scholarly journals The Formin Inhibitor, SMIFH2, Inhibits Members of the Myosin Superfamily

2020 ◽  
Author(s):  
Yukako Nishimura ◽  
Shidong Shi ◽  
Fang Zhang ◽  
Rong Liu ◽  
Yasuharu Takagi ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Careful Analysis ◽  
Retrograde Flow ◽  
Cultured Fibroblasts ◽  
Skeletal Muscle Myosin ◽  
Biological Studies ◽  
Chemical Screen ◽  
Muscle Myosin ◽  
Fiber Contraction

AbstractThe small molecular inhibitor of formin FH2 domains, SMIFH2, is widely used in cell biological studies. It was selected in a chemical screen as a compound inhibiting formin-driven actin polymerization in vitro, but not polymerization of pure actin, and found to be active against several types of formins from different species (Rizvi et al., 2009). Here, in experiments with cultured fibroblasts, we found that SMIFH2 inhibits retrograde flow of myosin 2 filaments and contraction of stress fibers. We further checked the effect of SMIFH2 on non-muscle myosin 2A and skeletal muscle myosin 2 in vitro and found that SMIFH2 inhibits myosin ATPase activity and ability to translocate actin filaments in the in vitro motility assay. While inhibition of myosin 2A in vitro required somewhat higher concentration of SMIFH2 than inhibition of retrograde flow and stress fiber contraction in cells, inhibition of several other non-muscle myosin types, e.g. mammalian myosin 10, Drosophila myosin 7a and Drosophila myosin 5 by SMIFH2, was equally or more efficient than inhibition of formins. Since actin polymerization and myosin contractility are linked in many cytoskeleton processes, additional careful analysis is needed in each case when function of formins was proposed solely on the basis of experiment with SMIFH2.

scholarly journals The Formin Inhibitor, SMIFH2, Inhibits Members of the Myosin Superfamily

2021 ◽  
pp. jcs.253708
Author(s):  
Yukako Nishimura ◽  
Shidong Shi ◽  
Fang Zhang ◽  
Rong Liu ◽  
Yasuharu Takagi ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Careful Analysis ◽  
Stress Fiber ◽  
Retrograde Flow ◽  
Skeletal Muscle Myosin ◽  
Biological Studies ◽  
Muscle Myosin ◽  
Myosin Superfamily ◽  
Fiber Contraction

The small molecular inhibitor of formin FH2 domains, SMIFH2, is widely used in cell biological studies. It inhibits formin-driven actin polymerization in vitro, but not polymerization of pure actin. It is active against several types of formins from different species (Rizvi et al., 2009). Here, we found that SMIFH2 inhibits retrograde flow of myosin 2 filaments and contraction of stress fibers. We further checked the effect of SMIFH2 on non-muscle myosin 2A and skeletal muscle myosin 2 in vitro and found that SMIFH2 inhibits myosin ATPase activity and ability to translocate actin filaments in the in vitro motility assay. The inhibition of non-muscle myosin 2A in vitro required a higher concentration of SMIFH2 than for the inhibition of retrograde flow and stress fiber contraction in cells. We also found that SMIFH2 inhibits several other non-muscle myosin types, e.g. mammalian myosin 10, Drosophila myosin 7a and Drosophila myosin 5, more efficient than inhibition of formins. These off-target inhibitions demand additional careful analysis in each case when solely SMIFH2 is used to probe formin functions.

scholarly journals ATP-dependent movement of myosin in vitro: characterization of a quantitative assay.

1984 ◽  
Vol 99 (5) ◽  
pp. 1867-1871 ◽  
Author(s):  
M P Sheetz ◽  
R Chasan ◽  
J A Spudich
Keyword(s):  
Skeletal Muscle ◽  
Actin Filaments ◽  
Quantitative Assay ◽  
Vitro Assay ◽  
Skeletal Muscle Myosin ◽  
In Vitro Characterization ◽  
Actin Cables ◽  
Muscle Myosin

Sheetz and Spudich (1983, Nature (Lond.), 303:31-35) showed that ATP-dependent movement of myosin along actin filaments can be measured in vitro using myosin-coated beads and oriented actin cables from Nitella. To establish this in vitro movement as a quantitative assay and to understand better the basis for the movement, we have defined the factors that affect the myosin-bead velocity. Beads coated with skeletal muscle myosin move at a rate of 2-6 micron/s, depending on the myosin preparation. This velocity is independent of myosin concentration on the bead surface for concentrations above a critical value (approximately 20 micrograms myosin/2.5 X 10(9) beads of 1 micron in diameter). Movement is optimal between pH 6.8 and 7.5, at KCl concentrations less than 70 mM, at ATP concentrations greater than 0.1 mM, and at Mg2+ concentrations between 2 and 6 mM. From the temperature dependence of bead velocity, we calculate activation energies of 90 kJ/mol below 22 degrees C and 40 kJ/mol above 22 degrees C. Different myosin species move at their own characteristic velocities, and these velocities are proportional to their actin-activated ATPase activities. Further, the velocities of beads coated with smooth or skeletal muscle myosin correlate well with the known in vivo rates of myosin movement along actin filaments in these muscles. This in vitro assay, therefore, provides a rapid, reproducible method for quantitating the ATP-dependent movement of myosin molecules on actin.

scholarly journals Assembly of smooth muscle myosin into side-polar filaments.

1977 ◽  
Vol 75 (3) ◽  
pp. 990-996 ◽  
Author(s):  
R Craig ◽  
J Megerman
Keyword(s):  
Smooth Muscle ◽  
Opposite Polarity ◽  
Skeletal Muscle Myosin ◽  
Myosin Filaments ◽  
Skeletal Myosin ◽  
Cross Bridges ◽  
Bipolar Filament ◽  
Muscle Myosin

The in vitro assembly of myosin purified from calf aorta muscle has been studied by electron microscopy. Two types of filament are formed: short bipolar filament similar to those formed from skeletal muscle myosin, and longer "side-polar" filaments having cross bridges with a single polarity along the entire length of one side and the opposite polarity along the other side. Unlike the case with skeletal myosin filaments, antiparallel interactions between myosin molecules occur along the whole length of side-polar filaments. The side-polar structure may be related to the in vivo form of myosin in vertebrate smooth muscle.

scholarly journals Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments.

1985 ◽  
Vol 101 (5) ◽  
pp. 1897-1902 ◽  
Author(s):  
J R Sellers ◽  
J A Spudich ◽  
M P Sheetz
Keyword(s):  
Smooth Muscle ◽  
Actin Filaments ◽  
Smooth Muscles ◽  
Smooth Muscle Myosin ◽  
Vitro Assay ◽  
Skeletal Muscle Myosin ◽  
Myosin Filaments ◽  
Muscle Myosin ◽  
Rate Of Movement

In smooth muscles there is no organized sarcomere structure wherein the relative movement of myosin filaments and actin filaments has been documented during contraction. Using the recently developed in vitro assay for myosin-coated bead movement (Sheetz, M.P., and J.A. Spudich, 1983, Nature (Lond.)., 303:31-35), we were able to quantitate the rate of movement of both phosphorylated and unphosphorylated smooth muscle myosin on ordered actin filaments derived from the giant alga, Nitella. We found that movement of turkey gizzard smooth muscle myosin on actin filaments depended upon the phosphorylation of the 20-kD myosin light chains. About 95% of the beads coated with phosphorylated myosin moved at velocities between 0.15 and 0.4 micron/s, depending upon the preparation. With unphosphorylated myosin, only 3% of the beads moved and then at a velocity of only approximately 0.01-0.04 micron/s. The effects of phosphorylation were fully reversible after dephosphorylation with a phosphatase prepared from smooth muscle. Analysis of the velocity of movement as a function of phosphorylation level indicated that phosphorylation of both heads of a myosin molecule was required for movement and that unphosphorylated myosin appears to decrease the rate of movement of phosphorylated myosin. Mixing of phosphorylated smooth muscle myosin with skeletal muscle myosin which moves at 2 microns/s resulted in a decreased rate of bead movement, suggesting that the more slowly cycling smooth muscle myosin is primarily determining the velocity of movement in such mixtures.

In Vitro Motility of Skeletal Muscle Myosin and Its Proteolytic Fragments1

1990 ◽  
Vol 107 (5) ◽  
pp. 671-679 ◽  
Author(s):  
Kingo Takiguchi ◽  
Hiroshi Hayashi ◽  
Eiji Kurimoto ◽  
Sugie Higasshi-Fujime
Keyword(s):  
Skeletal Muscle ◽  
Skeletal Muscle Myosin ◽  
In Vitro Motility ◽  
Muscle Myosin

Troponin in embryonic chick skeletal muscle cell in vitro: an immunoelectron microscopic study

1978 ◽  
Vol 36 (2) ◽  
pp. 162-163
Author(s):  
Yutaka Shimada ◽  
Takashi Obinata
Keyword(s):  
Skeletal Muscle ◽  
Chicken Breast ◽  
Thin Filaments ◽  
Structural Localization ◽  
Adult Chicken ◽  
Skeletal Muscle Myosin ◽  
Ammonium Sulphate Fractionation ◽  
The Right ◽  
Muscle Myosin

In developing skeletal muscle, myosin and actin are synthesized and polymerized into filamentous forms, thick and thin filaments, respectively. These myofilaments of two varieties have been shown, with the use of "decoration with heavy meromyosin" technique, to be arranged at the right polarity and spatial position as those seen in mature myofibrils from the initial phases of myofibrillogenesis. The question arises as to whether regulatory proteins are distributed along embryonic thin filaments from such early stages of development. In order to clarify this problem, the fine structural localization of troponin was investigated by the use of immunoelectron microscopy.Troponin and its components (troponin C [TN-C], I [TN-I] and T [TN-T]) were prepared from adult chicken breast muscles. Rabbit antisera against each of these troponin components were prepared. The serum was fractioned by ethanol or ammonium sulphate fractionation. Myogenic cells from 12-day chick embryonic thigh muscles were grown in monolayer and, after 2-8 days in vitro, the cultures were treated as follows: (i) They were immersed in 50% glycerol. The suspension of separated thin filaments was obtained by gentle homogenizing the cells.

scholarly journals Highly selective inhibition of myosin motors provides the basis of potential therapeutic application

2016 ◽  
Vol 113 (47) ◽  
pp. E7448-E7455 ◽  
Author(s):  
Serena Sirigu ◽  
James J. Hartman ◽  
Vicente José Planelles-Herrero ◽  
Virginie Ropars ◽  
Sheila Clancy ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Muscle Relaxation ◽  
Selective Inhibition ◽  
Pharmacological Agents ◽  
Skeletal Muscle Myosin ◽  
Starting Point ◽  
Myosin Motors ◽  
Muscle Myosin

Direct inhibition of smooth muscle myosin (SMM) is a potential means to treat hypercontractile smooth muscle diseases. The selective inhibitor CK-2018571 prevents strong binding to actin and promotes muscle relaxation in vitro and in vivo. The crystal structure of the SMM/drug complex reveals that CK-2018571 binds to a novel allosteric pocket that opens up during the “recovery stroke” transition necessary to reprime the motor. Trapped in an intermediate of this fast transition, SMM is inhibited with high selectivity compared with skeletal muscle myosin (IC50 = 9 nM and 11,300 nM, respectively), although all of the binding site residues are identical in these motors. This structure provides a starting point from which to design highly specific myosin modulators to treat several human diseases. It further illustrates the potential of targeting transition intermediates of molecular machines to develop exquisitely selective pharmacological agents.

scholarly journals Effect of low pH on single skeletal muscle myosin mechanics and kinetics

2008 ◽  
Vol 295 (1) ◽  
pp. C173-C179 ◽  
Author(s):  
E. P. Debold ◽  
S. E. Beck ◽  
D. M. Warshaw
Keyword(s):  
Skeletal Muscle ◽  
Single Molecule ◽  
Low Ph ◽  
Muscular Fatigue ◽  
Step Size ◽  
Skeletal Muscle Myosin ◽  
In Vitro Motility ◽  
Adp Release ◽  
Muscle Myosin

Acidosis (low pH) is the oldest putative agent of muscular fatigue, but the molecular mechanism underlying its depressive effect on muscular performance remains unresolved. Therefore, the effect of low pH on the molecular mechanics and kinetics of chicken skeletal muscle myosin was studied using in vitro motility (IVM) and single molecule laser trap assays. Decreasing pH from 7.4 to 6.4 at saturating ATP slowed actin filament velocity ( Vactin) in the IVM by 36%. Single molecule experiments, at 1 μM ATP, decreased the average unitary step size of myosin ( d) from 10 ± 2 nm (pH 7.4) to 2 ± 1 nm (pH 6.4). Individual binding events at low pH were consistent with the presence of a population of both productive (average d = 10 nm) and nonproductive (average d = 0 nm) actomyosin interactions. Raising the ATP concentration from 1 μM to 1 mM at pH 6.4 restored d (9 ± 3 nm), suggesting that the lifetime of the nonproductive interactions is solely dependent on the [ATP]. Vactin, however, was not restored by raising the [ATP] (1–10 mM) in the IVM assay, suggesting that low pH also prolongs actin strong binding ( ton). Measurement of ton as a function of the [ATP] in the single molecule assay suggested that acidosis prolongs ton by slowing the rate of ADP release. Thus, in a detachment limited model of motility (i.e., Vactin ∼ d/ ton), a slowed rate of ADP release and the presence of nonproductive actomyosin interactions could account for the acidosis-induced decrease in Vactin, suggesting a molecular explanation for this component of muscular fatigue.

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