scholarly journals Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies.

1984 ◽  
Vol 99 (3) ◽  
pp. 1024-1033 ◽  
Author(s):  
D P Kiehart ◽  
T D Pollard
Keyword(s):  
Monoclonal Antibodies ◽  
Atpase Activity ◽  
Conformational Changes ◽  
Binding Sites ◽  
Polyclonal Antibodies ◽  
Myosin Ii ◽  
Antigenic Site ◽  
Actin Binding ◽  
Filamentous Form ◽  
Antibody Effects

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.

A Dynamic Escape Problem of Molecular Motors

2020 ◽  
Vol 142 (5) ◽  
Author(s):  
Dean Culver ◽  
Bryan Glaz ◽  
Samuel Stanton
Keyword(s):  
Molecular Motors ◽  
Binding Sites ◽  
Molecular Motor ◽  
Myosin Ii ◽  
Three Dimensional ◽  
Production Capacity ◽  
Force Production ◽  
Actin Binding ◽  
Three Dimensional Model ◽  
Thermal Agitation

Abstract Animal skeletal muscle exhibits very interesting behavior at near-stall forces (when the muscle is loaded so strongly that it can barely contract). Near this physical limit, the myosin II proteins may be unable to reach advantageous actin binding sites through simple attractive forces. It has been shown that the advantageous utilization of thermal agitation is a likely source for an increased force-production capacity and reach in myosin-V (a processing motor protein), and here we explore the dynamics of a molecular motor without hand-over-hand motion including Brownian motion to show how local elastic energy well boundaries may be overcome. We revisit a spatially two-dimensional mechanical model to illustrate how thermal agitation can be harvested for useful mechanical work in molecular machinery inspired by this biomechanical phenomenon without rate functions or empirically inspired spatial potential functions. Additionally, the model accommodates variable lattice spacing, and it paves the way for a full three-dimensional model of cross-bridge interactions where myosin II may be azimuthally misaligned with actin binding sites. With potential energy sources based entirely on realizable components, this model lends itself to the design of artificial, molecular-scale motors.

Association Between Ligand-Induced Conformational Changes of Integrin IIbβ3 and IIbβ3-Mediated Intracellular Ca2+ Signaling

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3675-3683 ◽  
Author(s):  
Shigenori Honda ◽  
Yoshiaki Tomiyama ◽  
Toshiaki Aoki ◽  
Masamichi Shiraga ◽  
Yoshiyuki Kurata ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Conformational Changes ◽  
Binding Sites ◽  
Critical Role ◽  
Thromboxane A2 ◽  
Fibrinogen Binding ◽  
Group I ◽  
Group Ii ◽  
Inhibition Studies

Platelet IIbβ3 is a prototypic integrin and plays a critical role in platelet aggregation. Occupancy of IIbβ3 with multivalent RGD ligands, such as fibrinogen, induces both expression of ligand-induced binding sites (LIBS) and IIbβ3 clustering, which are thought to be necessary for outside-in signaling. However, the association between LIBS expression and outside-in signaling remains elusive. In this study, we used various IIbβ3-specific peptidomimetic compounds as a monovalent ligand instead of fibrinogen and examined the association between LIBS expression and outside-in signaling such as IIbβ3-mediated intracellular Ca2+ signaling. Using a set of monoclonal antibodies (MoAbs) against LIBS, we showed that antagonists can be divided into two groups. In group I, antagonists can induce LIBS on both IIb and β3 subunits. In group II, antagonists can induce LIBS on the IIb subunit, but not on the β3 subunit. Inhibition studies suggested that group I and group II antagonists interact with distinct but mutually exclusive sites on IIbβ3. Neither group I nor group II antagonist increased intracellular Ca2+concentrations ([Ca2+]i) in nonactivated platelets. All antagonists at nanomolar concentrations abolished the increase in [Ca2+]i in 0.03 U/mL thrombin-stimulated platelets, which is dependent on both fibrinogen-binding to IIbβ3 and platelet-aggregation. However, only group I antagonists at higher concentrations dose-dependently augmented the [Ca2+]i increase, which is due to aggregation-independent thromboxane A2 production. This increase in [Ca2+]i was not observed in thrombasthenic platelets, which express no detectable IIbβ3. Thus, only the group I antagonists, albeit a monovalent ligand, can initiate IIbβ3-mediated intracellular Ca2+ signaling in the presence of thrombin stimulation. Our findings strongly suggest the association between β3LIBS expression and IIbβ3-mediated intracellular Ca2+ signaling in platelets.

scholarly journals Cytokinesis Depends on the Motor Domains of Myosin-II in Fission Yeast but Not in Budding Yeast

2005 ◽  
Vol 16 (11) ◽  
pp. 5346-5355 ◽  
Author(s):  
Matthew Lord ◽  
Ellen Laves ◽  
Thomas D. Pollard
Keyword(s):  
Atpase Activity ◽  
Fission Yeast ◽  
Mechanism Of Action ◽  
Budding Yeast ◽  
Myosin Ii ◽  
Cellular Function ◽  
Motor Domain ◽  
Actin Binding ◽  
Contractile Ring ◽  
Head Domain

Budding yeast possesses one myosin-II, Myo1p, whereas fission yeast has two, Myo2p and Myp2p, all of which contribute to cytokinesis. We find that chimeras consisting of Myo2p or Myp2p motor domains fused to the tail of Myo1p are fully functional in supporting budding yeast cytokinesis. Remarkably, the tail alone of budding yeast Myo1p localizes to the contractile ring, supporting both its constriction and cytokinesis. In contrast, fission yeast Myo2p and Myp2p require both the catalytic head domain as well as tail domains for function, with the tails providing distinct functions ( Bezanilla and Pollard, 2000 ). Myo1p is the first example of a myosin whose cellular function does not require a catalytic motor domain revealing a novel mechanism of action for budding yeast myosin-II independent of actin binding and ATPase activity.

scholarly journals An N-terminal, 830 residues intrinsically disordered region of the cytoskeleton-regulatory protein supervillin contains Myosin II- and F-actin-binding sites

2013 ◽  
Vol 31 (10) ◽  
pp. 1150-1159 ◽  
Author(s):  
Stanislav O. Fedechkin ◽  
Jacob Brockerman ◽  
Elizabeth J. Luna ◽  
Michail Yu. Lobanov ◽  
Oxana V. Galzitskaya ◽  
...  
Keyword(s):  
Binding Sites ◽  
Myosin Ii ◽  
Regulatory Protein ◽  
Actin Binding ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Region

Association Between Ligand-Induced Conformational Changes of Integrin IIbβ3 and IIbβ3-Mediated Intracellular Ca2+ Signaling

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3675-3683 ◽  
Author(s):  
Shigenori Honda ◽  
Yoshiaki Tomiyama ◽  
Toshiaki Aoki ◽  
Masamichi Shiraga ◽  
Yoshiyuki Kurata ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Conformational Changes ◽  
Binding Sites ◽  
Critical Role ◽  
Thromboxane A2 ◽  
Fibrinogen Binding ◽  
Group I ◽  
Group Ii ◽  
Inhibition Studies

Abstract Platelet IIbβ3 is a prototypic integrin and plays a critical role in platelet aggregation. Occupancy of IIbβ3 with multivalent RGD ligands, such as fibrinogen, induces both expression of ligand-induced binding sites (LIBS) and IIbβ3 clustering, which are thought to be necessary for outside-in signaling. However, the association between LIBS expression and outside-in signaling remains elusive. In this study, we used various IIbβ3-specific peptidomimetic compounds as a monovalent ligand instead of fibrinogen and examined the association between LIBS expression and outside-in signaling such as IIbβ3-mediated intracellular Ca2+ signaling. Using a set of monoclonal antibodies (MoAbs) against LIBS, we showed that antagonists can be divided into two groups. In group I, antagonists can induce LIBS on both IIb and β3 subunits. In group II, antagonists can induce LIBS on the IIb subunit, but not on the β3 subunit. Inhibition studies suggested that group I and group II antagonists interact with distinct but mutually exclusive sites on IIbβ3. Neither group I nor group II antagonist increased intracellular Ca2+concentrations ([Ca2+]i) in nonactivated platelets. All antagonists at nanomolar concentrations abolished the increase in [Ca2+]i in 0.03 U/mL thrombin-stimulated platelets, which is dependent on both fibrinogen-binding to IIbβ3 and platelet-aggregation. However, only group I antagonists at higher concentrations dose-dependently augmented the [Ca2+]i increase, which is due to aggregation-independent thromboxane A2 production. This increase in [Ca2+]i was not observed in thrombasthenic platelets, which express no detectable IIbβ3. Thus, only the group I antagonists, albeit a monovalent ligand, can initiate IIbβ3-mediated intracellular Ca2+ signaling in the presence of thrombin stimulation. Our findings strongly suggest the association between β3LIBS expression and IIbβ3-mediated intracellular Ca2+ signaling in platelets.

scholarly journals Monoclonal antibodies against seven sites on the head and tail of Dictyostelium myosin.

1985 ◽  
Vol 100 (4) ◽  
pp. 1016-1023 ◽  
Author(s):  
G Peltz ◽  
J A Spudich ◽  
P Parham
Keyword(s):  
Monoclonal Antibodies ◽  
Atpase Activity ◽  
Heavy Chain ◽  
Light Chain ◽  
Binding Sites ◽  
Myosin Molecule ◽  
Dictyostelium Myosin ◽  
Antigenic Sites ◽  
Myosin Filaments ◽  
A Site

Ten monoclonal antibodies (My1-10) against Dictyostelium discoideum myosin were prepared and characterized. Nine bound to the 210-kD heavy chain and one (My8) bound to the 18-kD light chain. They defined six topographically distinct antigenic sites of the heavy chain. Five binding sites (the My1, My5, My10 site, and the My2, My3, My4, and My9 sites) are located on the rod portion of the myosin molecule. The position of the sixth site (the My6 and My7 site) is less certain, but it appears to be near the junction of the globular heads and the rod. Three of the antibodies (My2, My3, and My6) bound to myosin filaments in solution and could be sedimented in stoichiometric amounts with the filamentous myosin. In contrast, My4, which recognized a site on the rod, inhibited the polymerization of monomeric myosin into filaments. A single antibody (My6) affected the actin-activated ATPase of myosin. The nature of the effect depended on the valency of the antibody and the myosin. Bivalent IgG and F(ab')2 fragments of My6 inhibited the actin-activated ATPase of filamentous myosin by 50% whereas univalent Fab' fragments increased the activity by 50%. The actin-activated ATPase activity of the soluble chymotryptic fragment of myosin was increased 80-90% by both F(ab')2 and Fab' of My6.

Myosin II isoforms in smooth muscle: heterogeneity and function

2007 ◽  
Vol 293 (2) ◽  
pp. C493-C508 ◽  
Author(s):  
Thomas J. Eddinger ◽  
Daniel P. Meer
Keyword(s):  
Smooth Muscle ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Expression Profiles ◽  
Myosin Ii ◽  
Thick Filament ◽  
Actin Binding ◽  
Head Region ◽  
Organ Systems ◽  
Species Specific

Both smooth muscle (SM) and nonmuscle class II myosin molecules are expressed in SM tissues comprising hollow organ systems. Individual SM cells may express one or more of multiple myosin II isoforms that differ in myosin heavy chain (MHC) and myosin light chain (MLC) subunits. Although much has been learned, the expression profiles, organization within contractile filaments, localization within cells, and precise roles in various contractile functions of these different myosin molecules are still not well understood. However, data supporting unique physiological roles for certain isoforms continues to build. Isoform differences located in the S1 head region of the MHC can alter actin binding and rates of ATP hydrolysis. Differences located in the MHC tail can alter the formation, stability, and size of the myosin thick filament. In these distinct ways, both head and tail isoform differences can alter force generation and muscle shortening velocities. The MLCs that are associated with the lever arm of the S1 head can affect the flexibility and range of motion of this domain and possibly the motion of the S2 and motor domains. Phosphorylation of MLC20 has been associated with conformational changes in the S1 and/or S2 fragments regulating enzymatic activity of the entire myosin molecule. A challenge for the future will be delineation of the physiological significance of the heterogeneous expression of these isoforms in developmental, tissue-specific, and species-specific patterns and or the intra- and intercellular heterogeneity of myosin isoform expression in SM cells of a given organ.

scholarly journals Monoclonal antibodies as probes of the distribution of ZP-2, the major sulfated glycoprotein of the murine zona pellucida.

1984 ◽  
Vol 98 (3) ◽  
pp. 795-800 ◽  
Author(s):  
I J East ◽  
J Dean
Keyword(s):  
Extracellular Matrix ◽  
Monoclonal Antibodies ◽  
Zona Pellucida ◽  
Binding Sites ◽  
Antigenic Site ◽  
Matrix Proteins ◽  
Mouse Tissue ◽  
Cell Embryo ◽  
Embryo Stage ◽  
Mouse Egg

Three sulfated glycoproteins (ZP-1, ZP-2, and ZP-3) make up the zona pellucida, an extracellular glycocalyx that surrounds mouse oocytes. We have produced five monoclonal antibodies specific to the zona. All five immunoprecipitated ZP-2, and in addition, two of the antibodies immunoprecipitated ZP-3. This suggests the presence of either a common antigenic site or one made up in part by each of the two glycoproteins. The monoclonal antibodies bound to approximately 1.3 X 10(8) binding sites per ovulated mouse egg which represents 2% of the total number of ZP-2 molecules present in the zona. ZP-2 appeared to be present throughout the zona and indirect immunofluorescence revealed a fibrous pattern with no evidence of localization. Furthermore, this pattern of distribution, which was identical for all five monoclones, remained constant after fertilization at the two-cell embryo stage. Laser photobleaching demonstrated that ZP-2 is stably integrated in the extracellular matrix of the zona pellucida. No mouse tissue other than the ovary contained ZP-2 and ZP-2 is antigenically distinct from other previously described extracellular matrix proteins.

scholarly journals Interaction of a Dictyostelium member of the plastin/fimbrin family with actin filaments and actin-myosin complexes.

1997 ◽  
Vol 8 (1) ◽  
pp. 83-95 ◽  
Author(s):  
J Prassler ◽  
S Stocker ◽  
G Marriott ◽  
M Heidecker ◽  
J Kellermann ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Binding Sites ◽  
Actin Filaments ◽  
Myosin Ii ◽  
Actin Binding ◽  
Protein Protein Interactions ◽  
Actin Isoforms ◽  
Ef Hands ◽  
Immunofluorescence Labeling ◽  
Alpha Actin

A protein purified from cytoskeletal fractions of Dictyostelium discoideum proved to be a member of the fimbrin/plastin family of actin-bundling proteins. Like other family members, this Ca(2+)-inhibited 67-kDa protein contains two EF hands followed by two actin-binding sites of the alpha-actinin/beta-spectrin type. Dd plastin interacted selectively with actin isoforms: it bound to D. discoideum actin and to beta/gamma-actin from bovine spleen but not to alpha-actin from rabbit skeletal muscle. Immunofluorescence labeling of growth phase cells showed accumulation of Dd plastin in cortical structures associated with cell surface extensions. In the elongated, streaming cells of the early aggregation stage, Dd plastin was enriched in the front regions. To examine how the bundled actin filaments behave in myosin II-driven motility, complexes of F-actin and Dd plastin were bound to immobilized heavy meromyosin, and motility was started by photoactivating caged ATP. Actin filaments were immediately propelled out of bundles or even larger aggregates and moved on the myosin as separate filaments. This result shows that myosin can disperse an actin network when it acts as a motor and sheds light on the dynamics of protein-protein interactions in the cortex of a motile cell where myosin II and Dd plastin are simultaneously present.

Sign in / Sign up

Export Citation Format

Share Document