Single molecule optical trapping assays have now been applied to a great number of macromolecular systems including DNA, RNA, cargo motors, restriction enzymes, DNA helicases, chromosome remodelers, DNA polymerases and both viral and bacterial RNA polymerases. The advantages of the technique are the ability to observe dynamic, unsynchronized molecular processes, to determine the distributions of experimental quantities and to apply force to the system while monitoring the response over time. Here, we describe the application of these powerful techniques to study the dynamics of transcription elongation by RNA polymerase II from Saccharomyces cerevisiae.
Monday, August 31, 2009
Single molecule transcription elongation
Eric A. Galburt, Stephan W. Grill and Carlos Bustamante
Single molecule optical trapping assays have now been applied to a great number of macromolecular systems including DNA, RNA, cargo motors, restriction enzymes, DNA helicases, chromosome remodelers, DNA polymerases and both viral and bacterial RNA polymerases. The advantages of the technique are the ability to observe dynamic, unsynchronized molecular processes, to determine the distributions of experimental quantities and to apply force to the system while monitoring the response over time. Here, we describe the application of these powerful techniques to study the dynamics of transcription elongation by RNA polymerase II from Saccharomyces cerevisiae.
Single molecule optical trapping assays have now been applied to a great number of macromolecular systems including DNA, RNA, cargo motors, restriction enzymes, DNA helicases, chromosome remodelers, DNA polymerases and both viral and bacterial RNA polymerases. The advantages of the technique are the ability to observe dynamic, unsynchronized molecular processes, to determine the distributions of experimental quantities and to apply force to the system while monitoring the response over time. Here, we describe the application of these powerful techniques to study the dynamics of transcription elongation by RNA polymerase II from Saccharomyces cerevisiae.
Protein Friction Limits Diffusive and Directed Movements of Kinesin Motors on Microtubules
Friction limits the operation of macroscopic engines and is critical to the performance of micromechanical devices. We report measurements of friction in a biological nanomachine. Using optical tweezers, we characterized the frictional drag force of individual kinesin-8 motor proteins interacting with their microtubule tracks. At low speeds and with no energy source,the frictional drag was related to the diffusion coefficient by the Einstein relation. At higher speeds, the frictional drag force increased nonlinearly, consistent with the motor jumping 8 nanometers between adjacent tubulin dimers along the microtubule, and was asymmetric, reflecting the structural polarity of the microtubule. We argue that these frictional forces arise from breaking bonds between the motor domains and the microtubule, and they limit the speed and efficiency of kinesin.
Friday, August 28, 2009
Variety in intracellular diffusion during the cell cycle
Christine Selhuber-Unkel, Pernille Yde, Kirstine Berg-Sørensen and Lene B Oddershede
During the cell cycle, the organization of the cytoskeletal network undergoes dramatic changes. In order to reveal possible changes of the viscoelastic properties in the intracellular space during the cell cycle we investigated the diffusion of endogenous lipid granules within the fission yeast Schizosaccharomyces Pombeusing optical tweezers. The cell cycle was divided into interphase and mitotic cell division, and the mitotic cell division was further subdivided in its stages. During all stages of the cell cycle, the granules predominantly underwent subdiffusive motion, characterized by an exponent α that is also linked to the viscoelastic moduli of the cytoplasm. The exponent α was significantly smaller during interphase than during any stage of the mitotic cell division, signifying that the cytoplasm was more elastic during interphase than during division. We found no significant differences in the subdiffusive exponents from granules measured in different stages of cell division. Also, our results for the exponent displayed no significant dependence on the position of the granule within the cell. The observation that the cytoplasm is more elastic during interphase than during mitotic cell division is consistent with the fact that elastic cytoskeletal elements such as microtubules are less abundantly present during cell division than during interphase.
During the cell cycle, the organization of the cytoskeletal network undergoes dramatic changes. In order to reveal possible changes of the viscoelastic properties in the intracellular space during the cell cycle we investigated the diffusion of endogenous lipid granules within the fission yeast Schizosaccharomyces Pombeusing optical tweezers. The cell cycle was divided into interphase and mitotic cell division, and the mitotic cell division was further subdivided in its stages. During all stages of the cell cycle, the granules predominantly underwent subdiffusive motion, characterized by an exponent α that is also linked to the viscoelastic moduli of the cytoplasm. The exponent α was significantly smaller during interphase than during any stage of the mitotic cell division, signifying that the cytoplasm was more elastic during interphase than during division. We found no significant differences in the subdiffusive exponents from granules measured in different stages of cell division. Also, our results for the exponent displayed no significant dependence on the position of the granule within the cell. The observation that the cytoplasm is more elastic during interphase than during mitotic cell division is consistent with the fact that elastic cytoskeletal elements such as microtubules are less abundantly present during cell division than during interphase.
Simulation and analysis of single-ribosome translation
Ignacio Tinoco Jr and Jin-Der Wen
In the cell, proteins are synthesized by ribosomes in a multi-step process called translation. The ribosome translocates along the messenger RNA to read the codons that encode the amino acid sequence of a protein. Elongation factors, including EF-G and EF-Tu, are used to catalyze the process. Recently, we have shown that translation can be followed at the single-molecule level using optical tweezers; this technique allows us to study the kinetics of translation by measuring the lifetime the ribosome spends at each codon. Here, we analyze the data from single-molecule experiments and fit the data with simple kinetic models. We also simulate the translation kinetics based on a multi-step mechanism from ensemble kinetic measurements. The mean lifetimes from the simulation were consistent with our experimental single-molecule measurements. We found that the calculated lifetime distributions were fit in general by equations with up to five rate-determining steps. Two rate-determining steps were only obtained at low concentrations of elongation factors. These analyses can be used to design new single-molecule experiments to better understand the kinetics and mechanism of translation.
In the cell, proteins are synthesized by ribosomes in a multi-step process called translation. The ribosome translocates along the messenger RNA to read the codons that encode the amino acid sequence of a protein. Elongation factors, including EF-G and EF-Tu, are used to catalyze the process. Recently, we have shown that translation can be followed at the single-molecule level using optical tweezers; this technique allows us to study the kinetics of translation by measuring the lifetime the ribosome spends at each codon. Here, we analyze the data from single-molecule experiments and fit the data with simple kinetic models. We also simulate the translation kinetics based on a multi-step mechanism from ensemble kinetic measurements. The mean lifetimes from the simulation were consistent with our experimental single-molecule measurements. We found that the calculated lifetime distributions were fit in general by equations with up to five rate-determining steps. Two rate-determining steps were only obtained at low concentrations of elongation factors. These analyses can be used to design new single-molecule experiments to better understand the kinetics and mechanism of translation.
Analytical calculation of axial optical force on a Rayleigh particle illuminated by Gaussian beams beyond the paraxial approximation
Jun Chen, Jack Ng, Shiyang Liu, and Zhifang Lin
We investigate the optical trapping of a Rayleigh particle by a linearly or radially polarized Gaussian beam. The Mie theory is applied to obtain a full solution, with the incident beam being described by the mixed dipole model, which is beyond the paraxial approximation. We then obtain approximate analytical expressions for the optical force, equilibrium position, and trap stiffness for a Rayleigh particle. At equilibrium, the displacement for the particle from the focus scales like a3 (where a is the radius) for a transparent particle, owing to scattering, whereas for absorptive particles it scales like C+Da2, owing to absorption. The trap stiffness is found to be proportional to a3, in agreement with the recent experiment. The radially polarized beam is found to be superior to the linearly polarized beam in the Rayleigh regime in terms of its ability to trap. It is found that the larger the ratio of 
r/i, the closer the equilibrium to the focus, and thus higher stability.
r/i, the closer the equilibrium to the focus, and thus higher stability.NanoPen: Dynamic, Low-Power, and Light-Actuated Patterning of Nanoparticles
Arash Jamshidi, Steven L. Neale, Kyoungsik Yu, Peter J. Pauzauskie, Peter James Schuck, Justin K. Valley, Hsan-Yin Hsu, Aaron T. Ohta and Ming C. Wu
We introduce NanoPen, a novel technique for low optical power intensity, flexible, real-time reconfigurable, and large-scale light-actuated patterning of single or multiple nanoparticles, such as metallic spherical nanocrystals, and one-dimensional nanostructures, such as carbon nanotubes. NanoPen is capable of dynamically patterning nanoparticles over an area of thousands of square micrometers with light intensities <10 style="vertical-align: 0.4em; font-size: 0.8em; line-height: 0; ">2 (using a commercial projector) within seconds. Various arbitrary nanoparticle patterns and arrays (including a 10 × 10 array covering a 0.025 mm2 area) are demonstrated using this capability. One application of NanoPen is presented through the creation of surface-enhanced Raman spectroscopy hot-spots by patterning gold nanoparticles of 90 nm diameter with enhancement factors exceeding 107 and picomolar concentration sensitivities.
Thursday, August 27, 2009
Single Centrosome Manipulation Reveals Its Electric Charge and Associated Dynamic Structure
S. Hormeño, B. Ibarra, F.J. Chichón, K. Habermann, B.M.H. Lange, J.M. Valpuesta, J.L. Carrascosa and J.R. Arias-Gonzalez
The centrosome is the major microtubule-organizing center in animal cells and consists of a pair of centrioles surrounded by a pericentriolar material. We demonstrate laser manipulation of individual early Drosophila embryo centrosomes in between two microelectrodes to reveal that it is a net negatively charged organelle with a very low isoelectric region (3.1 ± 0.1). From this single-organelle electrophoresis, we infer an effective charge smaller than or on the order of 103 electrons, which corresponds to a surface-charge density significantly smaller than that of microtubules. We show, however, that the charge of the centrosome has a remarkable influence over its own structure. Specifically, we investigate the hydrodynamic behavior of the centrosome by measuring its size by both Stokes law and thermal-fluctuation spectral analysis of force. We find, on the one hand, that the hydrodynamic size of the centrosome is 60% larger than its electron microscopy diameter, and on the other hand, that this physiological expansion is produced by the electric field that drains to the centrosome, a self-effect that modulates its structural behavior via environmental pH. This methodology further proves useful for studying the action of different environmental conditions, such as the presence of Ca2+, over the thermally induced dynamic structure of the centrosome.
The centrosome is the major microtubule-organizing center in animal cells and consists of a pair of centrioles surrounded by a pericentriolar material. We demonstrate laser manipulation of individual early Drosophila embryo centrosomes in between two microelectrodes to reveal that it is a net negatively charged organelle with a very low isoelectric region (3.1 ± 0.1). From this single-organelle electrophoresis, we infer an effective charge smaller than or on the order of 103 electrons, which corresponds to a surface-charge density significantly smaller than that of microtubules. We show, however, that the charge of the centrosome has a remarkable influence over its own structure. Specifically, we investigate the hydrodynamic behavior of the centrosome by measuring its size by both Stokes law and thermal-fluctuation spectral analysis of force. We find, on the one hand, that the hydrodynamic size of the centrosome is 60% larger than its electron microscopy diameter, and on the other hand, that this physiological expansion is produced by the electric field that drains to the centrosome, a self-effect that modulates its structural behavior via environmental pH. This methodology further proves useful for studying the action of different environmental conditions, such as the presence of Ca2+, over the thermally induced dynamic structure of the centrosome.
Wednesday, August 26, 2009
Experimental studies of the transient fluctuation theorem using liquid crystals
Soma Datta and Arun Roy
In a thermodynamical process, the dissipation or production of entropy can only be positive or zero, according to the second law of thermodynamics. However, the laws of thermodynamics are applicable to large systems in the thermodynamic limit. Recently a fluctuation theorem, known as the transient fluctuation theorem (TFT), which generalizes the second law of thermodynamics to small systems has been proposed. This theorem has been tested in small systems such as a colloidal particle in an optical trap. We report for the first time an analogous experimental study of TFT in a spatially extended system using liquid crystals.
In a thermodynamical process, the dissipation or production of entropy can only be positive or zero, according to the second law of thermodynamics. However, the laws of thermodynamics are applicable to large systems in the thermodynamic limit. Recently a fluctuation theorem, known as the transient fluctuation theorem (TFT), which generalizes the second law of thermodynamics to small systems has been proposed. This theorem has been tested in small systems such as a colloidal particle in an optical trap. We report for the first time an analogous experimental study of TFT in a spatially extended system using liquid crystals.
Monday, August 24, 2009
Drug Effect Unveils Inter-head Cooperativity and Strain-dependent ADP Release in Fast Skeletal Actomyosin
Amrinone is a bipyridine compound with characteristic effects on the force-velocity relationship of fast skeletal muscle,including a reduction in the maximum shortening velocity and increased maximum isometric force. Here we performed experiments to elucidate the molecular mechanisms for these effects, with the additional aim to gain insight into the molecular mechanisms underlying the force-velocity relationship. In vitro motility assays established that amrinone reduces the sliding velocity of heavy meromyosin-propelled actin filaments by 30% at different ionic strengths of the assay solution. Stopped-flow studies of myofibrils, heavy meromyosin and myosin subfragment 1, showed that the effects on sliding speed were not because of a reduced rate of ATP-induced actomyosin dissociation because the rateof this process was increased by amrinone. Moreover, optical tweezers studies could not detect any amrinone-induced changes in the working stroke length. In contrast, the ADP affinity of acto-heavy meromyosin was increased about 2-fold by 1 mM amrinone. Similar effects were not observed for acto-subfragment 1. Together with the other findings, this suggests that the amrinone-induced reduction in sliding velocity is attributed to inhibition of a strain-dependent ADP release step. Modeling results show that such an effect may account for the amrinone-induced changes of the force-velocity relationship. The data emphasize the importance of the rate of a strain-dependent ADP release step in influencing the maximum sliding velocity in fast skeletal muscle. The data also lead us to discuss the possible importance of cooperative interactions between the two myosin heads in muscle contraction.
The influence of resonant absorption and heating on the equilibrium size of aqueous-solute aerosol droplets
Rachael E. H. Miles, Marc Guillon, Laura Mitchem, David McGloin and Jonathan P. Reid
The time-dependent evolution in the equilibrium size of an optically trapped aqueous sodium chloride droplet (>2
m radius) within an environment of varying relative humidity (RH) is shown to depend on both the depression in vapour pressure due to the presence of the solute and the elevation in temperature due to optical absorption. In particular, the level of optical absorption is highly dependent on the size of the droplet relative to the wavelength of the absorbed light. Thus, as the droplet size tunes into a Mie resonance at the trapping laser wavelength, the increased level of optical absorption leads to an elevation in droplet temperature. This increase in resonant heating can balance a continual increase in RH, leading to only marginal growth in droplet size and change in solute concentration. Once the RH is sufficiently high that the resonance condition can be surpassed, the droplet cools instantaneously and the solute concentration again dominates in determining the vapour pressure, with a rapid increase in size and a decrease in solute concentration returning the droplet to equilibrium with the gas phase RH. Thus, a growing droplet is observed to pass through periods of apparent size stability followed by instantaneous growth, consistent with the variation in absorption efficiency with droplet size. This provides a clear example of the coupling between the optical and physical properties of an aerosol and their influence on the equilibrium state.
The time-dependent evolution in the equilibrium size of an optically trapped aqueous sodium chloride droplet (>2
m radius) within an environment of varying relative humidity (RH) is shown to depend on both the depression in vapour pressure due to the presence of the solute and the elevation in temperature due to optical absorption. In particular, the level of optical absorption is highly dependent on the size of the droplet relative to the wavelength of the absorbed light. Thus, as the droplet size tunes into a Mie resonance at the trapping laser wavelength, the increased level of optical absorption leads to an elevation in droplet temperature. This increase in resonant heating can balance a continual increase in RH, leading to only marginal growth in droplet size and change in solute concentration. Once the RH is sufficiently high that the resonance condition can be surpassed, the droplet cools instantaneously and the solute concentration again dominates in determining the vapour pressure, with a rapid increase in size and a decrease in solute concentration returning the droplet to equilibrium with the gas phase RH. Thus, a growing droplet is observed to pass through periods of apparent size stability followed by instantaneous growth, consistent with the variation in absorption efficiency with droplet size. This provides a clear example of the coupling between the optical and physical properties of an aerosol and their influence on the equilibrium state.
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