Marco Grison, Ulrich Merkel, Julius Kostan, Kristina Djinović-Carugo, and Matthias Rief
Stable anchoring of titin within the muscle Z-disk is essential for preserving muscle integrity during passive stretching. One of the main candidates for anchoring titin in the Z-disk is the actin cross-linker α-actinin. The calmodulin-like domain of α-actinin binds to the Z-repeats of titin. However, the mechanical and kinetic properties of this important interaction are still unknown. Here, we use a dual-beam optical tweezers assay to study the mechanics of this interaction at the single-molecule level. A single interaction of α-actinin and titin turns out to be surprisingly weak if force is applied. Depending on the direction of force application, the unbinding forces can more than triple. Our results suggest a model where multiple α-actinin/Z-repeat interactions cooperate to ensure long-term stable titin anchoring while allowing the individual components to exchange dynamically.
DOI
Friday, January 27, 2017
Optical sorting and cultivation of denitrifying anaerobic methane oxidation archaea
Xiaoqiong Qi, David M. Carberry, Chen Cai, Shihu Hu, Zhiguo Yuan, Halin Rubinsztein Dunlop, and Jianhua Guo
Denitrifying anaerobic methane oxidizing (DAMO) microorganisms play an important role in the global carbon and nitrogen cycles as they are able to mediate methane oxidation using nitrite/nitrate under anoxic conditions. However, the physiological properties of DAMO microorganisms remain poorly understood, partially since the organisms are difficult to isolate or cultivate in pure culture and partially because of their long cultivation time. In this study, DAMO cell sorting has been conducted by integrating optical tweezers within enclosed microfluidic chips. This integrated cell sorting method has high purity, low infection rates, and causes no discernable harm to cell viability. The purity of the sorted cells was controlled by the microfluidic chip structure design and operation, while the cell viability was verified by imaging the cultured DAMO archaea after 420 days.
DOI
Denitrifying anaerobic methane oxidizing (DAMO) microorganisms play an important role in the global carbon and nitrogen cycles as they are able to mediate methane oxidation using nitrite/nitrate under anoxic conditions. However, the physiological properties of DAMO microorganisms remain poorly understood, partially since the organisms are difficult to isolate or cultivate in pure culture and partially because of their long cultivation time. In this study, DAMO cell sorting has been conducted by integrating optical tweezers within enclosed microfluidic chips. This integrated cell sorting method has high purity, low infection rates, and causes no discernable harm to cell viability. The purity of the sorted cells was controlled by the microfluidic chip structure design and operation, while the cell viability was verified by imaging the cultured DAMO archaea after 420 days.
DOI
Optical manipulation for studies of collisional dynamics of micron-sized droplets under gravity
Maksym Ivanov, Kelken Chang, Ivan Galinskiy, Bernhard Mehlig, and Dag Hanstorp
A new experimental technique for creating and imaging collisions of micron-sized droplets settling under gravity is presented. A pair of glycerol droplets is suspended in air by means of two optical traps. The droplet relative velocities are determined by the droplet sizes. The impact parameter is precisely controlled by positioning the droplets using the two optical traps. The droplets are released by turning off the trapping light using electro-optical modulators. The motion of the sedimenting droplets is then captured by two synchronized high-speed cameras, at a frame rate of up to 63 kHz. The method allows the direct imaging of the collision of droplets without the influence of the optical confinement imposed by the trapping force. The method will facilitate efficient studies of the microphysics of neutral, as well as charged, liquid droplets and their interactions with light, electric field and thermodynamic environment, such as temperature or vapor concentration.
DOI
A new experimental technique for creating and imaging collisions of micron-sized droplets settling under gravity is presented. A pair of glycerol droplets is suspended in air by means of two optical traps. The droplet relative velocities are determined by the droplet sizes. The impact parameter is precisely controlled by positioning the droplets using the two optical traps. The droplets are released by turning off the trapping light using electro-optical modulators. The motion of the sedimenting droplets is then captured by two synchronized high-speed cameras, at a frame rate of up to 63 kHz. The method allows the direct imaging of the collision of droplets without the influence of the optical confinement imposed by the trapping force. The method will facilitate efficient studies of the microphysics of neutral, as well as charged, liquid droplets and their interactions with light, electric field and thermodynamic environment, such as temperature or vapor concentration.
DOI
A Clostridium difficile-Specific, Gel-Forming Protein Required for Optimal Spore Germination
M. Lauren Donnelly, William Li, Yong-qing Li, Lauren Hinkel, Peter Setlow, Aimee Shen
Clostridium difficile is a Gram-positive spore-forming obligate anaerobe that is a leading cause of antibiotic-associated diarrhea worldwide. In order for C. difficile to initiate infection, its aerotolerant spore form must germinate in the gut of mammalian hosts. While almost all spore-forming organisms use transmembrane germinant receptors to trigger germination, C. difficile uses the pseudoprotease CspC to sense bile salt germinants. CspC activates the related subtilisin-like protease CspB, which then proteolytically activates the cortex hydrolase SleC. Activated SleC degrades the protective spore cortex layer, a step that is essential for germination to proceed. Since CspC incorporation into spores also depends on CspA, a related pseudoprotease domain, Csp family proteins play a critical role in germination. However, how Csps are incorporated into spores remains unknown. In this study, we demonstrate that incorporation of the CspC, CspB, and CspA germination regulators into spores depends on CD0311 (renamed GerG), a previously uncharacterized hypothetical protein. The reduced levels of Csps in gerG spores correlate with reduced responsiveness to bile salt germinants and increased germination heterogeneity in single-spore germination assays. Interestingly, asparagine-rich repeat sequences in GerG’s central region facilitate spontaneous gel formation in vitro even though they are dispensable for GerG-mediated control of germination. Since GerG is found exclusively in C. difficile, our results suggest that exploiting GerG function could represent a promising avenue for developing C. difficile-specific anti-infective therapies.
DOI
Clostridium difficile is a Gram-positive spore-forming obligate anaerobe that is a leading cause of antibiotic-associated diarrhea worldwide. In order for C. difficile to initiate infection, its aerotolerant spore form must germinate in the gut of mammalian hosts. While almost all spore-forming organisms use transmembrane germinant receptors to trigger germination, C. difficile uses the pseudoprotease CspC to sense bile salt germinants. CspC activates the related subtilisin-like protease CspB, which then proteolytically activates the cortex hydrolase SleC. Activated SleC degrades the protective spore cortex layer, a step that is essential for germination to proceed. Since CspC incorporation into spores also depends on CspA, a related pseudoprotease domain, Csp family proteins play a critical role in germination. However, how Csps are incorporated into spores remains unknown. In this study, we demonstrate that incorporation of the CspC, CspB, and CspA germination regulators into spores depends on CD0311 (renamed GerG), a previously uncharacterized hypothetical protein. The reduced levels of Csps in gerG spores correlate with reduced responsiveness to bile salt germinants and increased germination heterogeneity in single-spore germination assays. Interestingly, asparagine-rich repeat sequences in GerG’s central region facilitate spontaneous gel formation in vitro even though they are dispensable for GerG-mediated control of germination. Since GerG is found exclusively in C. difficile, our results suggest that exploiting GerG function could represent a promising avenue for developing C. difficile-specific anti-infective therapies.
DOI
Force generation by titin folding
Zsolt Mártonfalvi, Pasquale Bianco, Katalin Naftz, György G. Ferenczy, Miklós Kellermayer
Titin is a giant protein that provides elasticity to muscle. As the sarcomere is stretched, titin extends hierarchically according to the mechanics of its segments. Whether titin's globular domains unfold during this process and how such unfolded domains might contribute to muscle contractility are strongly debated. To explore the force-dependent folding mechanisms, here we manipulated skeletal-muscle titin molecules with high-resolution optical tweezers. In force-clamp mode, after quenching the force (<10 pN), extension fluctuated without resolvable discrete events. In position-clamp experiments the time-dependent force trace contained rapid fluctuations and a gradual increase of average force, indicating that titin can develop force via dynamic transitions between its structural states en route to the native conformation. In 4 M urea, which destabilizes H-bonds hence the consolidated native domain structure, the net force increase disappeared but the fluctuations persisted. Thus, whereas net force generation is caused by the ensemble folding of the elastically-coupled domains, force fluctuations arise due to a dynamic equilibrium between unfolded and molten-globule states. Monte-Carlo simulations incorporating a compact molten-globule intermediate in the folding landscape recovered all features of our nanomechanics results. The ensemble molten-globule dynamics delivers significant added contractility that may assist sarcomere mechanics, and it may reduce the dissipative energy loss associated with titin unfolding/refolding during muscle contraction/relaxation cycles.
DOI
Titin is a giant protein that provides elasticity to muscle. As the sarcomere is stretched, titin extends hierarchically according to the mechanics of its segments. Whether titin's globular domains unfold during this process and how such unfolded domains might contribute to muscle contractility are strongly debated. To explore the force-dependent folding mechanisms, here we manipulated skeletal-muscle titin molecules with high-resolution optical tweezers. In force-clamp mode, after quenching the force (<10 pN), extension fluctuated without resolvable discrete events. In position-clamp experiments the time-dependent force trace contained rapid fluctuations and a gradual increase of average force, indicating that titin can develop force via dynamic transitions between its structural states en route to the native conformation. In 4 M urea, which destabilizes H-bonds hence the consolidated native domain structure, the net force increase disappeared but the fluctuations persisted. Thus, whereas net force generation is caused by the ensemble folding of the elastically-coupled domains, force fluctuations arise due to a dynamic equilibrium between unfolded and molten-globule states. Monte-Carlo simulations incorporating a compact molten-globule intermediate in the folding landscape recovered all features of our nanomechanics results. The ensemble molten-globule dynamics delivers significant added contractility that may assist sarcomere mechanics, and it may reduce the dissipative energy loss associated with titin unfolding/refolding during muscle contraction/relaxation cycles.
DOI
Thursday, January 26, 2017
Single Upconversion Nanoparticle–Bacterium Cotrapping for Single-Bacterium Labeling and Analysis
Hongbao Xin, Yuchao Li, Dekang Xu, Yueli Zhang, Chia-Hung Chen, Baojun Li
Detecting and analyzing pathogenic bacteria in an effective and reliable manner is crucial for the diagnosis of acute bacterial infection and initial antibiotic therapy. However, the precise labeling and analysis of bacteria at the single-bacterium level are a technical challenge but very important to reveal important details about the heterogeneity of cells and responds to environment. This study demonstrates an optical strategy for single-bacterium labeling and analysis by the cotrapping of single upconversion nanoparticles (UCNPs) and bacteria together. A single UCNP with an average size of ≈120 nm is first optically trapped. Both ends of a single bacterium are then trapped and labeled with single UCNPs emitting green light. The labeled bacterium can be flexibly moved to designated locations for further analysis. Signals from bacteria of different sizes are detected in real time for single-bacterium analysis. This cotrapping method provides a new approach for single-pathogenic-bacterium labeling, detection, and real-time analysis at the single-particle and single-bacterium level.
Detecting and analyzing pathogenic bacteria in an effective and reliable manner is crucial for the diagnosis of acute bacterial infection and initial antibiotic therapy. However, the precise labeling and analysis of bacteria at the single-bacterium level are a technical challenge but very important to reveal important details about the heterogeneity of cells and responds to environment. This study demonstrates an optical strategy for single-bacterium labeling and analysis by the cotrapping of single upconversion nanoparticles (UCNPs) and bacteria together. A single UCNP with an average size of ≈120 nm is first optically trapped. Both ends of a single bacterium are then trapped and labeled with single UCNPs emitting green light. The labeled bacterium can be flexibly moved to designated locations for further analysis. Signals from bacteria of different sizes are detected in real time for single-bacterium analysis. This cotrapping method provides a new approach for single-pathogenic-bacterium labeling, detection, and real-time analysis at the single-particle and single-bacterium level.
Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers
Yongli Zhang
Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are universal molecular engines that drive membrane fusion. Particularly, synaptic SNAREs mediate fast calcium-triggered fusion of neurotransmitter-containing vesicles with plasma membranes for synaptic transmission, the basis of all thought and action. During membrane fusion, complementary SNAREs located on two apposed membranes (often called t- and v-SNAREs) join together to assemble into a parallel four-helix bundle, releasing the energy to overcome the energy barrier for fusion. A long-standing hypothesis suggests that SNAREs act like a zipper to draw the two membranes into proximity and thereby force them to fuse. However, a quantitative test of this SNARE zippering hypothesis was hindered by difficulties to determine the energetics and kinetics of SNARE assembly and to identify the relevant folding intermediates. Here, we first review different approaches that have been applied to study SNARE assembly and then focus on high-resolution optical tweezers (OTs). We summarize the folding energies, kinetics, and pathways of both wild-type and mutant SNARE complexes derived from this new approach. These results show that synaptic SNAREs assemble in four distinct stages with different functions: slow N-terminal domain (NTD) association initiates SNARE assembly; a middle domain (MD) suspends and controls SNARE assembly; and rapid sequential zippering of the C-terminal domain (CTD) and the linker domain (LD) directly drive membrane fusion. In addition, the kinetics and pathway of the stage-wise assembly are shared by other SNARE complexes. These measurements prove the SNARE zippering hypothesis and suggest new mechanisms for SNARE assembly regulated by other proteins.
Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are universal molecular engines that drive membrane fusion. Particularly, synaptic SNAREs mediate fast calcium-triggered fusion of neurotransmitter-containing vesicles with plasma membranes for synaptic transmission, the basis of all thought and action. During membrane fusion, complementary SNAREs located on two apposed membranes (often called t- and v-SNAREs) join together to assemble into a parallel four-helix bundle, releasing the energy to overcome the energy barrier for fusion. A long-standing hypothesis suggests that SNAREs act like a zipper to draw the two membranes into proximity and thereby force them to fuse. However, a quantitative test of this SNARE zippering hypothesis was hindered by difficulties to determine the energetics and kinetics of SNARE assembly and to identify the relevant folding intermediates. Here, we first review different approaches that have been applied to study SNARE assembly and then focus on high-resolution optical tweezers (OTs). We summarize the folding energies, kinetics, and pathways of both wild-type and mutant SNARE complexes derived from this new approach. These results show that synaptic SNAREs assemble in four distinct stages with different functions: slow N-terminal domain (NTD) association initiates SNARE assembly; a middle domain (MD) suspends and controls SNARE assembly; and rapid sequential zippering of the C-terminal domain (CTD) and the linker domain (LD) directly drive membrane fusion. In addition, the kinetics and pathway of the stage-wise assembly are shared by other SNARE complexes. These measurements prove the SNARE zippering hypothesis and suggest new mechanisms for SNARE assembly regulated by other proteins.
The frequency-dependent response of single aerosol particles to vapour phase oscillations and its application in measuring diffusion coefficients
Thomas C. Preston, James F. Davies and Kevin R. Wilson
A new method for measuring diffusion in the condensed phase of single aerosol particles is proposed and demonstrated. The technique is based on the frequency-dependent response of a binary particle to oscillations in the vapour phase of one of its chemical components. We discuss how this physical situation allows for what would typically be a non-linear boundary value problem to be approximately reduced to a linear boundary value problem. For the case of aqueous aerosol particles, we investigate the accuracy of the closed-form analytical solution to this linear problem through a comparison with the numerical solution of the full problem. Then, using experimentally measured whispering gallery modes to track the frequency-dependent response of aqueous particles to relative humidity oscillations, we determine diffusion coefficients as a function of water activity. The measured diffusion coefficients are compared to previously reported values found using the two common experiments: (i) the analysis of the sorption/desorption of water from a particle after a step-wise change to the surrounding relative humidity and (ii) the isotopic exchange of water between a particle and the vapour phase. The technique presented here has two main strengths: first, when compared to the sorption/desorption experiment, it does not require the numerical evaluation of a boundary value problem during the fitting process as a closed-form expression is available. Second, when compared to the isotope exchange experiment, it does not require the use of labeled molecules. Therefore, the frequency-dependent experiment retains the advantages of these two commonly used methods but does not suffer from their drawbacks.
DOI
A new method for measuring diffusion in the condensed phase of single aerosol particles is proposed and demonstrated. The technique is based on the frequency-dependent response of a binary particle to oscillations in the vapour phase of one of its chemical components. We discuss how this physical situation allows for what would typically be a non-linear boundary value problem to be approximately reduced to a linear boundary value problem. For the case of aqueous aerosol particles, we investigate the accuracy of the closed-form analytical solution to this linear problem through a comparison with the numerical solution of the full problem. Then, using experimentally measured whispering gallery modes to track the frequency-dependent response of aqueous particles to relative humidity oscillations, we determine diffusion coefficients as a function of water activity. The measured diffusion coefficients are compared to previously reported values found using the two common experiments: (i) the analysis of the sorption/desorption of water from a particle after a step-wise change to the surrounding relative humidity and (ii) the isotopic exchange of water between a particle and the vapour phase. The technique presented here has two main strengths: first, when compared to the sorption/desorption experiment, it does not require the numerical evaluation of a boundary value problem during the fitting process as a closed-form expression is available. Second, when compared to the isotope exchange experiment, it does not require the use of labeled molecules. Therefore, the frequency-dependent experiment retains the advantages of these two commonly used methods but does not suffer from their drawbacks.
DOI
Rigorous analysis of optical forces between two dielectric planar waveguides immersed in dielectric fluid media
Janderson R. Rodrigues, Vilson R. Almeida
This work presents a rigorous analysis of optical forces between planar waveguides immersed in an arbitrary background medium. This approach exploits the Minkowski stress tensor formulation, which is compared with a normalized version of the dispersion relation method, showing excellent results agreement for different dielectric fluid media. Due to slot-waveguide effect, optical forces from TM modes are more sensitive to changes in the fluid refractive index than the TE counterparts. Furthermore, the repulsive optical force from the antisymmetric TM1 mode becomes stronger for higher refractive indexes, whereas the attractive force of the symmetric TM0 mode becomes weaker. The methodology and results presented in this work provide a rigorous analysis of nano-optomechanical devices actuated by optical forces in a broad range of materials and applications. Therefore, this study may impact areas of light-induced interactions presenting novel optofluidic and optomechanical functionalities, thus finding applications in nanoscale transport,
DOI
This work presents a rigorous analysis of optical forces between planar waveguides immersed in an arbitrary background medium. This approach exploits the Minkowski stress tensor formulation, which is compared with a normalized version of the dispersion relation method, showing excellent results agreement for different dielectric fluid media. Due to slot-waveguide effect, optical forces from TM modes are more sensitive to changes in the fluid refractive index than the TE counterparts. Furthermore, the repulsive optical force from the antisymmetric TM1 mode becomes stronger for higher refractive indexes, whereas the attractive force of the symmetric TM0 mode becomes weaker. The methodology and results presented in this work provide a rigorous analysis of nano-optomechanical devices actuated by optical forces in a broad range of materials and applications. Therefore, this study may impact areas of light-induced interactions presenting novel optofluidic and optomechanical functionalities, thus finding applications in nanoscale transport,
DOI
Rotation and deformation of human red blood cells with light from tapered fiber probes
Xiaoshuai Liu, Jianbin Huang, Yuchao Li, Yao Zhang, Baojun Li
Dynamic rotation and deformation of human red blood cells (RBCs) are extremely important to investigate the survival and mechanical features of cells, which will be of great physiological and pathological significance. Here, we report an optical approach that is capable of both rotating and deforming RBCs with light from two tapered fiber probes (TFPs). With laser beams at the wavelength of 980 nm injected into the TFPs, a single RBC was rotated around different axes while single or multiple RBCs were stretched by adjusting the points of action and magnitude of the optical forces from the TFPs. The biological safety of the approach was also discussed by taking the laser power required into account.
DOI
Dynamic rotation and deformation of human red blood cells (RBCs) are extremely important to investigate the survival and mechanical features of cells, which will be of great physiological and pathological significance. Here, we report an optical approach that is capable of both rotating and deforming RBCs with light from two tapered fiber probes (TFPs). With laser beams at the wavelength of 980 nm injected into the TFPs, a single RBC was rotated around different axes while single or multiple RBCs were stretched by adjusting the points of action and magnitude of the optical forces from the TFPs. The biological safety of the approach was also discussed by taking the laser power required into account.
DOI
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