Differential localization of Acanthamoeba myosin I isoforms (original) (raw)
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Journal of Cell Biology, 1984
Electron microscopy of myosin-II molecules and filaments reacted with monoclonal antibodies demonstrates directly where the antibodies bind and shows that certain antibodies can inhibit the polymerization of myosin-II into filaments. The binding sites of seven of 23 different monoclonal antibodies were localized by platinum shadowing of myosin monomer-antibody complexes. The antibodies bind to a variety of sites on the myosin-II molecule, including the heads, the proximal end of the tail near the junction of the heads and tail, and the tip of the tail. The binding sites of eight of the 23 antibodies were also localized on myosin filaments by negative staining. Antibodies that bind to either the myosin heads or to the proximal end of the tail decorate the ends of the bipolar filaments. Some of the antibodies that bind to the tip of the myosin-II tail decorate the bare zone of the myosin-II thin filament with 14-nm periodicity. By combining the data from these electron microscope stud...
Journal of Cell Biology, 1984
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 ...
Purification and characterization of a third isoform of myosin I from Acanthamoeba castellanii
Journal of Biological Chemistry
A third isoform of myosin I has been isolated from Acanthamoeba and designated myosin IC. Peptide maps and immunoassays indicate that myosin IC is not a modified form of myosin IA, IB, or 11. However, myosin IC has most of the distinctive properties of a myosin I. It is a globular protein of native M, -162,000, apparently composed of a single 130-kDa heavy chain and a pair of 14-kDa light chains. It is soluble in MgATP at low ionic strength, conditions favoring filament assembly by myosin 11. Myosin IC has high Ca2+-and (K+,EDTA)-ATPase activities. Its low Mg2+-ATPase activity is stimulated to a maximum rate of 20 s-l by the addition of F-actin if its heavy chain has been phosphorylated by myosin I heavy chain kinase. The dependence of the Mg2+-ATPase activity of myosin IC on F-actin concentration is triphasic; and, at fixed concentrations of F-actin, this activity increases cooperatively as the concentration of myosin IC is increased. These unusual kinetics were first demonstrated for myosins IA and IB and shown to be due to the presence of two actin-binding sites on each heavy chain which enable those myosins I to cross-link actin filaments. Myosin IC is also capable of cross-linking Factin, which, together with the kinetics of its actinactivated Mg2+-ATPase activity, suggests that it, like myosins IA and IB, possesses two independent actinbinding domains.
Journal of Cell Biology, 1989
We used 90 ° light scattering, analytical ultracentrifugation, and electron microscopy to deduce that Acanthamoeba myosin-II minifilaments, composed of eight molecules each, assemble by a novel mechanism consisting of three successive dimerization steps rather than by the addition of monomers or parallel dimers to a nucleus. Above 200 mM KCI, Acanthamoeba myosin-II is monomeric. At low ionic strength (<100 mM KCI), myosin-II polymerizes into bipolar minifilaments. Between 100 and 200 mM KCI, plots of light scattering vs. myosin concentration all extrapolate to the origin but have slopes which decrease with increasing KCI. This indicates that structures intermediate in size between monomers and full length minifilaments are formed, and that the critical concentrations for assembly of these structures is very low. Analytical ultracentrifugation has confirmed that
1989
To study the in vivo role of myosin-I1 in Acanthamoeba castellunii, motile cells were microinjected with monoclonal antibodies raised against the myosin-I1 heavy chain. All injected cells underwent a transient shock response. It was found that although injection of buffer alone or of an endogenous Acanthamoeba protein decreased the motility of injected cells from 7 pm/min to-3 p d m i n , injection of monoclonal antibodies specific for myosin-I1 decreased motility further to-0.8 pm/min. This effect was seen whether or not the monoclonal antibody to myosin-I1 inhibited the actomyosin-I1 MgATPase activity in vitro. Levels of antibody far in excess of endogenous myosin-I1 concentrations could not completely block amoeboid movement. The morphology of moving antimyosin-11-injected cells was unusual, suggesting a greater defect in the ability to retract the trailing edge of the cell rather than to extend the leading edge. Endosomes frequently disappeared from injected cells, and although buffer-injected cells rapidly recovered visible endosomes (50% recovery at 5 min), endosomes were not seen in antimyosin-11-injected cells until, on the average,-50 min after injection. Injection of a nonspecific antibody or of a nonspecific exogenous protein (ovalbumin) also decreased the mobility of the injected cells beyond that of buffer-injected cells (to-1 pdmin). These cells tended to recover endosomes more rapidly (-25 min) than cells injected with antimyosin-I1 monoclonal antibodies. The inability of antibodies to myosin-I1 to inhibit completely any of the movements studied suggests that although myosin-I1 probably plays a role in these motilities, the cell either routinely uses or can draw upon another cytoplasmic motor to maintain locomotion, organelle movement, contractile vacuole activity, and endocytosis.
Journal of Biological Chemistry
The three isoforms of Acanthamoeba myosin I (nonfilamentous myosin with only a single heavy chain) express actin-activated M@+-ATPase activity only when phosphorylated at a single site by myosin I heavy chain kinase. The kinase is activated by autophosphorylation that is greatly stimulated by acidic phospholipids. Substantial fractions of the three myosins I and the kinase are associated in s i t u with membranes, and all four enzymes bind to purified membranes in vitro. W e now report that when kinase and myosin I are incubated together with phosphatidylserine vesicles not only does the kinase autophosphorylate more rapidly than soluble kinase in the absence of phosphatidylserine but that, probably as a result, the kinase phosphorylates myosin I more rapidly than soluble kinase phosphorylates soluble myosin I. Similarly, plasma membrane-bound kinase phosphorylates membrane-bound myosin I and activates its actin-activated Mg2"ATPase activity more rapidly than soluble kinase phosphorylates and activates soluble myosin I in the absence of membranes. However, the enhanced activity of membrane-bound kinase (which is comparable to the activity of kinase in the presence of phosphatidylserine) is not due to autophosphorylation of the membrane-bound kinase, which is very much slower than for kinase activated by phosphatidylserine vesicles.
The Journal of Cell Biology, 1991
The actin-activated Mg"-ATPase activities of Acanthamoeba myosins I are known to be maximally expressed only when a single threonine (myosin IA) or serine (myosins IB and IC) is phosphorylated by myosin I heavy chain kinase. The purified kinase is highly activated by autophosphorylation and the rate of autophosphorylation is greatly enhanced by the presence of acidic phospholipids . In this paper, we show by immunofluorescence and immunoelectron microscopy of permeabilized cells that myosin I heavy chain kinase is highly concentrated, but not exclusively, at the plasma membrane. Judged by their electrophoretic mobilities, kinase associated with purified plasma membranes may differ from the cytoplasmic kinase, possibly in the extent of its phosphorylation . Purified kinase binds to
Identification of Novel Myosin-V Binding Partners by Immunoprecipitation and Column Chromatography
The Biological Bulletin, 2004
Fucus zygotes can be polarized-as shown by their later outgrowth directions-by many environmental influences. These include those exerted by all tested intertidal marine plants (including all of the main algal phyla as well as the flowering plant Phyllospadix) at distances of up to 5-10 mm away from a piece of the plant in a laboratory dish. Earlier studies inferred action via diffusing molecules. A reinvestigation indicates that such actions can be exerted through glass barriers and suggests action via luminescent and, perhaps, infrared signals. Since this polarizing influence is exerted by pieces of all tested plants, the infrared light signals might be emitted by luminescent bacteria growing in biofilms on the surfaces of all intertidal plants.
The Journal of Cell Biology, 1984
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 ap...