Yi-Ju Wu, Cheng-Han Wu, Athena Yi-Chun Yeh and Jin-Der Wen
Folding messenger RNA into specific structures is a common regulatory mechanism involved in translation. In Escherichia coli, the operator of the rpsO gene transcript folds into a pseudoknot or double-hairpin conformation. S15, the gene product, binds only to the pseudoknot, thereby repressing its own synthesis when it is present in excess in the cell. The two RNA conformations have been proposed to exist in equilibrium. However, it remained unclear how structural changes can be achieved between these two topologically distinct conformations. We used optical tweezers to study the structural dynamics and rearrangements of the rpsO operator RNA at the single-molecule level. We discovered that the two RNA structures can be interchanged spontaneously and the pseudoknot can exist in conformations that exhibit various levels of stability. Conversion from the double hairpin to a pseudoknot through potential hairpin–hairpin interactions favoured the high-stability conformation. By contrast, mutations that blocked the formation of a hairpin typically resulted in alternative low-stability pseudoknots. These results demonstrate that specific tertiary interactions of RNA can be established and modulated based on the interactions and rearrangements between secondary structural components. Our findings provide new insight into the RNA folding pathway that leads to a regulatory conformation for target protein binding.
DOI
Friday, January 31, 2014
Optimal beam diameter for lateral optical forces on microspheres at a water-air interface
Mincheng Zhong, Xi Wang, Jinhua Zhou, Ziqiang Wang, and Yinmei Li
Optical tweezers with a low numerical aperture microscope objective is used to manipulate the microspheres at the water-air interface. In this letter, we determine the optimal optical trap for the lateral manipulation of microspheres at a water-air interface. The experimental results show that the trapping force is influenced by the expansion of the trapping beam at the back aperture of the objective. The optimal filling ratio of 0.65 is suggested for lateral optical manipulation at the water-air interface. The lateral trapping forces at the water-air interface are theoretically investigated with the ray-optics model. The numerical results show that the lateral trapping forces can be changed by shrinking the diameter of the trapping laser beam. The numerical results are in accordance with the experimental results.
DOI
Optical tweezers with a low numerical aperture microscope objective is used to manipulate the microspheres at the water-air interface. In this letter, we determine the optimal optical trap for the lateral manipulation of microspheres at a water-air interface. The experimental results show that the trapping force is influenced by the expansion of the trapping beam at the back aperture of the objective. The optimal filling ratio of 0.65 is suggested for lateral optical manipulation at the water-air interface. The lateral trapping forces at the water-air interface are theoretically investigated with the ray-optics model. The numerical results show that the lateral trapping forces can be changed by shrinking the diameter of the trapping laser beam. The numerical results are in accordance with the experimental results.
DOI
Size-dependent position of a single aerosol droplet in a Bessel beam trap
Thomas C Preston, Bernard J Mason, Jonathan P Reid, David Luckhaus and Ruth Signorell
The equilibrium position of an aerosol droplet trapped in a counter-propagating Bessel beam and gas flow is studied both experimentally and theoretically. This provides an aerosol analogue to the separation of particles of differing size and refractive index in counter-propagating laser beam and liquid flow, referred to as optical chromatography. Using the model system of a pure glycerol droplet it is found that, as evaporation takes place and the size of the droplet decreases, the size-dependent equilibrium position does not change in a simple monotonic fashion. Instead, the position of the droplet is greatly affected by the excitation of whispering gallery modes. This leads to sharp peaks in the equilibrium position curve, not unlike those that occur in single particle spectroscopy. The conditions necessary to excite whispering gallery modes are thoroughly investigated.
DOI
DOI
Single-Molecule Fluorescence and in Vivo Optical Traps: How Multiple Dyneins and Kinesins Interact
Benjamin H. Blehm and Paul R. Selvin
In this review, we describe experimental systems at multiple levels of complexity, including single-motor-type in vitro assays, multimotor in vitro assays, purified-organelle in vitro assays, and finally in vivo cellular assays (Figure 1). This spread of experiments allows an unprecedented view of the transport complex, as kinesin and dynein can be observed with differing components of the transport complex (i.e., different levels of accessory proteins) and in different environments. Through the combination of measurements at all of these levels of complexity, the ability to parse out the function of parts of the transport complex, and reconstitute it in vitro, becomes a real possibility.
DOI
In this review, we describe experimental systems at multiple levels of complexity, including single-motor-type in vitro assays, multimotor in vitro assays, purified-organelle in vitro assays, and finally in vivo cellular assays (Figure 1). This spread of experiments allows an unprecedented view of the transport complex, as kinesin and dynein can be observed with differing components of the transport complex (i.e., different levels of accessory proteins) and in different environments. Through the combination of measurements at all of these levels of complexity, the ability to parse out the function of parts of the transport complex, and reconstitute it in vitro, becomes a real possibility.
DOI
Tuesday, January 28, 2014
Reconstruction and Identification of DNA Sequence Landscapes from Unzipping Experiments at Equilibrium
Carlo Barbieri, Simona Cocco, Thomas Jorg, Rémi Monasson
Two methods for reconstructing the free-energy landscape of a DNA molecule from the knowledge of the equilibrium unzipping force versus extension signal are introduced: a simple and fast procedure, based on a parametric representation of the experimental force signal, and a maximum-likelihood inference of coarse-grained free-energy parameters. In addition, we propose a force alignment procedure to correct for the drift in the experimental measure of the opening position, a major source of error. For unzipping data obtained by Huguet et al., the reconstructed basepair (bp) free energies agree with the running average of the true free energies on a 20–50 bp scale, depending on the region in the sequence. Features of the landscape at a smaller scale (5–10 bp) could be recovered in favorable regions at the beginning of the molecule. Based on the analysis of synthetic data corresponding to the 16S rDNA gene of bacteria, we show that our approach could be used to identify specific DNA sequences among thousands of homologous sequences in a database.
DOI
Two methods for reconstructing the free-energy landscape of a DNA molecule from the knowledge of the equilibrium unzipping force versus extension signal are introduced: a simple and fast procedure, based on a parametric representation of the experimental force signal, and a maximum-likelihood inference of coarse-grained free-energy parameters. In addition, we propose a force alignment procedure to correct for the drift in the experimental measure of the opening position, a major source of error. For unzipping data obtained by Huguet et al., the reconstructed basepair (bp) free energies agree with the running average of the true free energies on a 20–50 bp scale, depending on the region in the sequence. Features of the landscape at a smaller scale (5–10 bp) could be recovered in favorable regions at the beginning of the molecule. Based on the analysis of synthetic data corresponding to the 16S rDNA gene of bacteria, we show that our approach could be used to identify specific DNA sequences among thousands of homologous sequences in a database.
DOI
Anti-frameshifting ligand reduces the conformational plasticity of the SARS virus pseudoknot
Dustin B Ritchie , Jingchyuan Soong , William Sikkema , and Michael T. Woodside
Programmed −1 ribosomal frameshifting (−1 PRF) stimulated by mRNA pseudoknots regulates gene expression in many viruses, making pseudoknots potential targets for anti-viral drugs. The mechanism by which pseudoknots trigger −1 PRF, however, remains controversial, with several competing models. Recent work showed that high −1 PRF efficiency was linked to high pseudoknot conformational plasticity via the formation of alternate conformers. We tested whether pseudoknots bound with an anti-frameshifting ligand exhibited a similar correlation between conformational plasticity and −1 PRF efficiency, by measuring the effects of a ligand which was found to inhibit −1 PRF in the SARS coronavirus on the conformational dynamics of the SARS pseudoknot. Using single-molecule force spectroscopy to unfold pseudoknots mechanically, we found that the ligand binding effectively abolished the formation of alternate conformers. This result extends the connection between −1 PRF and conformational dynamics, and moreover suggests that targeting the conformational dynamics of pseudoknots may be an effective strategy for anti-viral drug design.
DOI
Programmed −1 ribosomal frameshifting (−1 PRF) stimulated by mRNA pseudoknots regulates gene expression in many viruses, making pseudoknots potential targets for anti-viral drugs. The mechanism by which pseudoknots trigger −1 PRF, however, remains controversial, with several competing models. Recent work showed that high −1 PRF efficiency was linked to high pseudoknot conformational plasticity via the formation of alternate conformers. We tested whether pseudoknots bound with an anti-frameshifting ligand exhibited a similar correlation between conformational plasticity and −1 PRF efficiency, by measuring the effects of a ligand which was found to inhibit −1 PRF in the SARS coronavirus on the conformational dynamics of the SARS pseudoknot. Using single-molecule force spectroscopy to unfold pseudoknots mechanically, we found that the ligand binding effectively abolished the formation of alternate conformers. This result extends the connection between −1 PRF and conformational dynamics, and moreover suggests that targeting the conformational dynamics of pseudoknots may be an effective strategy for anti-viral drug design.
DOI
Multi-Dimensional Manipulation of Yeast Cells Using a LP_11 Mode Beam
Zhang, Y.; Liang, P. ; Lei, J. ; Wang, L. ; Liu, Z. ; Yang, J. ; Yuan, L.
We report a new method for constructing a single fiber optical tweezers, which can realize multi-dimensional manipulation of trapped yeast cells by using a LP$_{bf 11}$ mode beam excited in a normal communication single core optical fiber. This allows a simple and convenient orientation control on the trapped yeast cells. The LP$_{bf 11}$ mode beam, both for generating trap and orientation manipulation, has been modulated by using the tension and twisting loaded on the fiber. We present experimental results of controllable deflection and orientation manipulation of the yeast cells. To the best of our knowledge, it is the first report about the trapped yeast cells being driven by the normal single fiber optical tweezers in multi dimensions, and it constitutes a new development for single fiber optical trapping and makes possible of more practical applications in the biomedical research fields.
DOI
We report a new method for constructing a single fiber optical tweezers, which can realize multi-dimensional manipulation of trapped yeast cells by using a LP$_{bf 11}$ mode beam excited in a normal communication single core optical fiber. This allows a simple and convenient orientation control on the trapped yeast cells. The LP$_{bf 11}$ mode beam, both for generating trap and orientation manipulation, has been modulated by using the tension and twisting loaded on the fiber. We present experimental results of controllable deflection and orientation manipulation of the yeast cells. To the best of our knowledge, it is the first report about the trapped yeast cells being driven by the normal single fiber optical tweezers in multi dimensions, and it constitutes a new development for single fiber optical trapping and makes possible of more practical applications in the biomedical research fields.
DOI
Dynamically reconfigurable Fibre Optical Spanner
Thorsten Kolb, Sahradha Albert, Michael Haug and Graeme Whyte
In this paper we describe a pneumatically actuated fibre-optic spanner integrated into a microfluidic Lab-on-a-Chip device for the controlled trapping and rotation of living cells. The dynamic nature of the system allows interactive control over the rotation speed with the same optical power. The use of a multi-layer device makes it possible to rotate a cell both in the imaging plane and also in a perpendicular plane allowing tomographic imaging of the trapped living cell. The integrated device allows easy operation and by combining it with high-resolution confocal microscopy we show for the first time that the pattern of rotation can give information regarding the sub-cellular composition of a rotated cell.
DOI
In this paper we describe a pneumatically actuated fibre-optic spanner integrated into a microfluidic Lab-on-a-Chip device for the controlled trapping and rotation of living cells. The dynamic nature of the system allows interactive control over the rotation speed with the same optical power. The use of a multi-layer device makes it possible to rotate a cell both in the imaging plane and also in a perpendicular plane allowing tomographic imaging of the trapped living cell. The integrated device allows easy operation and by combining it with high-resolution confocal microscopy we show for the first time that the pattern of rotation can give information regarding the sub-cellular composition of a rotated cell.
DOI
Controlled Particle Collision Leads to Direct Observation of Docking and Fusion of Lipid Droplets in an Optical Trap
Chiran Ghimire , Deepak Koirala , Malcom B. Mathis , Edgar Eduard Kooijman , and Hanbin Mao
As an intracellular organelle, phospholipid coated lipid droplets have shown increasing importance due to their expanding biological functions other than the lipid storage. The growing biological significance necessitates a close scrutiny on lipid droplets, which have been proposed to mature in a cell through processes such as fusions. Unlike phospholipid vesicles that are well known to fuse through docking and hemifusion steps, little is known on the fusion of lipid droplets. Herein, we used laser tweezers to capture two micrometer-sized 1,2,3-trioleoylglycerol (triolein) droplets coated with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) that closely resemble intracellular lipid droplets. We started the fusion processes by a well-controlled collision between the two lipid droplets in phosphate buffers at pH 7.4. By monitoring the change in the pathway of a trapping laser that captures the collided lipid droplets, docking and fusion events were clearly distinguished for the first time and their lifetimes were determined with a resolution of 10 microseconds after post-synchronization analyses. Our method revealed that the rate limiting docking process is affected by anions according to a Hofmeister series, which sheds light on the important role of interfacial water shedding during the process. During the actual fusion, the kinetics between bare triolein droplets is faster than lipid droplets, suggesting that breaking of phospholipid coating is involved in the process. This scenario was further supported by direct observation of a short-lived hemifusion state with ~ 46 milliseconds lifetime in POPC coated lipid droplets, but not in bare triolein droplets.
DOI
As an intracellular organelle, phospholipid coated lipid droplets have shown increasing importance due to their expanding biological functions other than the lipid storage. The growing biological significance necessitates a close scrutiny on lipid droplets, which have been proposed to mature in a cell through processes such as fusions. Unlike phospholipid vesicles that are well known to fuse through docking and hemifusion steps, little is known on the fusion of lipid droplets. Herein, we used laser tweezers to capture two micrometer-sized 1,2,3-trioleoylglycerol (triolein) droplets coated with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) that closely resemble intracellular lipid droplets. We started the fusion processes by a well-controlled collision between the two lipid droplets in phosphate buffers at pH 7.4. By monitoring the change in the pathway of a trapping laser that captures the collided lipid droplets, docking and fusion events were clearly distinguished for the first time and their lifetimes were determined with a resolution of 10 microseconds after post-synchronization analyses. Our method revealed that the rate limiting docking process is affected by anions according to a Hofmeister series, which sheds light on the important role of interfacial water shedding during the process. During the actual fusion, the kinetics between bare triolein droplets is faster than lipid droplets, suggesting that breaking of phospholipid coating is involved in the process. This scenario was further supported by direct observation of a short-lived hemifusion state with ~ 46 milliseconds lifetime in POPC coated lipid droplets, but not in bare triolein droplets.
DOI
Optical tweezers assisted imaging of the Z-ring in Escherichia coli: measuring its radial width
G Carmon, P Kumar and M Feingold
Using single-beam, oscillating optical tweezers we can trap and rotate rod-shaped bacterial cells with respect to the optical axis. This technique allows imaging fluorescently labeled three-dimensional sub-cellular structures from different, optimized viewpoints. To illustrate our method we measure D, the radial width of the Z-ring in unconstricted Escherichia coli. We use cells that express FtsZ-GFP and have their cytoplasmic membrane stained with FM4-64. In a vertically oriented cell, both the Z-ring and the cytoplasmic membrane images appear as symmetric circular structures that lend themselves to quantitative analysis. We found that D cong 100 nm, much larger than expected.
DOI
Using single-beam, oscillating optical tweezers we can trap and rotate rod-shaped bacterial cells with respect to the optical axis. This technique allows imaging fluorescently labeled three-dimensional sub-cellular structures from different, optimized viewpoints. To illustrate our method we measure D, the radial width of the Z-ring in unconstricted Escherichia coli. We use cells that express FtsZ-GFP and have their cytoplasmic membrane stained with FM4-64. In a vertically oriented cell, both the Z-ring and the cytoplasmic membrane images appear as symmetric circular structures that lend themselves to quantitative analysis. We found that D cong 100 nm, much larger than expected.
DOI
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