| 1. |
- Balaz, Martina, et al.
(författare)
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Protein-surface Interactions and Functional Geometry of Surface-adsorbed Myosin Motor Fragments
- 2009
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Ingår i: Biophysical Journal. - : Biophysical Society. - 0006-3495 .- 1542-0086. ; 96:3 Suppl. 1, s. 495A-495A
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Biophysical studies with myosin motor fragments (heavy meromyosin; HMM and subfragment 1; S1) adsorbed to artificial surfaces, are important for elucidation of actomyosin function. In spite of the widespread use of such in vitro motility assays and single molecule studies, little is known about the adsorption geometry and effects of protein-surface interactions on the motor properties. Here, we investigate these factors with focus on HMM using quartz crystal microbalance with dissipation (QCM-D) and total internal reflection fluorescence (TIRF) spectroscopy based ATPase assays. In the latter, we monitored the turnover of Alexa-fluor647-ATP (Alexa-ATP) by surface adsorbed HMM. Studies were performed with HMM/S1 adsorbed to model hydrophilic (SiO2) or hydrophobic (trimethyl-chlorosilane [TMCS] - derivatized) surfaces. The results suggest that adsorption of HMM is weakened on SiO2 (but not on TMCS) at high (245 mM) compared to low (65 mM) ionic strengths. The changes in ionic strength were also associated with structural changes in the protein layer according to QCM-D studies. Moreover, the TIRF based ATPase assay suggested a larger fraction of HMM molecules with low catalytic activity on SiO2. These and other TIRF and QCM-D results, suggest that HMM preferentially adsorbs to negatively charged hydrophilic surfaces via the actin-binding region. In contrast, the majority of the HMM molecules seem to adsorb via their C-terminal tail on moderately hydrophobic surfaces. In the latter case the catalytic sites appear to be close to, but not immobilized on the surface. The results with HMM were compared to, and found consistent with, QCM-D and TIRF-data obtained with S1 motor fragments.
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| 2. |
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| 3. |
- Bengtsson, Elina, et al.
(författare)
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Actomyosin Interactions and Different Structural States of Actin Filaments
- 2013
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Ingår i: Biophysical Journal. - : Biophysical Society. - 0006-3495 .- 1542-0086. ; 104:2 Suppl. 1, s. 480A-481A
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The persistence length (LP) of a polymer is proportional to its flexural rigidity and quantifies the decay length of its tangent angle (for a polymer freely suspended in solution). Further, it has been suggested that the decay length for the sliding direction of heavy meromyosin (HMM) propelled actin filaments in the in vitro motility assay (IVMA) is quantitatively identical to Lp of the free leading filament end. On this assumption we measured LP under different conditions to address a hypothesis that the actin filament exists in different metastable conformations, each characterized by a different flexural rigidity. The following values for Lp (mean 5 95 % confidence limits) were obtained: 1. with phalloidin (Ph) in solution: 12.61 5 0.65 mm (N=809). 2. without Phin solution: 9.07 5 1.06 mm (N=811), 3. with Ph and HMM in solution (rigor):10.21 5 0.75 mm (N=429), 4. without Ph (from IVMA paths; 1 mM MgATP):10.0850.66 mm (N=309), 5. with Ph, IVMA (1 mM MgATP): 11.41 5 0.57 mm (N=243), 6. with Ph, IVMA, 0.05 mM MgATP: 6.30 5 0.27 mm (N=383) and 7. without Ph, IVMA, 0.02-0.05 mM MgATP: 5.33 5 0.37 mm (N=161). The re-sults are consistent with different actin filament states where one is stabilized by phalloidin and one is favored by HMM binding and the absence of Ph. Effects of HMM are consistent with a possible role of the structural state of actin filaments in effective actomyosin motility. The very low LP found for IVMA at low [MgATP] (6-7) may reflect the presence of an actin filament state populated at low average cross-bridge strains, possibly with MgADP at the active site. Alternatively, it may be due to sideways forces produced by increased number of HMM-actin interactions close to the leading filament end.
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| 4. |
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| 5. |
- Bengtsson, Elina, et al.
(författare)
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Altered Structural State of Actin Filaments Upon MYOSIN II Binding
- 2015
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Ingår i: Biophysical Journal. - : Biophysical Society. - 0006-3495 .- 1542-0086. ; 108:2 Suppl. 1, s. 299A-300A
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The paths of actin filaments propelled over a heavy meromyosin (HMM) surface in the in vitro motility assay (IVMA) can statistically be described by a path persistence length (LPP) and has been hypothesized to be proportional to the flexural rigidity of the filaments. Here, we have studied the LPP at high (130 mM) ionic strength along with the persistence length of actin filaments in solution (LPS) to elucidate how HMM binding affects the flexural rigidity of actin filaments. Characterization and control of material properties, such as the path persistence length, is useful in engineered devices that takes advantages of the function of the muscle contractile proteins e.g. for biocomputation. It has been suggested that myosin binding reduces Lpp for phalloidin stabilizedact in filaments. This is consistent with the results presented here where the phalloidin stabilized actin filaments rigidity is reduced to the level of phalloidin free actin filaments in the IVMA. Further, reducing the MgATP concentration in the IVMA would increase the HMM head density along the actin filament hence making the effect of myosin binding more pronounced. A reduced [MgATP] from 1 mM to 0.02-0.05 mM did indeed reduce the LPP from 10-12 mm to 6-7 mm for both phalloidin-stabilized and phalloidin free actin filaments. Additionally, we found a negative correlation between the LPS and the [HMM]/actin ratio. However, this [HMM] dependent reduction observed in LPS was too small to account for the reduction in LPP seen with reduced [MgATP] in the IVMA. Monte-Carlo simulations and theoretical analysis revealed that the large reduction in LPP is consistent with the idea that every head attachment adds an extra angular displacement.(Support from EU-FP7-FET-ABACUS grant number 613044).
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| 6. |
- Bengtsson, Elina, et al.
(författare)
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Myosin-Induced Gliding Patterns at Varied [MgATP] Unveil a Dynamic Actin Filament
- 2016
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 111:7, s. 1465-1477
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Tidskriftsartikel (refereegranskat)abstract
- Actin filaments have key roles in cell motility but are generally claimed to be passive interaction partners in actin-myosin -based motion generation. Here, we present evidence against this static view based on an altered myosin-induced actin filament gliding pattern in an in vitro motility assay at varied [MgATP]. The statistics that characterize the degree of meandering of the actin filament paths suggest that for [MgATP] >= 0.25 mM, the flexural rigidity of heavy meromyosin (HMM)-propelled actin filaments is similar (without phalloidin) or slightly lower (with phalloidin) than that of HMM-free filaments observed in solution without surface tethering. When [MgATP] was reduced to <= 0.1 mM, the actin filament paths in the in vitro motility assay became appreciably more winding in both the presence and absence of phalloidin. This effect of lowered [MgATP] was qualitatively different from that seen when HMM was mixed with ATP-insensitive, N-ethylmaleimide-treated HMM (NEM-HMM; 25-30%). In particular, the addition of NEM-HMM increased a non-Gaussian tail in the path curvature distribution as well as the number of events in which different parts of an actin filament followed different paths. These effects were the opposite of those observed with reduced [MgATP]. Theoretical modeling suggests a 30-40% lowered flexural rigidity of the actin filaments at [MgATP] <= 0.1 mM and local bending of the filament front upon each myosin head attachment. Overall, the results fit with appreciable structural changes in the actin filament during actomyosin-based motion generation, and modulation of the actin filament mechanical properties by the dominating chemomechanical actomyosin state.
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| 7. |
- Bengtsson, Elina, et al.
(författare)
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Winding Actin Filament Paths Provide Mechanistic Insights Into Actomyosin Function
- 2012
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Ingår i: Biophysical Journal. - : Biophysical Society. - 0006-3495 .- 1542-0086. ; 102:3 Suppl. 1, s. 146A-146A
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The statistics of heavy meromyosin (HMM) driven actin filament paths in vitro, and thermal fluctuations of actin filaments suspended in a pseudo 2D-space in solution, can be described by the cosine correlation equation (CCE): = exp(-s/[2*Lp]). Here, q0) and qs) represent tangent angles at distance 0 and s, respectively from one filament end (in solution) or from the starting point of the path. The quantity Lp is the persistence length (proportional to flexural rigidity) of the filament/path. In vitro motility assay (IVMA) studies (27-29oC) were performed along with studies of actin filaments suspended between two cover-slips in solution. Fits to the CCE gave LP = 16.5 5 1.7 mm (mean 5 95 % confidence interval) and 11.1 5 0.6 mm for phalloidin stabilized filaments in solution and propelled by HMM, respectively. In contrast, phalloidin free actin filaments (NHS-rhodamine labeled) exhibited similar LP in solution 10.1 52.1 mm and during HMM propulsion (9.8 5 0.9 mm). The filament paths were modeled using a Monte-Carlo approach updating angular changes in sliding direction at short time intervals (dt) assuming 1. lateral displacements due to cross-bridge forces and 2. thermal fluctuations of the leading filament end. The results suggest that > 3nm average lateral displacement during each actomyosin interaction would reduce LP by > 30 % compared to that of filaments without HMM. The findings are consistent with the following ideas: 1. Actin filaments exist in two different flexural rigidity states, one favored by myosin binding and the other by phalloidin stabilization, 2. Changes in actin filament flexural rigidity is not required for motion generation. 3. The myosin cross-bridges produce minimal lateral movements (< 3 nm) during the power-stroke.
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| 8. |
- Lard, Mercy, et al.
(författare)
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Detection of Single Actin Filaments at Fluorescence Interference Contrast Checkpoints
- 2012
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Ingår i: Biophysical Journal. - : Biophysical Society. - 0006-3495 .- 1542-0086. ; 102:3 Suppl. 1, s. 727A-727A
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- A number of emerging concepts for on-chip biotechnologies replace micro-fluidic flow by active, molecular-motor driven transport of filaments. Examples include applications in bio-simulation, diagnostics, and drug screening. Here we employ actomyosin molecular motors, embedded in nanostructures, as a platform for bio-simulation of the time evolution of motile objects in complex networks. A specific need for this type of application is detection of filaments at specific checkpoints in the device with high signal-to-noise ratio, for example to record the number and speed of filaments at a certain location in the device. To serve this need, we make use of fluorescence interference contrast (FLIC) at thin gold lines running perpendicular to nano-sized polymer resist channels that guide filament motion. We have demonstrated that it is possible to track single or multiple filaments passing over these gold lines, using either an enhanced or quenched fluorescence signal. We will discuss the fine-tuning of the device design, development of an algorithm for analyzing the optical readout signal from these detectors, and explo-ration of the error limits of detection. The results will help establish the viability of active, motor-driven on-chip applications which, among other advantages, offer substantial potential for miniaturization due to the absence of a need for pumps. The results also open for automatic read-out of velocity inhigh-throughput motility assays e.g. for drug discovery or fundamental bio-physical investigations. This work is supported by MONAD, an EU-FP7 collaborative effort.
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| 9. |
- Månsson, Alf, et al.
(författare)
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Nonlinear Actomyosin Elasticity in Muscle?
- 2019
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Ingår i: Biophysical Journal. - : Cell Press. - 0006-3495 .- 1542-0086. ; 116:2, s. 330-346
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Tidskriftsartikel (refereegranskat)abstract
- Cyclic interactions between myosin II motor domains and actin filaments that are powered by turnover of ATP underlie muscle contraction and have key roles in motility of nonmuscle cells. The elastic characteristics of actin-myosin cross-bridges are central in the force-generating process, and disturbances in these properties may lead to disease. Although the prevailing paradigm is that the cross-bridge elasticity is linear (Hookean), recent single-molecule studies suggest otherwise. Despite convincing evidence for substantial nonlinearity of the cross-bridge elasticity in the single-molecule work, this finding has had limited influence on muscle physiology and physiology of other ordered cellular actin-myosin ensembles. Here, we use a biophysical modeling approach to close the gap between single molecules and physiology. The model is used for analysis of available experimental results in the light of possible nonlinearity of the cross-bridge elasticity. We consider results obtained both under rigor conditions (in the absence of ATP) and during active muscle contraction. Our results suggest that a wide range of experimental findings from mechanical experiments on muscle cells are consistent with nonlinear actin-myosin elasticity similar to that previously found in single molecules. Indeed, the introduction of nonlinear cross-bridge elasticity into the model improves the reproduction of key experimental results and eliminates the need for force dependence of the ATP-induced detachment rate, consistent with observations in other single-molecule studies. The findings have significant implications for the understanding of key features of actin-myosin-based production of force and motion in living cells, particularly in muscle, and for the interpretation of experimental results that rely on stiffness measurements on cells or myofibrils.
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| 10. |
- Persson, Malin, et al.
(författare)
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Comparison of electron paramagnetic resonance methods to determine distances between spin labels on human carbonic anhydrase II
- 2001
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 80:6, s. 2886-2897
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Tidskriftsartikel (refereegranskat)abstract
- Four doubly spin-labeled variants of human carbonic anhydrase II and corresponding singly labeled variants were prepared by site-directed spin labeling. The distances between the spin labels were obtained from continuous-wave electron paramagnetic resonance spectra by analysis of the relative intensity of the half-field transition, Fourier deconvolution of line-shape broadening, and computer simulation of line-shape changes. Distances also were determined by four-pulse double electron-electron resonance. For each variant, at least two methods were applicable and reasonable agreement between methods was obtained. Distances ranged from 7 to 24 W. The doubly spin-labeled samples contained some singly labeled protein due to incomplete labeling. The sensitivity of each of the distance determination methods to the noninteracting component was compared.
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