scholarly journals Characterization of monoclonal antibodies to Acanthamoeba myosin-I that cross-react with both myosin-II and low molecular mass nuclear proteins.

1986 ◽  
Vol 103 (6) ◽  
pp. 2121-2128 ◽  
Author(s):  
S J Hagen ◽  
D P Kiehart ◽  
D A Kaiser ◽  
T D Pollard
Keyword(s):  
Monoclonal Antibodies ◽  
Atpase Activity ◽  
Heavy Chain ◽  
Fluorescent Antibody ◽  
Myosin Ii ◽  
Nuclear Staining ◽  
Cytoplasmic Matrix ◽  
Isolated Nuclei ◽  
Whole Cells ◽  
Myosin I

We characterized nine monoclonal antibodies that bind to the heavy chain of Acanthamoeba myosin-IA. Eight of these antibodies bind to myosin-IB and eight cross-react with Acanthamoeba myosin-II. All but one of the antibodies bind to a 30-kD chymotryptic peptide of myosin-IA that derives from the COOH terminus of the molecule, and to tryptic peptides as small as 17 kD, hence these epitopes are clustered closely together on the heavy chain. None of the antibodies prevent heavy chain phosphorylation by myosin-I heavy chain kinase. One antibody inhibits the K+-EDTA ATPase activity and three antibodies inhibit the actin-activated Mg++-ATPase activity of myosin-I under the set of conditions that we tested. When fluorescent antibody staining of both whole cells and isolated nuclei is done, several of these monoclonal antibodies react strongly with nuclei. These antibodies also stain the cytoplasmic matrix, especially the cortex near the plasma membrane. All nine of the monoclonal antibodies bind to polypeptides of 30-34 kD that are highly enriched in nuclei isolated from Acanthamoeba. There is no myosin-I in the isolated nuclei, so the 30-34-kD polypeptides, not myosin-I, are responsible for the nuclear staining.

scholarly journals Monoclonal antibodies demonstrate limited structural homology between myosin isozymes from Acanthamoeba.

1984 ◽  
Vol 99 (3) ◽  
pp. 1002-1014 ◽  
Author(s):  
D P Kiehart ◽  
D A Kaiser ◽  
T D Pollard
Keyword(s):  
Monoclonal Antibodies ◽  
Heavy Chain ◽  
Solid Phase ◽  
Polyclonal Antibodies ◽  
Myosin Ii ◽  
Light Chains ◽  
Homologous Region ◽  
Myosin Isozymes ◽  
Antibody Staining ◽  
Myosin I

We used a library of 31 monoclonal and six polyclonal antibodies to compare the structures of the two classes of cytoplasmic myosin isozymes isolated from Acanthamoeba: myosin-I, a 150,000-mol-wt, globular molecule; and myosin-II, a 400,000-mol-wt molecule with two heads and a 90-nm tail. This analysis confirms that myosin-I and -II are unique gene products and provides the first evidence that these isozymes have at least one structurally homologous region functionally important for myosin's role in contractility. Characterization of the 23 myosin-II monoclonal antibody binding sites by antibody staining of one-dimensional peptide maps and solid phase, competitive binding assays demonstrate that they bind to at least 15 unique sites on the myosin-II heavy chain. The antibodies can be grouped into six families, whose members bind close to one another. None of the monoclonal antibodies bind to myosin-II light chains and polyclonal antibodies against myosin-II light or heavy chain bind only to myosin-II light or heavy chains, respectively: no antibody binds both heavy and light chains. Six of eight monoclonal antibodies and one of two polyclonal sera that react with the myosin-I heavy chain also bind to determinants on the myosin-II heavy chain. The cross-reactive monoclonal antibodies bind to the region of myosin-II recognized by the largest family of myosin-II monoclonal antibodies. In the two papers that immediately follow, we show that this family of monoclonal antibodies to myosin-II binds to the myosin-II tail near the junction with the heads and inhibits both the actin-activated ATPase of myosin-II and contraction of gelled cytoplasmic extracts of Acanthamoeba cytoplasm. Further, this structurally homologous region may play a key role in energy transduction by cytoplasmic myosins.

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.

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.

scholarly journals Biochemical and immunological characterization of p190-calmodulin complex from vertebrate brain: a novel calmodulin-binding myosin.

1992 ◽  
Vol 118 (2) ◽  
pp. 359-368 ◽  
Author(s):  
F S Espindola ◽  
E M Espreafico ◽  
M V Coelho ◽  
A R Martins ◽  
F R Costa ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Brush Border ◽  
Myosin Ii ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Mammalian Brain ◽  
Rat Tissues ◽  
Tail Region ◽  
Calmodulin Binding ◽  
Myosin I

We have recently identified a novel 190-kD calmodulin-binding protein (p190) associated with the actin-based cytoskeleton from mammalian brain (Larson, R. E., D. E. Pitta, and J. A. Ferro. 1988. Braz. J. Med. Biol. Res. 21:213-217; Larson, R. E., F. S. Espindola, and E. M. Espreafico. 1990. J. Neurochem. 54:1288-1294). These studies indicated that p190 is a phosphoprotein substrate for calmodulin-dependent kinase II and has calcium- and calmodulin-stimulated MgATPase activity. We now have biochemical and immunological evidence that this protein is a novel calmodulin-binding myosin whose properties include (a) Ca2+ dependent action activation of its Mg-ATPase activity, which seems to be mediated by Ca2+ binding directly to calmodulin(s) associated with p190 (maximal activation by actin requires the presence of Ca2+ and is further augmented by addition of exogenous calmodulin); (b) ATP-sensitive cross-linking of skeletal muscle F-actin, as demonstrated by the low-speed actin sedimentation assay; and (c) cross-reactivity with mAbs specific for epitopes in the head of brush border myosin I. We also show that p190 has properties distinct from conventional brain myosin II and brush border myosin I, including (a) separation of p190 from brain myosin II by gel filtration on a Sephacryl S-500 column; (b) lack by p190 of K(+)-stimulated EDTA ATPase activity characteristic of most myosins; (c) lack of immunological cross-reactivity of polyclonal antibodies which recognize p190 and brain myosin II, respectively; (d) lack of immunological recognition of p190 by mAbs against an epitope in the tail region of brush border myosin I; and (e) distinctive proteolytic susceptibility to calpain. A survey of rat tissues by immunoblotting indicated that p190 is expressed predominantly in the adult forebrain and cerebellum, and could be detected in embryos 11 d post coitus. Immunocytochemical studies showed p190 to be present in the perikarya and dendritic extensions of Purkinje cells of the cerebellum.

scholarly journals UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II

2004 ◽  
Vol 167 (2) ◽  
pp. 315-325 ◽  
Author(s):  
Matthew Lord ◽  
Thomas D. Pollard
Keyword(s):  
Atpase Activity ◽  
Heavy Chain ◽  
Actin Filaments ◽  
Myosin Ii ◽  
Gliding Motility ◽  
Light Chains ◽  
Temperature Sensitive ◽  
Contractile Ring ◽  
In Vitro Motility

We purified native Myo2p/Cdc4p/Rlc1p (Myo2), the myosin-II motor required for cytokinesis by Schizosaccharomyces pombe. The Myo2p heavy chain associates with two light chains, Cdc4p and Rlc1p. Although crude Myo2 supported gliding motility of actin filaments in vitro, purified Myo2 lacked this activity in spite of retaining full Ca-ATPase activity and partial actin-activated Mg-ATPase activity. Unc45-/Cro1p-/She4p-related (UCS) protein Rng3p restored the full motility and actin-activated Mg-ATPase activity of purified Myo2. The COOH-terminal UCS domain of Rng3p alone restored motility to pure Myo2. Thus, Rng3p contributes directly to the motility activity of native Myo2. Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring. The absence of Rlc1p or mutations in the Myo2p head or Rng3p compromise the in vitro motility of Myo2 and explain the defects in cytokinesis associated with some of these mutations. In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis.

The localization of myosin I and myosin II in Acanthamoeba by fluorescence microscopy

1992 ◽  
Vol 102 (3) ◽  
pp. 629-642 ◽  
Author(s):  
S. Yonemura ◽  
T.D. Pollard
Keyword(s):  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Myosin Ii ◽  
Leading Edge ◽  
Limited Range ◽  
Contractile Vacuole ◽  
Cytoplasmic Vacuole ◽  
Cytoplasmic Staining ◽  
Myosin I ◽  
Dividing Cells

We used several fixation protocols and a panel of monoclonal antibodies to re-examine the localization of myosin I and myosin II in Acanthamoeba. Two monoclonal antibodies that bind to the head of myosin II stain a range of particles in the cytoplasm. The smallest and most numerous cytoplasmic particles are about the same size and intensity as myosin II minifilaments and are distributed throughout the endoplasm. The largest particles stain like myosin II thick filaments and are concentrated in the cleavage furrow of dividing cells and in the tail of locomoting cells. Five different monoclonal antibodies that bind to the myosin II tail also stain cytoplasmic particles but with a limited range of intensity. None of the myosin II monoclonal antibodies stains the contractile vacuole or plasma membrane. Two monoclonal antibodies to myosin I gave punctate cytoplasmic staining that did not correspond clearly to any of the phase-dense particles in the cytoplasm. In many, but not all, locomoting cells, the myosin I staining was concentrated at the leading edge. Both myosin I antibodies stained a single cytoplasmic vacuole of variable size that was presumed to be the contractile vacuole. The antibody that binds myosin IA but not myosin IB stained novel intercellular contacts and the antibody that binds both myosin IA and myosin IB stained the plasma membrane, especially the tips of filopodia.

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