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| 2. |
- 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. |
- 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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| 5. |
- 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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| 6. |
- 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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| 7. |
- Persson, Malin, 1983-, et al.
(författare)
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In Vitro Motility Assay Studies at Low [MgATP] - Evidence For Inter-Head Cooperativity in Fast Skeletal Myosin II
- 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 idea that fast skeletal myosin II exhibits processivity with sequential actions of the two myosin heads during muscle contraction has long been a topic of discussion but with appreciable difficulties to obtain conclusive experimental results. The difficulties may be related to a limited processivity (few sequential head actions) that is of greatest importance at low velocity (e.g. high resistive load), difficult to study accurately in vitro. In order to aid the investigations we here make use of recent evidence that the bipyridine drug amrinone inhibits the strain-dependent ADP-release step of myosin II with expected enhancement of processivity (Albet-Torres et al., J Biol Chem., 2009); Månsson, Biophys J., 2010). For accurate velocity measurements, the recombinant expressed capping protein CapZ (Soeno et. al., J Muscle Res Cell Motil., 1998) was fluorescence labeled. Nanometer tracking was achieved by two-dimensional Gaussian fits to single molecule fluorescence intensity profiles representing CapZ attached to the trailing actin filament end. Importantly, the heavy meromyosin (HMM) propelled actin filament sliding velocity (1mM MgATP) for CapZ-capped filaments was comparable to that of uncapped filaments at different ionic strengths and HMM surface densities. Studies at low [MgATP] (5-30mM) suggests a non-linearity in the [MgATP]-velocity plot that was enhanced by 1mM amrinone. The result is in contrast to the linearity expected for independent myosin heads and could be interpreted as an apparent velocity dependence of the myosin step length. This is in accordance with the idea of limited processivity of myosin II if it is assumed that a doubled apparent step length corresponds to sequential action of the two heads. Further insight into the mechanism will be obtained using proteolytically prepared one-headed HMM and e.g. studies with external loads on actin.
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| 8. |
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| 9. |
- Persson, Malin, et al.
(författare)
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Nonlinear Cross-Bridge Elasticity and Post-Power-Stroke Events in Fast Skeletal Muscle Actomyosin
- 2013
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 105:8, s. 1871-1881
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Tidskriftsartikel (refereegranskat)abstract
- Generation-of force and movement by actomyosin cross-bridges is the molecular basis of muscle contraction, but generally accepted ideas about cross-bridge properties have recently been questioned. Of the utmost significance, evidence for nonlinear cross-bridge elasticity has been presented. We here investigate how this and other newly discovered or postulated phenomena would modify cross-bridge operation, with focus on post-power-stroke events. First, as an experimental basis, we present evidence for a hyperbolic [MgATP]-velocity relationship of heavy-meromyosin-propelled actin filaments in the in vitro motility assay using fast rabbit skeletal muscle myosin (28-29 degrees C). As the hyperbolic [MgATP]-velocity relationship was not consistent with interhead cooperativity, we developed a cross-bridge model with independent myosin heads and strain-dependent interstate transition rates. The model, implemented with inclusion of MgATP-independent detachment from the rigor state, as suggested by previous single-molecule mechanics experiments, accounts well for the [MgATP]-velocity relationship if nonlinear cross-bridge elasticity is assumed, but not if linear cross-bridge elasticity is assumed. In addition, a better fit is obtained with load-independent than with load-dependent MgATP-induced detachment rate. We discuss our results in relation to previous data showing a nonhyperbolic [MgATP1-velocity relationship when actin filaments are propelled by myosin subfragment 1 or full-length myosin. We also consider the implications of our results for characterization of the cross-bridge elasticity in the filament lattice of muscle.
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| 10. |
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