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.

Myosin II-dependent cylindrical protrusions induced by quinine inDictyostelium: antagonizing effects of actin polymerization at the leading edge

2001 ◽  
Vol 114 (11) ◽  
pp. 2155-2165
Author(s):  
Kunito Yoshida ◽  
Kei Inouye
Keyword(s):  
Cell Membrane ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Cell Movement ◽  
Leading Edge ◽  
Contractile Vacuole ◽  
Cell Periphery ◽  
Slowing Down ◽  
On Line ◽  
Cylindrical Protrusion

We found that amoeboid cells of Dictyostelium are induced by a millimolar concentration of quinine to form a rapidly elongating, cylindrical protrusion, which often led to sustained locomotion of the cells. Formation of the protrusion was initiated by fusion of a contractile vacuole with the cell membrane. During protrusion extension, a patch of the contractile vacuole membrane stayed undiffused on the leading edge of the protrusion for over 30 seconds. Protrusion formation was not inhibited by high osmolarity of the external medium (at least up to 400 mosM). By contrast, mutant cells lacking myosin II (mhc− cells) failed to extend protrusions upon exposure to quinine. When GFP-myosin-expressing cells were exposed to quinine, GFP-myosin was accumulated in the cell periphery forming a layer under the cell membrane, but a newly formed protrusion was initially devoid of a GFP-myosin layer, which gradually formed and extended from the base of the protrusion. F-actin was absent in the leading front of the protrusion during the period of its rapid elongation, and the formation of a layer of F-actin in the front was closely correlated with its slowing-down or retraction. Periodical or continuous detachment of the F-actin layer from the apical membrane of the protrusion, accompanied by a transient increase in the elongation speed at the site of detachment, was observed in some of the protrusions. The detached F-actin layers, which formed a spiral layer of F-actin in the case of continuous detachment, moved in the opposite direction of protrusion elongation. In the presence of both cytochalasin A and quinine, the protrusions formed were not cylindrical but spherical, which swallowed up the entire cellular contents. The estimated bulk flux into the expanding spherical protrusions of such cells was four-times higher than the flux into the elongating cylindrical protrusions of the cells treated with quinine alone. These results indicate that the force responsible for the quinine-induced protrusion is mainly due to contraction of the cell body, which requires normal myosin II functions, while actin polymerization is important in restricting the direction of its expansion. We will discuss the possible significance of tail contraction in cell movement in the multicellular phase of Dictyostelium development, where cell locomotion similar to that induced by quinine is often observed without quinine treatment, and in protrusion elongation in general.Movies available on-line

The nullo protein is a component of the actin-myosin network that mediates cellularization in Drosophila melanogaster embryos

1994 ◽  
Vol 107 (7) ◽  
pp. 1863-1873 ◽  
Author(s):  
M.A. Postner ◽  
E.F. Wieschaus
Keyword(s):  
Drosophila Melanogaster ◽  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Nuclear Division ◽  
Leading Edge ◽  
The Other ◽  
Drosophila Embryogenesis ◽  
Zygotic Transcription ◽  
Hexagonal Network

After the 13th nuclear division cycle of Drosophila embryogenesis, cortical microfilaments are reorganized into a hexagonal network that drives the subsequent cellularization of the syncytial embryo. Zygotic transcription of the nullo and serendipity-alpha genes is required for normal structuring of the microfilament network. When either gene is deleted, the network assumes an irregular configuration leading to the formation of multinucleate cells. To investigate the role of these genes during cellularization, we have made monoclonal antibodies to both proteins. The nullo protein is present from cycle 13 through the end of cellularization. During cycle 13, it localizes between interphase actin caps and within metaphase furrows. In cellularizing embryos, nullo co-localizes with the actin-myosin network and invaginates along with the leading edge of the plasma membrane. The serendipity-alpha (sry-alpha) protein co-localizes with nullo protein to the hexagonal network but, unlike the nullo protein, it localizes to the sides rather than the vertices of each hexagon. Mutant embryos demonstrate that neither protein translationally regulates the other, but the localization of the sry-alpha protein to the hexagonal network is dependent upon nullo.

scholarly journals Differential localization of Acanthamoeba myosin I isoforms.

1992 ◽  
Vol 119 (5) ◽  
pp. 1193-1203 ◽  
Author(s):  
I C Baines ◽  
H Brzeska ◽  
E D Korn
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Contractile Vacuole ◽  
Membrane Domains ◽  
Cytoplasmic Vesicle ◽  
Large Vacuole ◽  
Anionic Phospholipids ◽  
Myosin I ◽  
Cytoplasmic Vesicles ◽  
Phagocytic Cups

Acanthamoeba myosins IA and IB were localized by immunofluorescence and immunoelectron microscopy in vegetative and phagocytosing cells and the total cell contents of myosins IA, IB, and IC were quantified by immunoprecipitation. The quantitative distributions of the three myosin I isoforms were then calculated from these data and the previously determined localization of myosin IC. Myosin IA occurs almost exclusively in the cytoplasm, where it accounts for approximately 50% of the total myosin I, in the cortex beneath phagocytic cups and in association with small cytoplasmic vesicles. Myosin IB is the predominant isoform associated with the plasma membrane, large vacuole membranes and phagocytic membranes and accounts for almost half of the total myosin I in the cytoplasm. Myosin IC accounts for a significant fraction of the total myosin I associated with the plasma membrane and large vacuole membranes and is the only myosin I isoform associated with the contractile vacuole membrane. These data suggest that myosin IA may function in cytoplasmic vesicle transport and myosin I-mediated cortical contraction, myosin IB in pseudopod extension and phagocytosis, and myosin IC in contractile vacuole function. In addition, endogenous and exogenously added myosins IA and IB appeared to be associated with the cytoplasmic surface of different subpopulations of purified plasma membranes implying that the different myosin I isoforms are targeted to specific membrane domains through a mechanism that involves more than the affinity of the myosins for anionic phospholipids.

Immunohistochemical Analysis of Human Lung Cancers with Anti-ras p21 Monoclonal Antibodies

1987 ◽  
Vol 2 (2) ◽  
pp. 75-82 ◽  
Author(s):  
Hirotoshi Dosaka ◽  
Masao Harada ◽  
Noboru Kuzumaki ◽  
Hitoshi Kobayashi ◽  
Hiroshi Isobe ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Cancer Cells ◽  
Positive Reaction ◽  
Primary Lung Cancer ◽  
Cytoplasmic Staining ◽  
Lung Cancers ◽  
Ras P21 ◽  
Malignant Pleurisy

The expression of ras oncogene product p21 in human malignant pleurisy and primary lung cancer was studied immunocyto-histochemically with monoclonal antibodies (MoAbs) rp-28 and rp-35 against ras p21. In pleural effusion cells, cancer cells revealed more intensively positive reaction with MoAb rp-35 than with MoAb rp-28, especially in the plasma membrane, and no positive reaction was obtained in any kind of inflammation cells with the exception of faintly positive reaction in the cytoplasm of macrophages. In primary lung cancers, well or moderately differentiated adenocarcinoma tissues showed higher reactivity with MoAb rp-28 than those of poorly differentiated adenocarcinoma or any other histological subtype of lung cancer. With MoAb rp-35, intensively positive reaction was obtained in most of cases with all different histological subtypes of lung cancer. The staining in cancer cells was usually localized intensively to the plasma membrane and weakly to the cytoplasm with both MoAbs. Normal bronchial epithelial and glandular tissues showed only cytoplasmic staining. These two MoAbs, especially MoAb rp-35, may be useful in clinicopathological applications for the diagnosis of malignant pleurisy and primary lung cancer.

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 Relative distribution of actin, myosin I, and myosin II during the wound healing response of fibroblasts.

1993 ◽  
Vol 120 (6) ◽  
pp. 1381-1391 ◽  
Author(s):  
P A Conrad ◽  
K A Giuliano ◽  
G Fisher ◽  
K Collins ◽  
P T Matsudaira ◽  
...  
Keyword(s):  
Myosin Ii ◽  
Leading Edge ◽  
Relative Distribution ◽  
Linear Arrays ◽  
3T3 Fibroblasts ◽  
Myosin I ◽  
Wound Healing Response ◽  
Correct Location ◽  
Swiss 3T3 Fibroblasts ◽  
Migrating Cells

Myosin I is present in Swiss 3T3 fibroblasts and its localization reflects a possible involvement in the extension and/or retraction of protrusions at the leading edge of locomoting cells and the transport of vesicles, but not in the contraction of stress fibers or transverse fibers. An affinity-purified polyclonal antibody to brush border myosin I colocalizes with a polypeptide of 120 kD in fibroblast extracts. Within initial protrusions of polarized, migrating fibroblasts, myosin I exhibits a punctate distribution, whereas actin is diffuse and myosin II is absent. Myosin I also exists in linear arrays parallel to the direction of migration in filopodia and microspikes, established protrusions, and within the leading lamellae of migrating cells. Myosin II and actin colocalize along transverse fibers in the lamellae of migrating cells, while myosin I displays no definitive organization along these fibers. During contractions of actin-based fibers, myosin II is concentrated in the center of the cell, while the distribution of myosin I does not change. Thus, myosin I is found at the correct location and time to be involved in the extension and/or retraction of protrusions and the transport of vesicles. Myosin II-based contractions in more posterior cellular regions could generate forces to separate cells, maintain a polarized cell shape, maintain the direction of locomotion, maximize the rate of locomotion, and/or aid in the delivery of cytoskeletal/contractile subunits to the leading edge.

scholarly journals Membrane tension controls adhesion positioning at the leading edge of cells

2017 ◽  
Vol 216 (9) ◽  
pp. 2959-2977 ◽  
Author(s):  
Bruno Pontes ◽  
Pascale Monzo ◽  
Laurent Gole ◽  
Anabel-Lise Le Roux ◽  
Anita Joanna Kosmalska ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Migration ◽  
Actin Cytoskeleton ◽  
Myosin Ii ◽  
Leading Edge ◽  
Membrane Tension ◽  
Independent Manner ◽  
Mechanical Signal ◽  
Cytoskeleton Remodeling ◽  
Upstream Control

Cell migration is dependent on adhesion dynamics and actin cytoskeleton remodeling at the leading edge. These events may be physically constrained by the plasma membrane. Here, we show that the mechanical signal produced by an increase in plasma membrane tension triggers the positioning of new rows of adhesions at the leading edge. During protrusion, as membrane tension increases, velocity slows, and the lamellipodium buckles upward in a myosin II–independent manner. The buckling occurs between the front of the lamellipodium, where nascent adhesions are positioned in rows, and the base of the lamellipodium, where a vinculin-dependent clutch couples actin to previously positioned adhesions. As membrane tension decreases, protrusion resumes and buckling disappears, until the next cycle. We propose that the mechanical signal of membrane tension exerts upstream control in mechanotransduction by periodically compressing and relaxing the lamellipodium, leading to the positioning of adhesions at the leading edge of cells.

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.

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