A simple method for automatic tracking of actin filaments in the motility assay

1996 ◽  
Vol 17 (4) ◽  
pp. 497-506 ◽  
Author(s):  
Steven B. Marston ◽  
Iain D. C. Fraser ◽  
Wu Bing ◽  
Giles Roper
Keyword(s):  
Actin Filaments ◽  
Motility Assay ◽  
Simple Method ◽  
Automatic Tracking

scholarly journals A simple method for measuring the relative force exerted by myosin on actin filaments in the in vitro motility assay: evidence that tropomyosin and troponin increase force in single thin filaments

2000 ◽  
Vol 350 (3) ◽  
pp. 693-699 ◽  
Author(s):  
Wu BING ◽  
Adam KNOTT ◽  
Steven B. MARSTON
Keyword(s):  
Actin Filaments ◽  
Isometric Force ◽  
Critical Concentration ◽  
Isometric Tension ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
Simple Method ◽  
In Vitro Motility ◽  
Muscle Tropomyosin

We have studied the effect of an internal load on the movement of actin filaments over a bed of heavy meromyosin (HMM) in the invitro motility assay. Immobilized α-actinin can bind to actin filaments reversibly and ultimately stop the filaments from moving. Above a critical concentration of α-actinin, thin filament velocity rapidly diminished to zero. The fraction of thin motile filaments decreased linearly to zero with increasing α-actinin concentration. The concentration of α-actinin needed to stop all filaments from moving (0.8µg/ml with actin) was very consistent both within and between experiments. In the present study we have defined the ‘index of retardation’ as the concentration of α-actinin needed to stop all filament movement, and we propose that this index is a measure of the isometric force exerted by HMM on actin filaments. When we measured the effect of immobilized α-actinin on motility in the presence of 10mM Pi we found that the index of retardation was 0.62±0.07 (n = 3) times that in the absence of Pi. This observation is in agreement with the reduction of isometric tension in chemically-skinned muscle due to Pi. In a series of comparative experiments we observed that tropomyosin and troponin increase the index of retardation and that the degree of increase depends upon the tropomyosin isoform studied. The index of retardation of actin is increased 1.8-fold by skeletal-muscle tropomyosin, and 3-fold by both cardiac-muscle and smooth-muscle tropomyosin. In the presence of troponin the index of retardation is 2.9–3.4-fold greater than that of actin with all tropomyosin isoforms.

The in vitro motility assay parameters of actin filaments from Mytilus edulis exposed in vivo to copper ions

2009 ◽  
Vol 491 (1-2) ◽  
pp. 32-38 ◽  
Author(s):  
Natalia N. Vikhoreva ◽  
Petr G. Vikhorev ◽  
Maria A. Fedorova ◽  
Ralf Hoffmann ◽  
Alf Månsson ◽  
...  
Keyword(s):  
Mytilus Edulis ◽  
Actin Filaments ◽  
Copper Ions ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility

scholarly journals Collective and contractile filament motions in the myosin motility assay

Soft Matter ◽  
2020 ◽  
Vol 16 (6) ◽  
pp. 1548-1559
Author(s):  
Wonyeong Jung ◽  
Luke A. Fillenwarth ◽  
Atsushi Matsuda ◽  
Jing Li ◽  
Yasuhiro Inoue ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Computational Study ◽  
Motility Assay ◽  
Cross Linking ◽  
Collective Behaviors

In this computational study of the myosin motility assay, we demonstrated that volume-exclusion effects lead to distinct collective behaviors of actin filaments, whereas actin cross-linking proteins induce contractile behaviors of actin filaments.

scholarly journals How VASP enhances actin-based motility

2003 ◽  
Vol 163 (1) ◽  
pp. 131-142 ◽  
Author(s):  
Stanislav Samarin ◽  
Stéphane Romero ◽  
Christine Kocks ◽  
Dominique Didry ◽  
Dominique Pantaloni ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Living Cells ◽  
Catalytic Cycle ◽  
Motility Assay ◽  
Regulatory Proteins ◽  
Capping Proteins ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

The function of vasodilator-stimulated phosphoprotein (VASP) in motility is analyzed using a biomimetic motility assay in which ActA-coated microspheres propel themselves in a medium containing actin, the Arp2/3 complex, and three regulatory proteins in the absence or presence of VASP. Propulsion is linked to cycles of filament barbed end attachment-branching-detachment-growth in which the ActA-activated Arp2/3 complex incorporates at the junctions of branched filaments. VASP increases the velocity of beads. VASP increases branch spacing of filaments in the actin tail, as it does in lamellipodia in living cells. The effect of VASP on branch spacing of Arp2/3-induced branched actin arrays is opposed to the effect of capping proteins. However, VASP does not compete with capping proteins for binding barbed ends of actin filaments. VASP enhances branched actin polymerization only when ActA is immobilized on beads or on Listeria. VASP increases the rate of dissociation of the branch junction from immobilized ActA, which is the rate-limiting step in the catalytic cycle of site-directed filament branching.

Force on Single Actin Filaments in a Motility Assay Measured with an Optical Trap

1993 ◽  
pp. 331-337 ◽  
Author(s):  
R. M. Simmonst ◽  
J. T. Finer ◽  
H. M. Warrick ◽  
B. Kralik ◽  
S. Chu ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Optical Trap ◽  
Motility Assay

Actin-based motor properties of native myosin VIIa

2002 ◽  
Vol 115 (2) ◽  
pp. 445-450 ◽  
Author(s):  
Igor P. Udovichenko ◽  
Daniel Gibbs ◽  
David S. Williams
Keyword(s):  
Atpase Activity ◽  
Inner Ear ◽  
Actin Filaments ◽  
Maximal Activity ◽  
Motility Assay ◽  
Calmodulin Binding ◽  
Retinal Tissue ◽  
Protein Functions ◽  
Bona Fide ◽  
Myosin Viia

Myosin VIIa has critical roles in the inner ear and the retina. To help understand how this protein functions, native myosin VIIa was tested for mechanoenzymatic properties. Myosin VIIa was immunoprecipitated from retinal tissue and found to be associated with calmodulin in a Ca2+-sensitive manner. Myosin VIIa Mg-ATPase activity was detected; in the absence of Ca2+ (i.e. with bound calmodulin), it was stimulated by f-actin with a Kcat of 4.3 s–1 and with 7 μM actin required for half-maximal activity. In a sliding filament motility assay, myosin VIIa moved actin filaments with a velocity of 190 nm s–1. These results demonstrate that myosin VIIa is a calmodulin-binding protein and a bona fide actin-based motor.

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