Johanna Forsting, Julia Kraxner, Hannes Witt, Andreas Janshoff, Sarah Köster
Intermediate filaments (IFs) are part of the cytoskeleton of eukaryotic cells and, therefore, are largely responsible for the cell’s mechanical properties. IFs are characterized by a pronounced extensibility and remarkable resilience that enable them to support cells in extreme situations. Previous experiments showed that, under strain, α-helices in vimentin IFs might unfold to β-sheets. Upon repeated stretching, the filaments soften; however, the remaining plastic strain is negligible. Here, we observe that vimentin IFs do not recover their original stiffness on reasonable time scales, and we explain these seemingly contradicting results by introducing a third, less well-defined conformational state. Reversibility on the nanoscale can be fully rescued by introducing cross-linkers that prevent transition to the β-sheet. Our results classify IFs as a nanomaterial with intriguing mechanical properties, which is likely to play a major role for the cell’s local adaption to external stimuli.
DOI
Thursday, October 31, 2019
Direct measurement of electrostatic interactions between poly(methyl methacrylate) microspheres with optical laser tweezers
Kyu Hwan Choi, Dong Woo Kang, Kyung Hak Kim, Jiwon Kim, Youngbok Lee, Sang Hyuk Im and Bum Jun Park
In this study, we measured the force of electrostatic interactions between poly(methyl methacrylate) (PMMA) particles dispersed in organic solvent mixtures of cyclohexyl bromide (CHB) and n-decane. Optical laser tweezers were employed to directly measure interactive forces between paired PMMA particles in a CHB medium that contained n-decane in various volume ratios. CHB, having a moderate dielectric constant, provided an environment with a high charge storage capacity. The addition of n-decane lowered the effective refractive index of the medium, which increased the optical trapping efficiency. We also fabricated microscope flow cells with a commonly used UV-curable adhesive and quantified the effects of dissolved adhesive compounds through interactive force measurements and nuclear magnetic resonance analysis. In addition, we studied the impact of CHB dissociation into H+ and Br− ions, which could screen electrostatic interactions.
DOI
In this study, we measured the force of electrostatic interactions between poly(methyl methacrylate) (PMMA) particles dispersed in organic solvent mixtures of cyclohexyl bromide (CHB) and n-decane. Optical laser tweezers were employed to directly measure interactive forces between paired PMMA particles in a CHB medium that contained n-decane in various volume ratios. CHB, having a moderate dielectric constant, provided an environment with a high charge storage capacity. The addition of n-decane lowered the effective refractive index of the medium, which increased the optical trapping efficiency. We also fabricated microscope flow cells with a commonly used UV-curable adhesive and quantified the effects of dissolved adhesive compounds through interactive force measurements and nuclear magnetic resonance analysis. In addition, we studied the impact of CHB dissociation into H+ and Br− ions, which could screen electrostatic interactions.
DOI
Two-Stage Collapse of PNIPAM Brushes: Viscoelastic Changes Revealed by an Interferometric Laser Technique
David van Duinen, Hans-Jürgen Butt, Rüdiger Berger
Many temperature-responsive polymers exhibit a single-phase transition at the lower critical solution temperature (LCST). One exception is poly(N-isopropylacryamide) (PNIPAM). PNIPAM brush layers (51 ± 3 nm thick) that are end-grafted onto glass beads collapse in two stages. The viscoelastic changes of a PNIPAM brush layers were investigated with an interferometric laser method at different temperatures. This method is able to measure the two-stage collapse of beads coated with a polymer brush layer. When these beads are situated close to a hydrophilic glass surface, they exhibit Brownian motion. As this Brownian motion changes with temperature, it reveals the collapse of the polymer layer. The characteristic spectrum of the Brownian motion of beads are modelled by a damped harmonic oscillator, where the polymer layer acts as both spring and damping. The change of the Brownian motion spectrum with temperature indicates two transitions of the PNIPAM brush layer, one at 36 °C and one at 46 °C. We attribute the first transition to the LCST volume collapse of PNIPAM. Here, changes of the density and viscosity of the brush dominate. The second transition is dominated by a stiffening of the brush layer.
DOI
Many temperature-responsive polymers exhibit a single-phase transition at the lower critical solution temperature (LCST). One exception is poly(N-isopropylacryamide) (PNIPAM). PNIPAM brush layers (51 ± 3 nm thick) that are end-grafted onto glass beads collapse in two stages. The viscoelastic changes of a PNIPAM brush layers were investigated with an interferometric laser method at different temperatures. This method is able to measure the two-stage collapse of beads coated with a polymer brush layer. When these beads are situated close to a hydrophilic glass surface, they exhibit Brownian motion. As this Brownian motion changes with temperature, it reveals the collapse of the polymer layer. The characteristic spectrum of the Brownian motion of beads are modelled by a damped harmonic oscillator, where the polymer layer acts as both spring and damping. The change of the Brownian motion spectrum with temperature indicates two transitions of the PNIPAM brush layer, one at 36 °C and one at 46 °C. We attribute the first transition to the LCST volume collapse of PNIPAM. Here, changes of the density and viscosity of the brush dominate. The second transition is dominated by a stiffening of the brush layer.
DOI
Cardiomyopathy-associated mutations in tropomyosin differently affect actin–myosin interaction at single-molecule and ensemble levels
Galina V. Kopylova, Daniil V. Shchepkin, Salavat R. Nabiev, Alexander M. Matyushenko, Natalia A. Koubassova, Dmitrii I. Levitsky, Sergey Y. Bershitsky
In the heart, mutations in the TPM1 gene encoding the α-isoform of tropomyosin lead, in particular, to the development of hypertrophic and dilated cardiomyopathies. We compared the effects of hypertrophic, D175N and E180G, and dilated, E40K and E54K, cardiomyopathy mutations in TPM1 gene on the properties of single actin–myosin interactions and the characteristics of the calcium regulation in an ensemble of myosin molecules immobilised on a glass surface and interacting with regulated thin filaments. Previously, we showed that at saturating Ca2+ concentration the presence of Tpm on the actin filament increases the duration of the interaction. Here, we found that the studied Tpm mutations differently affected the duration: the D175N mutation reduced it compared to WT Tpm, while the E180G mutation increased it. Both dilated mutations made the duration of the interaction even shorter than with F-actin. The duration of the attached state of myosin to the thin filament in the optical trap did not correlate to the sliding velocity of thin filaments and its calcium sensitivity in the in vitro motility assay. We suppose that at the level of the molecular ensemble, the cooperative mechanisms prevail in the manifestation of the effects of cardiomyopathy-associated mutations in Tpm.
DOI
In the heart, mutations in the TPM1 gene encoding the α-isoform of tropomyosin lead, in particular, to the development of hypertrophic and dilated cardiomyopathies. We compared the effects of hypertrophic, D175N and E180G, and dilated, E40K and E54K, cardiomyopathy mutations in TPM1 gene on the properties of single actin–myosin interactions and the characteristics of the calcium regulation in an ensemble of myosin molecules immobilised on a glass surface and interacting with regulated thin filaments. Previously, we showed that at saturating Ca2+ concentration the presence of Tpm on the actin filament increases the duration of the interaction. Here, we found that the studied Tpm mutations differently affected the duration: the D175N mutation reduced it compared to WT Tpm, while the E180G mutation increased it. Both dilated mutations made the duration of the interaction even shorter than with F-actin. The duration of the attached state of myosin to the thin filament in the optical trap did not correlate to the sliding velocity of thin filaments and its calcium sensitivity in the in vitro motility assay. We suppose that at the level of the molecular ensemble, the cooperative mechanisms prevail in the manifestation of the effects of cardiomyopathy-associated mutations in Tpm.
DOI
Monday, October 28, 2019
Memory effects in single-molecule force spectroscopy measurements of biomolecular folding
Andrew G.T. Pyo and Michael T Woodside
Folding is generally assumed to be a Markov process, without memory. When the molecular motion is coupled to that of a probe as in single-molecule force spectroscopy (SMFS) experiments, however, theory predicts that the coupling to a second Markov process should induce memory when monitoring a projection of the full multi-dimensional motion onto a reduced coordinate. We developed a method to evaluate the time constant of the induced memory from its effects on the autocorrelation function, which can be readily determined from experimental data. Applying this method to both simulated SMFS measurements and experimental trajectories of DNA hairpin folding measured by optical tweezers as a model system, we validated the prediction that the linker induces memory. For these measurements, the timescale of the induced memory was found to be similar to the time required for the force probe to respond to changes in the molecule, and in the regime where the experimentally observed dynamics were not significantly perturbed by probe-molecule coupling artifacts. Memory effects are thus a general feature of SMFS measurements induced by the mechanical connection between the molecule and force probe that should be considered when interpreting experimental data.
DOI
Folding is generally assumed to be a Markov process, without memory. When the molecular motion is coupled to that of a probe as in single-molecule force spectroscopy (SMFS) experiments, however, theory predicts that the coupling to a second Markov process should induce memory when monitoring a projection of the full multi-dimensional motion onto a reduced coordinate. We developed a method to evaluate the time constant of the induced memory from its effects on the autocorrelation function, which can be readily determined from experimental data. Applying this method to both simulated SMFS measurements and experimental trajectories of DNA hairpin folding measured by optical tweezers as a model system, we validated the prediction that the linker induces memory. For these measurements, the timescale of the induced memory was found to be similar to the time required for the force probe to respond to changes in the molecule, and in the regime where the experimentally observed dynamics were not significantly perturbed by probe-molecule coupling artifacts. Memory effects are thus a general feature of SMFS measurements induced by the mechanical connection between the molecule and force probe that should be considered when interpreting experimental data.
DOI
Formation Mechanism and Fluorescence Characterization of a Transient Assembly of Nanoparticles Generated by Femtosecond Laser Trapping
Wei-Yi Chiang, Jim Jui-Kai Chen, Anwar Usman, Tetsuhiro Kudo, kangwei Xia, Jia Su, Teruki Sugiyama, Johan Hofkens, Hiroshi Masuhara
Femtosecond (fs) laser trapping of dielectric nanoparticles displays a unique trapping and directional ejection phenomenon which is never observed in continuous-wave (cw) laser trapping. We studied the fs laser trapping and considered its dynamics and mechanism in terms of gathering, association and ejection of nanoparticles. By tuning solution viscosity through adding ethylene glycol (EG), the trapping behavior by fs and cw lasers are examined and compared with each other. Viscous solution slows down the diffusional and rotational movement of nanoparticles and increases the chance of nanoparticle assembly in the trapping site, which eventually leads to the novel ejection. We took advantage of fluorescence measurement to confirm the formation of the nanoparticle assembly during fs laser trapping. A red emission irregularly appeared in the fluorescence spectra, which strongly supports the transient assembly formation in molecular level.
DOI
Femtosecond (fs) laser trapping of dielectric nanoparticles displays a unique trapping and directional ejection phenomenon which is never observed in continuous-wave (cw) laser trapping. We studied the fs laser trapping and considered its dynamics and mechanism in terms of gathering, association and ejection of nanoparticles. By tuning solution viscosity through adding ethylene glycol (EG), the trapping behavior by fs and cw lasers are examined and compared with each other. Viscous solution slows down the diffusional and rotational movement of nanoparticles and increases the chance of nanoparticle assembly in the trapping site, which eventually leads to the novel ejection. We took advantage of fluorescence measurement to confirm the formation of the nanoparticle assembly during fs laser trapping. A red emission irregularly appeared in the fluorescence spectra, which strongly supports the transient assembly formation in molecular level.
DOI
TGFβ1 single-nucleotide polymorphism C-509T alters mucosal cell function in pediatric eosinophilic esophagitis
L. D. Duong, R. Rawson, A. Bezryadina, M. C. Manresa, R. O. Newbury, R. Dohil, Z. Liu, K. Barrett, R. Kurten & S. S. Aceves
Eosinophilic esophagitis (EoE) is a chronic Th2 antigen-driven disorder associated with tissue remodeling. Inflammation and remodeling lead to esophageal rigidity, strictures, and dysphagia. TGFβ1 drives esophageal remodeling including epithelial barrier dysfunction and subepithelial fibrosis. A functional SNP in the TGFβ1 gene that increases its transcription (C-509T) is associated with elevated numbers of esophageal TGFβ1-expressing cells. We utilized esophageal biopsies and fibroblasts from TT-genotype EoE children to understand if TGFβ1 influenced fibroblast and epithelial cell function in vivo. Genotype TT EoE esophageal fibroblasts had higher baseline TGFβ1, collagen1α1, periostin, and MMP2 (p < 0.05) gene expression and distinct contractile properties compared with CC genotype (n = 6 subjects per genotype). In vitro TGFβ1 exposure caused greater induction of target gene expression in genotype CC fibroblasts (p < 0.05). Esophageal biopsies from TT-genotype subjects had significantly less epithelial membrane-bound E-cadherin (p < 0.01) and wider cluster distribution at nanometer resolution. TGFβ1 treatment of stratified primary human esophageal epithelial cells and spheroids disrupted transepithelial resistance (p < 0.001) and E-cadherin localization (p < 0.0001). A TGFβ1-receptor-I inhibitor improved TGFβ1-mediated E-cadherin mislocalization. These data suggest that EoE severity can depend on genotypic differences that increase in vivo exposure to TGFβ1. TGFβ1 inhibition may be a useful therapy in subsets of EoE patients.
DOI
Eosinophilic esophagitis (EoE) is a chronic Th2 antigen-driven disorder associated with tissue remodeling. Inflammation and remodeling lead to esophageal rigidity, strictures, and dysphagia. TGFβ1 drives esophageal remodeling including epithelial barrier dysfunction and subepithelial fibrosis. A functional SNP in the TGFβ1 gene that increases its transcription (C-509T) is associated with elevated numbers of esophageal TGFβ1-expressing cells. We utilized esophageal biopsies and fibroblasts from TT-genotype EoE children to understand if TGFβ1 influenced fibroblast and epithelial cell function in vivo. Genotype TT EoE esophageal fibroblasts had higher baseline TGFβ1, collagen1α1, periostin, and MMP2 (p < 0.05) gene expression and distinct contractile properties compared with CC genotype (n = 6 subjects per genotype). In vitro TGFβ1 exposure caused greater induction of target gene expression in genotype CC fibroblasts (p < 0.05). Esophageal biopsies from TT-genotype subjects had significantly less epithelial membrane-bound E-cadherin (p < 0.01) and wider cluster distribution at nanometer resolution. TGFβ1 treatment of stratified primary human esophageal epithelial cells and spheroids disrupted transepithelial resistance (p < 0.001) and E-cadherin localization (p < 0.0001). A TGFβ1-receptor-I inhibitor improved TGFβ1-mediated E-cadherin mislocalization. These data suggest that EoE severity can depend on genotypic differences that increase in vivo exposure to TGFβ1. TGFβ1 inhibition may be a useful therapy in subsets of EoE patients.
DOI
Probing the Functional Role of Physical Motion in Development
Moritz Kreysing
Spatiotemporal organization during development has frequently been proposed to be explainable by reaction-transport models, where biochemical reactions couple to physical motion. However, whereas genetic tools allow causality of molecular players to be dissected via perturbation experiments, the functional role of physical transport processes, such as diffusion and cytoplasmic streaming, frequently remains untestable. This Perspective explores the challenges of validating reaction-transport hypotheses and highlights new opportunities provided by perturbation approaches that specifically target physical transport mechanisms. Using these methods, experimental physics may begin to catch up with molecular biology and find ways to test roles of diffusion and flows in development.
DOI
Spatiotemporal organization during development has frequently been proposed to be explainable by reaction-transport models, where biochemical reactions couple to physical motion. However, whereas genetic tools allow causality of molecular players to be dissected via perturbation experiments, the functional role of physical transport processes, such as diffusion and cytoplasmic streaming, frequently remains untestable. This Perspective explores the challenges of validating reaction-transport hypotheses and highlights new opportunities provided by perturbation approaches that specifically target physical transport mechanisms. Using these methods, experimental physics may begin to catch up with molecular biology and find ways to test roles of diffusion and flows in development.
DOI
Friday, October 25, 2019
Emulsions Stabilized by Fine Dust Particles
Yong Woo Kima, Donghyun Lim, Hyerin Han, Seunghyun Lee, Kyu Hwan Choi, Bum Jun Park
We investigated the capability of fine dust particles (FDPs) to be used as stabilizers for preparing emulsions. With the aid of cationic surfactants, it was found that the FDPs formed oil in water emulsions that were extremely stable for a long time period. The stabilization mechanism was quantitatively analyzed via ζ-potential measurements of surface modified FDPs and interface adsorption experiments of FDP-coated microbeads by using optical laser tweezers.
DOI
We investigated the capability of fine dust particles (FDPs) to be used as stabilizers for preparing emulsions. With the aid of cationic surfactants, it was found that the FDPs formed oil in water emulsions that were extremely stable for a long time period. The stabilization mechanism was quantitatively analyzed via ζ-potential measurements of surface modified FDPs and interface adsorption experiments of FDP-coated microbeads by using optical laser tweezers.
DOI
Interaction of a Polyarginine Peptide with Membranes of Different Mechanical Properties
Matías A. Crosio, Matías A. Via, Candelaria I. Cámara, Agustin Mangiarotti, Mario G. Del Pópolo and Natalia Wilke
The membrane translocation efficiency of cell penetrating peptides (CPPs) has been largely studied, and poly-arginines have been highlighted as particularly active CPPs, especially upon negatively charged membranes. Here we inquire about the influence of membrane mechanical properties in poly-arginine adsorption, penetration and translocation, as well as the subsequent effect on the host membrane. For this, we selected anionic membranes exhibiting different rigidity and fluidity, and exposed them to the nona-arginine KR9C. Three different membrane compositions were investigated, all of them having 50% of the anionic lipid 1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DOPG), thus, ensuring a high affinity of the peptide for membrane surfaces. The remaining 50% was a saturated PC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), an unsaturated PC (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) or a mixture of DOPC with cholesterol. Peptide-membrane interactions were studied using four complementary models for membranes: Langmuir monolayers, Large Unilamellar Vesicles, Black Lipid Membranes and Giant Unilamellar Vesicles. The patterns of interaction of KR9C varied within the different membrane compositions. The peptide strongly adsorbed on membranes with cholesterol, but did not incorporate or translocate them. KR9C stabilized phase segregation in DPPC/DOPG films and promoted vesicle rupture. DOPC/DOPG appeared like the better host for peptide translocation: KR9C adsorbed, inserted and translocated these membranes without breaking them, despite softening was observed.
DOI
The membrane translocation efficiency of cell penetrating peptides (CPPs) has been largely studied, and poly-arginines have been highlighted as particularly active CPPs, especially upon negatively charged membranes. Here we inquire about the influence of membrane mechanical properties in poly-arginine adsorption, penetration and translocation, as well as the subsequent effect on the host membrane. For this, we selected anionic membranes exhibiting different rigidity and fluidity, and exposed them to the nona-arginine KR9C. Three different membrane compositions were investigated, all of them having 50% of the anionic lipid 1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DOPG), thus, ensuring a high affinity of the peptide for membrane surfaces. The remaining 50% was a saturated PC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), an unsaturated PC (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) or a mixture of DOPC with cholesterol. Peptide-membrane interactions were studied using four complementary models for membranes: Langmuir monolayers, Large Unilamellar Vesicles, Black Lipid Membranes and Giant Unilamellar Vesicles. The patterns of interaction of KR9C varied within the different membrane compositions. The peptide strongly adsorbed on membranes with cholesterol, but did not incorporate or translocate them. KR9C stabilized phase segregation in DPPC/DOPG films and promoted vesicle rupture. DOPC/DOPG appeared like the better host for peptide translocation: KR9C adsorbed, inserted and translocated these membranes without breaking them, despite softening was observed.
DOI
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