Leukosialin (CD43, sialophorin) redistribution in uropods of polarized neutrophils is induced by CD43 cross-linking by antibodies, by colchicine or by chemotactic peptides

1997 ◽  
Vol 110 (13) ◽  
pp. 1465-1475
Author(s):  
S. Seveau ◽  
S. Lopez ◽  
P. Lesavre ◽  
J. Guichard ◽  
E.M. Cramer ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Motility ◽  
Small Proportion ◽  
Cytochalasin B ◽  
Cell Polarization ◽  
Cross Linking ◽  
Chemotactic Peptides ◽  
Butanedione Monoxime ◽  
Major Surface ◽  
Triton X

We investigated a possible association of leukosialin (CD43), the major surface sialoglycoprotein of leukocytes, with neutrophil cytoskeleton. We first analysed the solubility of CD43 in Triton X-100 and observed that CD43 of resting neutrophils was mostly soluble. The small proportion of CD43 molecules, which ‘spontaneously’ precipitated in Triton, appeared associated with F-actin, as demonstrated by the fact that this insolubility did not occur when cells were incubated with cytochalasin B or when F-actin was depolymerized with DNase I in the Triton precipitate. Cell stimulation with anti-CD43 mAb (MEM59) enhanced this CD43-cytoskeleton association. By immunofluorescence as well as by electron microscopy, we observed a redistribution of CD43 on the neutrophil membrane, initially in patches followed by caps, during anti-CD43 cross-linking at 37 degrees C. This capping did not occur at 4 degrees C and was inhibited by cytochalasin B and by a myosin disrupting drug butanedione monoxime, thus providing evidence that the actomyosin contracile sytem is involved in the capping and further suggesting an association of CD43 with the cytoskeleton. Some of the capped cells exhibited a front-tail polarization with CD43 caps located in the uropod at the rear of the cell. Surprisingly, colchicine and the chemotactic factor fNLPNTL which induce neutrophil polarization associated with cell motility, also resulted in a clustering of CD43 in the uropod, independently of a cross-linking of the molecule by mAbs. An intracellular redistribution of F-actin, mainly at the leading front and of myosin in the tail, was observed during CD43 clustering induced by colchicine and in cells polarized by anti-CD43 mAbs cross-linking. We conclude that neutrophil CD43 interacts with the cytoskeleton, either directly or indirectly, to redistribute in the cell uropod under antibodies stimulation or during cell polarization by colchicine, thus highly suggesting that CD43 may be involved in cell polarization.

scholarly journals Human Coronavirus 229E Binds to CD13 in Rafts and Enters the Cell through Caveolae

2004 ◽  
Vol 78 (16) ◽  
pp. 8701-8708 ◽  
Author(s):  
Ryuji Nomura ◽  
Asuka Kiyota ◽  
Etsuko Suzaki ◽  
Katsuko Kataoka ◽  
Yoshihide Ohe ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Interference ◽  
Human Fibroblasts ◽  
Cross Linking ◽  
Subsequent Infection ◽  
Human Coronavirus ◽  
Caveolin 1 ◽  
Membrane Microdomain ◽  
Triton X ◽  
Human Coronavirus 229E

ABSTRACT CD13, a receptor for human coronavirus 229E (HCoV-229E), was identified as a major component of the Triton X-100-resistant membrane microdomain in human fibroblasts. The incubation of living fibroblasts with an anti-CD13 antibody on ice gave punctate labeling that was evenly distributed on the cell surface, but raising the temperature to 37°C before fixation caused aggregation of the labeling. The aggregated labeling of CD13 colocalized with caveolin-1 in most cells. The HCoV-229E virus particle showed a binding and redistribution pattern that was similar to that caused by the anti-CD13 antibody: the virus bound to the cell evenly when incubated on ice but became colocalized with caveolin-1 at 37°C; importantly, the virus also caused sequestration of CD13 to the caveolin-1-positive area. Electron microscopy confirmed that HCoV-229E was localized near or at the orifice of caveolae after incubation at 37°C. The depletion of plasmalemmal cholesterol with methyl β-cyclodextrin significantly reduced the HCoV-229E redistribution and subsequent infection. A caveolin-1 knockdown by RNA interference also reduced the HCoV-229E infection considerably. The results indicate that HCoV-229E first binds to CD13 in the Triton X-100-resistant microdomain, then clusters CD13 by cross-linking, and thereby reaches the caveolar region before entering cells.

scholarly journals Cryo-EM structure of NPF-bound human Arp2/3 complex and activation mechanism

2020 ◽  
Vol 6 (23) ◽  
pp. eaaz7651 ◽  
Author(s):  
Austin Zimmet ◽  
Trevor Van Eeuwen ◽  
Malgorzata Boczkowska ◽  
Grzegorz Rebowski ◽  
Kenji Murakami ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Motility ◽  
Binding Sites ◽  
Activation Mechanism ◽  
Actin Binding ◽  
Cross Linking ◽  
Specific Interactions ◽  
Related Protein ◽  
Actin Networks ◽  
Cryo Electron Microscopy

Actin-related protein (Arp) 2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including two actin-related subunits (Arp2 and Arp3). Two nucleation-promoting factors (NPFs) bind Arp2/3 complex during activation, but the order, specific interactions, and contribution of each NPF to activation are unresolved. Here, we report the cryo–electron microscopy structure of recombinantly expressed human Arp2/3 complex with two WASP family NPFs bound and address the mechanism of activation. A cross-linking assay that captures the transition of the Arps into the activated filament-like conformation shows that actin binding to NPFs favors this transition. Actin-NPF binding to Arp2 precedes binding to Arp3 and is sufficient to promote the filament-like conformation but not activation. Structure-guided mutagenesis of the NPF-binding sites reveals their distinct roles in activation and shows that, contrary to budding yeast Arp2/3 complex, NPF-mediated delivery of actin at the barbed end of both Arps is required for activation of human Arp2/3 complex.

Embedding Procedure for Water Swollen Samples

1970 ◽  
Vol 28 ◽  
pp. 504-505
Author(s):  
Ann M. Thomas ◽  
Virginia Shemeley
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Ion Exchange ◽  
Structural Changes ◽  
Cross Linking ◽  
Ion Exchange Resins ◽  
Transmission Electron ◽  
First Pass ◽  
Exchange Resins

Those samples which swell rapidly when exposed to water are, at best, difficult to section for transmission electron microscopy. Some materials literally burst out of the embedding block with the first pass by the knife, and even the most rapid cutting cycle produces sections of limited value. Many ion exchange resins swell in water; some undergo irreversible structural changes when dried. We developed our embedding procedure to handle this type of sample, but it should be applicable to many materials that present similar sectioning difficulties.The purpose of our embedding procedure is to build up a cross-linking network throughout the sample, while it is in a water swollen state. Our procedure was suggested to us by the work of Rosenberg, where he mentioned the formation of a tridimensional structure by the polymerization of the GMA biproduct, triglycol dimethacrylate.

The Current Situation in Chemical Stabilization of Biological Materials

1972 ◽  
Vol 30 ◽  
pp. 132-133
Author(s):  
John H. Luft
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Molecular Level ◽  
Cross Linking ◽  
Chemical Stabilization ◽  
Specimen Preparation ◽  
Sufficient Condition ◽  
Radio Telescopes

With information processing devices such as radio telescopes, microscopes or hi-fi systems, the quality of the output often is limited by distortion or noise introduced at the input stage of the device. This analogy can be extended usefully to specimen preparation for the electron microscope; fixation, which initiates the processing sequence, is the single most important step and, unfortunately, is the least well understood. Although there is an abundance of fixation mixtures recommended in the light microscopy literature, osmium tetroxide and glutaraldehyde are favored for electron microscopy. These fixatives react vigorously with proteins at the molecular level. There is clear evidence for the cross-linking of proteins both by osmium tetroxide and glutaraldehyde and cross-linking may be a necessary if not sufficient condition to define fixatives as a class.

CRYO-Electron Microscopy of the Acto-Myosin-ATP Complex

1990 ◽  
Vol 48 (1) ◽  
pp. 248-249
Author(s):  
John Trinickt ◽  
Howard White
Keyword(s):  
Electron Microscopy ◽  
Atp Hydrolysis ◽  
Myosin Head ◽  
Bound State ◽  
Cross Linking ◽  
Weakly Bound ◽  
Cryo Electron Microscopy ◽  
Myosin Subfragment 1 ◽  
Attached State ◽  
High Fraction

The primary force of muscle contraction is thought to involve a change in the myosin head whilst attached to actin, the energy coming from ATP hydrolysis. This change in attached state could either be a conformational change in the head or an alteration in the binding angle made with actin. A considerable amount is known about one bound state, the so-called strongly attached state, which occurs in the presence of ADP or in the absence of nucleotide. In this state, which probably corresponds to the last attached state of the force-producing cycle, the angle between the long axis myosin head and the actin filament is roughly 45°. Details of other attached states before and during power production have been difficult to obtain because, even at very high protein concentration, the complex is almost completely dissociated by ATP. Electron micrographs of the complex in the presence of ATP have therefore been obtained only after chemically cross-linking myosin subfragment-1 (S1) to actin filaments to prevent dissociation. But it is unclear then whether the variability in attachment angle observed is due merely to the cross-link acting as a hinge.We have recently found low ionic-strength conditions under which, without resorting to cross-linking, a high fraction of S1 is bound to actin during steady state ATP hydrolysis. The structure of this complex is being studied by cryo-electron microscopy of hydrated specimens. Most advantages of frozen specimens over ambient temperature methods such as negative staining have already been documented. These include improved preservation and fixation rates and the ability to observe protein directly rather than a surrounding stain envelope. In the present experiments, hydrated specimens have the additional benefit that it is feasible to use protein concentrations roughly two orders of magnitude higher than in conventional specimens, thereby reducing dissociation of weakly bound complexes.

scholarly journals Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy.

1975 ◽  
Vol 66 (1) ◽  
pp. 198-200 ◽  
Author(s):  
D Mazia ◽  
G Schatten ◽  
W Sale
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Mammalian Cells ◽  
Experimental Treatment ◽  
The Body ◽  
Transmission Electron ◽  
Coated Plate ◽  
Living Material ◽  
Coated Surfaces ◽  
Triton X

Cells of many kinds adhere firmly to glass or plastic surfaces which have been pretreated with polylysine. The attachment takes place as soon as the cells make contact with the surfaces, and the flattening of the cells against the surfaces is quite rapid. Cells which do not normally adhere to solid surfaces, such as sea urchin eggs, attach as well as cells which normally do so, such as amebas or mammalian cells in culture. The adhesion is interpreted simply as the interaction between the polyanionic cell surfaces and the polycationic layer of adsorbed polylysine. The attachment of cells to the polylysine-treated surfaces can be exploited for a variety of experimental manipulations. In the preparation of samples for scanning or transmission electron microscopy, the living material may first be attached to a polylysine-coated plate or grid, subjected to some experimental treatment (fertilization of an egg, for example), then transferred rapidly to fixative and further passed through processing for observation; each step involves only the transfer of the plate or grid from one container to the next. The cells are not detached. The adhesion of the cell may be so firm that the body of the cell may be sheared away, leaving attached a patch of cell surface, face up, for observation of its inner aspect. For example, one may observe secretory vesicles on the inner face of the surface (3) or may study the association of filaments with the inner surface (Fig. 1). Subcellular structures may attach to the polylysine-coated surfaces. So far, we have found this to be the case for nuclei isolated from sea urchin embryos and for the microtubules of flagella, which are well displayed after the membrane has been disrupted by Triton X-100 (Fig. 2).

scholarly journals Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell

1978 ◽  
Vol 77 (3) ◽  
pp. R27 ◽  
Author(s):  
M Osborn ◽  
RE Webster ◽  
K Weber
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Light Microscope ◽  
Immunofluorescence Microscopy ◽  
Uranyl Acetate ◽  
Tubulin Antibodies ◽  
Ptk2 Cell ◽  
Cytoplasmic Microtubules ◽  
Triton X ◽  
Microtubular Structures

PtK2 cells were grown on gold grids and treated with Triton X-100 in a microtubule stabilizing buffer. The resulting cytoskeletons were fixed with glutaraldehyde and subjected to the indirect immunofluorescence procedure using monospecific tubulin antibodies. Grids were examined first by fluorescence microscopy, and the display of fluorescent cytoplasmic microtubules was recorded. The grids were then stained with uranyl acetate and the display of fibrous structures recorded by electron microscopy. Thus the display of cytoplasmic microtubular structures in the light microscope and the electron microscope can be compared within the same cytoskeleton. The results show a direct correspondence of the fluorescent fibers in the light microscope with uninterrupted fibers of diameter approximately 550 A in the electron microscope. This is the diameter reported for a single microtubule decorated around its circumference by two layers of antibody molecules. Thus under optimal conditions immunofluorescence microscopy can visualize individual microtubules.

Cytochalasin B-induced pseudo-cleavage of mouse oocytes in vitro. II. Studies of the mechanism and morphological consequences of pseudocleavage

1977 ◽  
Vol 26 (1) ◽  
pp. 323-337
Author(s):  
P.M. Wassarman ◽  
T.E. Ukena ◽  
W.J. Josefowicz ◽  
G.E. Letourneau ◽  
M.J. Karnovsky
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Cytochalasin B ◽  
Oocyte Size ◽  
Contractile Ring ◽  
Subsequent Formation ◽  
Mouse Oocytes ◽  
Meiotic Competence ◽  
Scanning Electron

Mouse oocytes are induced by cytochalasin B to undergo ‘pseudocleavage’ in vitro into 2 compartments, only one of which possesses microvilli. It has been found that this particular response to cytochalasin B is related to oocyte size and, possibly, to the acquisition of meiotic competence by the oocyte during its growth phase. Certain of the morphological events which characterize pseudocleavage have been determined using transmission and scanning electron microscopy. These events include: (i) an initial withdrawal of microvilli from the surface of the oocyte, together with the concomitant disappearance of microfilaments normally associated with the microvilli; (ii) the subsequent formation of a pseudocleavage furrow and contractile ring; and (iii) the reappearance of microvilli and associated microfilaments in one of the two resulting oocyte compartments. These changes in surface architecture are reflected in the distribution of fluorescein-conjugated lectins bound to the oocyte surface during pseudocleavage.

Cyclic changes in amphibian egg microvilli occur during the division cycle: implication of MPF

Development ◽  
1991 ◽  
Vol 112 (1) ◽  
pp. 91-98
Author(s):  
C. Aimar
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Protein Synthesis ◽  
Cytochalasin B ◽  
Transmission Electron ◽  
Sensitive Factor ◽  
Cyclic Changes

The microvilli (MV) of Pleurodeles (amphibian) eggs were examined following fertilization and compared with those of artificially activated eggs and enucleated eggs using scanning and transmission electron microscopy. The MV pattern in fertilized eggs was found to undergo a cyclic transformation during the course of the first few division cycles. Similar changes also occurred in the MV of artificially activated eggs and enucleated eggs. The reorganization of the MV was sensitive to cycloheximide and cytochalasin B, but was unaffected by colchicine. Thus, this MV alteration requires protein synthesis and microfilaments but microtubules are not implicated in this process. In addition, the effects on the MV pattern of the maturation or mitosis promoting factor (MPF) were tested. Injection of MPF into eggs at different times during the first division cycle nearly always induced an elongation of the MV. This observation suggests that MPF could regulate either directly or indirectly, via a MPF-sensitive factor, the cyclic transformation of amphibian egg MV.

Direct visualization of the 10-nm (100-A)-filament network in whole and enucleated cultured cells

1978 ◽  
Vol 31 (1) ◽  
pp. 393-409
Author(s):  
J.V. Small ◽  
J.E. Celis
Keyword(s):  
Cytochalasin B ◽  
Cultured Cells ◽  
Marked Effect ◽  
High Electron ◽  
Perinuclear Region ◽  
Structural Elements ◽  
3T3 Cells ◽  
10 Nm Filaments ◽  
Triton X ◽  
Filament Network

Following extraction of actomyosin and tubulin from cultured cells treated with Triton X-100, a cytoskeleton remains which is composed predominantly of the cell nucleus encompassed by a network of 10-nm filaments. After negative staining the dense perinuclear region appears as a densely woven filament net punctuated by patches of high electron density. Enucleation of 3T3 cells with cytochalasin B gives rise to karyoplasts surronunded by 10-nm filaments and cytoplasts in which 10-nm filaments remain situated in the central region of the cytoplasm. While the 10-nm filaments occurred mainly as single filaments in human skin fibroblasts and 3T3 cells, in epithelioid PtK1 and PtK2 cells they were commonly associated in prominent meandering bundles. In addition, in these latter cells after Triton extraction the remaining ribosomes were bound specifically to the 10-nm-filament net. After exposure of 3T3 cells to cytochalasin B the 10-nm filaments formed branches that radiated from the perinuclear region into the immobile cell extensions. Concavalin A had no marked effect on the distribution of the 10-nm-filament net. The results suggest that the 10-nm filaments act primarily as structural elements, serving, in particular, to support and constrain the nucleus in its position in the cell.

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