electromagnetic scattering by a uniaxial anisotropic sphere, the analytical expressions of the radiation force and torque exerted on a homogeneous absorbing uniaxial anisotropic sphere by the arbitrary incident Gaussian beam. We numerically analyze and discuss the following: the effects of an anisotropic absorbing dielectric on the axial and transverse radiation forces exerted by an off-axis Gaussian beam on a uniaxial anisotropic sphere; the variations in the axial, transverse, and resultant radiation forces (with incident angle β and polarization angle α) imposed by an obliquely Gaussian beam on a uniaxial anisotropic sphere; and the results on the characteristics of the three components of the radiation forces versus the center-to-center distance between the sphere and beam. Selected numerically results on the radiation torque exerted on a stationary uniaxial anisotropic transparent or absorbing sphere by a linearly polarized Gaussian beam are shown, and the results are compared with those exerted an isotropic sphere. The accuracy of the theory and code is confirmed by comparing the axial radiation forces with the results obtained from references.
DOI
Friday, July 6, 2012
Phase, Morphology, and Hygroscopicity of Mixed Oleic Acid/Sodium Chloride/Water Aerosol Particles before and after Ozonolysis
Benjamin J. Dennis-Smither, Kate L. Hanford, Nana-Owusua A. Kwamena, Rachael E. H. Miles, and Jonathan P. Reid
Aerosol optical tweezers are used to probe the phase, morphology, and hygroscopicity of single aerosol particles consisting of an inorganic component, sodium chloride, and a water insoluble organic component, oleic acid. Coagulation of oleic acid aerosol with an optically trapped aqueous sodium chloride droplet leads to formation of a phase-separated particle with two partially engulfed liquid phases. The dependence of the phase and morphology of the trapped particle with variation in relative humidity (RH) is investigated by cavity enhanced Raman spectroscopy over the RH range <5% to >95%. The efflorescence and deliquescence behavior of the inorganic component is shown to be unaffected by the presence of the organic phase. Whereas efflorescence occurs promptly (<1 s), the deliquescence process requires both dissolution of the inorganic component and the adoption of an equilibrium morphology for the resulting two phase particle, occurring on a time-scale of <20 s. Comparative measurements of the hygroscopicity of mixed aqueous sodium chloride/oleic acid droplets with undoped aqueous sodium chloride droplets show that the oleic acid does not impact on the equilibration partitioning of water between the inorganic component and the gas phase or the time response of evaporation/condensation. The oxidative aging of the particles through reaction with ozone is shown to increase the hygroscopicity of the organic component.
DOI
DOI
Tomographic imaging of a suspending single live cell using optical tweezer-combined full-field optical coherence tomography
Woo June Choi, Kwan Seob Park, Tae Joong Eom, Myung-Kyu Oh, and Byeong Ha Lee
We propose a label-free depth-resolved tomographic scheme for imaging a single live cell in fluid. This approach utilizes a modified time-domain full-field optical coherence tomography (FF-OCT) system combined with an optical tweezer technique. The optical trap for holding a moving specimen is made by tightly focusing a 1064 nm Q-switching pulsed laser beam with a 1.0 NA microscope objective in the sample arm of the FF-OCT part. By cosharing the probe for both systems, the optical actions of trapping and cellular resolution tomographic imaging could be achieved simultaneously. Feasibility of the combined system is demonstrated by imaging micron-sized polystyrene beads and a living suspension cell in medium.
DOI
We propose a label-free depth-resolved tomographic scheme for imaging a single live cell in fluid. This approach utilizes a modified time-domain full-field optical coherence tomography (FF-OCT) system combined with an optical tweezer technique. The optical trap for holding a moving specimen is made by tightly focusing a 1064 nm Q-switching pulsed laser beam with a 1.0 NA microscope objective in the sample arm of the FF-OCT part. By cosharing the probe for both systems, the optical actions of trapping and cellular resolution tomographic imaging could be achieved simultaneously. Feasibility of the combined system is demonstrated by imaging micron-sized polystyrene beads and a living suspension cell in medium.
DOI
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