Proper tension maintenance in the cytoskeleton is essential for regulated cell polarity, cell motility, and division. Non-muscle myosin IIB (NMIIB) generates tension along actin filaments in many cell types, including neuronal, cardiac, and smooth muscle cells. Using a three-bead optical trapping assay, we recorded NMIIB interactions with actin filaments to determine if a NMIIB dimer cycles along an actin filament in a processive manner. Our results show that NMIIB is the first myosin II to exhibit evidence of processive stepping behavior. Analysis of these data reveals a forward displacement of 5.4 nm and, surprisingly, frequent backward steps of −5.9 nm. Processive stepping along the long pitch helix of actin may provide a mechanism for disassembly of fascin-actin bundles. Forward steps and detachment are weakly force-dependent at all forces, consistent with rate-limiting and force-dependent ADP release. However, backward steps are nearly force-independent. Our data support a model in which NMIIB can readily move in both directions at stall, which may be important for a general regulator of cytoskeleton tension.
Thursday, August 26, 2010
Unconventional Processive Mechanics of Non-muscle Myosin IIB
Melanie F. Norstrom, Philip A. Smithback and Ronald S. Rock
Proper tension maintenance in the cytoskeleton is essential for regulated cell polarity, cell motility, and division. Non-muscle myosin IIB (NMIIB) generates tension along actin filaments in many cell types, including neuronal, cardiac, and smooth muscle cells. Using a three-bead optical trapping assay, we recorded NMIIB interactions with actin filaments to determine if a NMIIB dimer cycles along an actin filament in a processive manner. Our results show that NMIIB is the first myosin II to exhibit evidence of processive stepping behavior. Analysis of these data reveals a forward displacement of 5.4 nm and, surprisingly, frequent backward steps of −5.9 nm. Processive stepping along the long pitch helix of actin may provide a mechanism for disassembly of fascin-actin bundles. Forward steps and detachment are weakly force-dependent at all forces, consistent with rate-limiting and force-dependent ADP release. However, backward steps are nearly force-independent. Our data support a model in which NMIIB can readily move in both directions at stall, which may be important for a general regulator of cytoskeleton tension.
Proper tension maintenance in the cytoskeleton is essential for regulated cell polarity, cell motility, and division. Non-muscle myosin IIB (NMIIB) generates tension along actin filaments in many cell types, including neuronal, cardiac, and smooth muscle cells. Using a three-bead optical trapping assay, we recorded NMIIB interactions with actin filaments to determine if a NMIIB dimer cycles along an actin filament in a processive manner. Our results show that NMIIB is the first myosin II to exhibit evidence of processive stepping behavior. Analysis of these data reveals a forward displacement of 5.4 nm and, surprisingly, frequent backward steps of −5.9 nm. Processive stepping along the long pitch helix of actin may provide a mechanism for disassembly of fascin-actin bundles. Forward steps and detachment are weakly force-dependent at all forces, consistent with rate-limiting and force-dependent ADP release. However, backward steps are nearly force-independent. Our data support a model in which NMIIB can readily move in both directions at stall, which may be important for a general regulator of cytoskeleton tension.
Insight into helicase mechanism and function revealed through single-molecule approaches
Jaya G. Yodh, Michael Schlierf and Taekjip Ha
Helicases are a class of nucleic acid (NA) motors that catalyze NTP-dependent unwinding of NA duplexes into single strands, a reaction essential to all areas of NA metabolism. In the last decade, single-molecule (sm) technology has proven to be highly useful in revealing mechanistic insight into helicase activity that is not always detectable via ensemble assays. A combination of methods based on fluorescence, optical and magnetic tweezers, and flow-induced DNA stretching has enabled the study of helicase conformational dynamics, force generation, step size, pausing, reversal and repetitive behaviors during translocation and unwinding by helicases working alone and as part of multiprotein complexes. The contributions of these sm investigations to our understanding of helicase mechanism and function will be discussed.
DOI
Helicases are a class of nucleic acid (NA) motors that catalyze NTP-dependent unwinding of NA duplexes into single strands, a reaction essential to all areas of NA metabolism. In the last decade, single-molecule (sm) technology has proven to be highly useful in revealing mechanistic insight into helicase activity that is not always detectable via ensemble assays. A combination of methods based on fluorescence, optical and magnetic tweezers, and flow-induced DNA stretching has enabled the study of helicase conformational dynamics, force generation, step size, pausing, reversal and repetitive behaviors during translocation and unwinding by helicases working alone and as part of multiprotein complexes. The contributions of these sm investigations to our understanding of helicase mechanism and function will be discussed.
DOI
Haptic Feedback of Piconewton Interactions with Optical Tweezers
Cécile Pacoret, Arvid Bergander and Stéphane Régnier
Haptic feedback for micro- and nanomanipulation is a research area of growing importance with many potential applications in micro- and biotechnology. Past research often involves the coupling of atomic force microscopes to haptic devices, but the results are not satisfactory. We propose to adopt a different approach, which consists of contactless manipulation, in particular by using optical tweezers, coupled with a haptic feedback device. In this article, we describe the potential of such a tool and show with some first experiments of stable interactions between micro-particles.
DOI
Haptic feedback for micro- and nanomanipulation is a research area of growing importance with many potential applications in micro- and biotechnology. Past research often involves the coupling of atomic force microscopes to haptic devices, but the results are not satisfactory. We propose to adopt a different approach, which consists of contactless manipulation, in particular by using optical tweezers, coupled with a haptic feedback device. In this article, we describe the potential of such a tool and show with some first experiments of stable interactions between micro-particles.
DOI
Friday, August 20, 2010
Confocal Raman Microscopy of Optical-Trapped Particles in Liquids
Daniel P. Cherney and Joel M. Harris
The in situ analysis of small, dispersed particles in liquids is a challenging problem, the successful solution to which influences diverse applications of colloidal particles in materials science, synthetic chemistry, and molecular biology. Optical trapping of small particles with a tightly focused laser beam can be combined with confocal Raman microscopy to provide molecular structure information about individual, femtogram-sized particles in liquid samples. In this review, we consider the basic principles of combining optical trapping and confocal Raman spectroscopy, then survey the applications that have been developed through the combination of these techniques and their use in the analysis of particles dispersed in liquids.
DOI
The in situ analysis of small, dispersed particles in liquids is a challenging problem, the successful solution to which influences diverse applications of colloidal particles in materials science, synthetic chemistry, and molecular biology. Optical trapping of small particles with a tightly focused laser beam can be combined with confocal Raman microscopy to provide molecular structure information about individual, femtogram-sized particles in liquid samples. In this review, we consider the basic principles of combining optical trapping and confocal Raman spectroscopy, then survey the applications that have been developed through the combination of these techniques and their use in the analysis of particles dispersed in liquids.
DOI
Light forces the pace: optical manipulation for biophotonics
David James Stevenson, Frank Gunn-Moore, Kishan Dholakia
The biomedical sciences have benefited immensely from photonics technologies in thelast 50 years. This includes the application of minute forces that enable the trapping and manipulation of cells and single molecules. In terms of the area of biophotonics, optical manipulation has made a seminal contribution to our understanding of the dynamics of single molecules and the microrheology of cells. Here we present a review of optical manipulation, emphasizing its impact on the areas of single-molecule studies and single-cell biology, and indicating some of the key experiments in the fields.
DOI
The biomedical sciences have benefited immensely from photonics technologies in thelast 50 years. This includes the application of minute forces that enable the trapping and manipulation of cells and single molecules. In terms of the area of biophotonics, optical manipulation has made a seminal contribution to our understanding of the dynamics of single molecules and the microrheology of cells. Here we present a review of optical manipulation, emphasizing its impact on the areas of single-molecule studies and single-cell biology, and indicating some of the key experiments in the fields.
DOI
Tuesday, August 17, 2010
Nonlinear Elasticity and an 8-nm Working Stroke of Single Myosin Molecules in Myofilaments
Motoshi Kaya and Hideo Higuchi
Using optical trapping and fluorescence imaging techniques, we measured the step size and stiffness of single skeletal myosins interacting with actin filaments and arranged on myosin-rodcofilaments that approximate myosin mechanics during muscle contraction. Stiffness is dramatically lower for negatively compared to positively strained myosins, consistent with buckling of myosin’s subfragment 2 rod domain. Low stiffness minimizes drag of negatively strained myosins during contraction at loaded conditions. Myosin's elastic portion is stretched during active force generation, reducing apparent step size with increasing load, even though the working stroke is approximately constant at about 8 nanometers. Taking account of the nonlinear nature of myosin elasticity is essential to relate myosin’s internal structural changes to physiological force generation and filament sliding.
DOI
Using optical trapping and fluorescence imaging techniques, we measured the step size and stiffness of single skeletal myosins interacting with actin filaments and arranged on myosin-rodcofilaments that approximate myosin mechanics during muscle contraction. Stiffness is dramatically lower for negatively compared to positively strained myosins, consistent with buckling of myosin’s subfragment 2 rod domain. Low stiffness minimizes drag of negatively strained myosins during contraction at loaded conditions. Myosin's elastic portion is stretched during active force generation, reducing apparent step size with increasing load, even though the working stroke is approximately constant at about 8 nanometers. Taking account of the nonlinear nature of myosin elasticity is essential to relate myosin’s internal structural changes to physiological force generation and filament sliding.
DOI
Monday, August 16, 2010
Flow-assisted Single-beam Optothermal Manipulation of Microparticles
Yangyang Liu and Andrew W. Poon
An optothermal tweezer was developed with a single-beam laser at 1550 nm for manipulation of colloidal microparticles. Strong absorption in water can thermally induce a localized flow, which exerts a Stokes’ drag on the particles that complements the gradient force. Long-range capturing of 6 μm polystyrene particles over ∼176 μm was observed with a tweezing power of ∼7 mW. Transportation and levitation, targeted deposition and selective levitation of particles were explored to experimentally demonstrate the versatility of the optothermal tweezer as a multipurpose particle manipulation tool.
DOI
An optothermal tweezer was developed with a single-beam laser at 1550 nm for manipulation of colloidal microparticles. Strong absorption in water can thermally induce a localized flow, which exerts a Stokes’ drag on the particles that complements the gradient force. Long-range capturing of 6 μm polystyrene particles over ∼176 μm was observed with a tweezing power of ∼7 mW. Transportation and levitation, targeted deposition and selective levitation of particles were explored to experimentally demonstrate the versatility of the optothermal tweezer as a multipurpose particle manipulation tool.
DOI
Holographic optical manipulation of motor-driven membranous structures in living NG-108 cells
Arnau Farré, Carol López-Quesada, Jordi Andilla, Estela Martín-Badosa, and Mario Montes-Usategui
Optical tweezer experiments have partially unveiled the mechanical properties of processive motor proteins while driving polystyrene or silica microbeads in vitro. However, the set of forces underlying the more complex transport mechanisms in living samples remains poorly understood. Several studies have shown that optical tweezers are capable of trapping vesicles and organelles in the cytoplasm of living cells, which can be used as handles to mechanically interact with engaged (active) motors, or other components regulating transport. This may ultimately enable the exploration of the mechanics of this trafficking mechanism in vivo. These cell manipulation experiments have been carried out using different strategies to achieve dynamic beam steering capable of trapping thesesubcellular structures. We report here the first trapping and manipulation, to our knowledge, of such small motor-propelled cargos in living cells using holographic technology.
DOI
Optical tweezer experiments have partially unveiled the mechanical properties of processive motor proteins while driving polystyrene or silica microbeads in vitro. However, the set of forces underlying the more complex transport mechanisms in living samples remains poorly understood. Several studies have shown that optical tweezers are capable of trapping vesicles and organelles in the cytoplasm of living cells, which can be used as handles to mechanically interact with engaged (active) motors, or other components regulating transport. This may ultimately enable the exploration of the mechanics of this trafficking mechanism in vivo. These cell manipulation experiments have been carried out using different strategies to achieve dynamic beam steering capable of trapping thesesubcellular structures. We report here the first trapping and manipulation, to our knowledge, of such small motor-propelled cargos in living cells using holographic technology.
DOI
Optical manipulators of microparticles using femtosecond laser radiation
V V Buchanov, V A Derzhavin, A D Zalesskii, I V Reshetov, Oleg M Sarkisov and A I Shushin
Two setups for manipulating (translocation, stretching, rotation) microscopic objects (with the dimensions from several nanometers to tens of micrometers) are developed based on optical trapping by femtosecond laser radiation. The possibility of single-cell translocation is shown. The possibility of destructing malignant cells as well as of cutting off a fragment from a malignant cell cluster due to the rapture of bonds is demonstrated in multiphoton absorption of femtosecond light pulses. The possibility of the holographic control to move several particles simultaneously is shown.
DOI
Two setups for manipulating (translocation, stretching, rotation) microscopic objects (with the dimensions from several nanometers to tens of micrometers) are developed based on optical trapping by femtosecond laser radiation. The possibility of single-cell translocation is shown. The possibility of destructing malignant cells as well as of cutting off a fragment from a malignant cell cluster due to the rapture of bonds is demonstrated in multiphoton absorption of femtosecond light pulses. The possibility of the holographic control to move several particles simultaneously is shown.
DOI
Thursday, August 12, 2010
Doppler Cooling a Microsphere
P. F. Barker
Doppler cooling the center-of-mass motion of an optically levitated microsphere via the velocity-dependent scattering force from narrow whispering gallery mode resonances is described. Light that is red detuned from the whispering gallery mode resonance can be used to damp the center-of-mass motion in a process analogous to the Doppler cooling of atoms. The scattering force is not limited by saturation but can be controlled by the incident power. Cooling times on the order of seconds are calculated for a 20 μm diameter silica microsphere trapped within optical tweezers.
DOI
Doppler cooling the center-of-mass motion of an optically levitated microsphere via the velocity-dependent scattering force from narrow whispering gallery mode resonances is described. Light that is red detuned from the whispering gallery mode resonance can be used to damp the center-of-mass motion in a process analogous to the Doppler cooling of atoms. The scattering force is not limited by saturation but can be controlled by the incident power. Cooling times on the order of seconds are calculated for a 20 μm diameter silica microsphere trapped within optical tweezers.
DOI
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