Actin filament destruction by osmium tetroxide

Maupin-Szamier, P.; Pollard, Thomas D.

doi:10.1083/jcb.77.3.837

Cited by 345 publications

(135 citation statements)

References 43 publications

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“…WITH TANNIC ACID: (The first two procedures [a and b] [4,5] were used to make the cell membrane permeable to tannic acid). (a) Exposure of cells to OsO4 vapor for 5 s, followed by immersion in 2% glutaraldehyde in 0,1 M HEPES buffer (pH 7.0) containing either 0.2% or 2% tannic acid for 30 rain followed with 0.05% or 0.1% OsO4 for l0 min; (b) 2% glutaraldehyde in 0.1 M HEPES buffer (pH 7.0) containing 0.05% saponin and 0.2% tannic acid for 30 min followed by 0.1% OsO4 for 10 rain; or (c) 1% glutaraldehyde, 50 mM KCI, 50 mM sodium phosphate buffer (pH 7.0), 5 mM MgCl2, 4% tannic acid (4).…”

Section: Fixationmentioning

confidence: 99%

The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections.

Ris¹

1985

The Journal of Cell Biology

163

110

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High voltage electron microscopy of intact cells prepared by the critical point drying (CPD) procedure has become an important tool in the study of three-dimensional relationships between cytoplasmic organelles. It has been claimed that critical point-dried specimens reveal a structure that is not visible in sections of plastic-embedded material; it has also been claimed that this structure, in association with known cytoplasmic filaments, forms a meshwork of tapering threads ("microtrabecular lattice"). Alternatively, this structure might be a surface tension artifact produced during CPD. To test possible sources of artifacts during CPD, model fiber systems of known structure were used. It was found that traces of water or ethanol in the CO2 caused distortions and fusion of fibers in pure muscle actin, fibrin, collagen, chromatin, and microtubules that produce a structure very similar to the proposed "microtrabecular lattice." These structures were, however, well preserved if water and ethanol were totally excluded from the CO2. The same results were obtained with whole mounts of cultured cells. A "microtrabecular lattice" was obtained if some water or ethanol was present in the pressure chamber. On the other hand, when water or ethanol were totally excluded from the CO2 during CPD, cytoplasmic filaments were uniform in thickness similar to their appearance in sections of plastic-embedded cells. It is concluded that the "microtrabecular lattice" is a distorted image of the cytoplasmic filament network produced during CPD by traces of water or ethanol in the CO2.

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Section: Fixationmentioning

confidence: 99%

The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections.

Ris¹

1985

The Journal of Cell Biology

163

110

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“…Antibody reaction was revealed by incubation with GARG l0 (l:10) for 2 h at room temperature. Labeled cryostat sections were washed in PBS, then in 0.2 M phosphate buffer pH 7.4, fixed with 1% glutaraldehyde/ 0.2% tannic acid, and postfixed with 0.5% OsO4 for 10 min at 0*C, to preserve actin from the destructive effects of the osmium tetroxide according to MaupinSzamier and Pollard (42). Cryostat sections were rapidly dehydrated with ethanol, embedded in Epon-Araldite, and cut with an LKB ultramicrotome into thin sections, which will be observed without any further staining.…”

Section: Immunogold Labelingmentioning

confidence: 99%

Evidence for a polarity in the distribution of proteins from the cytoskeleton in Torpedo marmorata electrocytes.

Kordeli¹,

Cartaud²,

Nghiêm³

et al. 1986

The Journal of Cell Biology

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Abstract. The subcellular distribution of the 43,000-D protein (43 kD or u~) and of some major cytoskeletal proteins was investigated in Torpedo marmorata electrocytes by immunocytochemical methods (immunofluorescence and immunogold at the electron microscope level) on frozen-fixed sections and homogenates of electric tissue. A monoclonal antibody directed against the 43-kD protein (Nghirm, H. O., J. Cartaud, C. Dubreuil, C. Kordeli, G. Buttin, and J. P. Changeux, 1983, Proc. Natl. Acad. Sci. USA, 80:6403-6407), selectively labeled the postsynaptic membrane on its cytoplasmic face. Staining by anti-actin and anti-desmin antibodies appeared evenly distributed within the cytoplasm: anti-desmin antibodies being associated with the network of intermediate-sized filaments that spans the electrocyte, and anti-actin antibodies making scattered clusters throughout the cytoplasm without preferential labeling of the postsynaptic membrane. On the other hand, a dense coating by anti-actin antibodies became apparent on the postsynaptic membrane in homogenates of electric tissue pointing to the possible artifactual redistribution of a soluble cytoplasmic actin pool.Anti-fodrin and anti-ankyrin antibodies selectively labeled the non-innervated membrane of the cell. F actin was also detected in this membrane. Filamin and vinculin, two actin-binding proteins recently localized at the rat neuromuscular junction (Bloch, R.J., and Z. W. Hall, 1983, J. CellBiol., 97:217-223), were detected in the electrocyte by the immunoblot technique but not by immunocytochemistry.The data are interpreted in terms of the functional polarity of the electrocyte and of the selective interaction of the cytoskeleton with the innervated and noninnervated domains of the plasma membrane.T HE postsynaptic membrane of the neuromuscular junction and of the electromotor synapse corresponds to a local differentiation of the plasma membrane characterized by an accumulation of the nicotinic acetylcholine receptor (Ach-R).t This membrane specialization persists for a long period after denervation (reviewed in references 10 and 18), indicating that physical constraints maintain the Ach-R molecules in place and, in particular, prevent against their lateral diffusion. Interactions of the Ach-R with extrinsic components from the extracellular matrix (13, 55) and/or with the cytoskeleton (17, 48, 67) have been postulated to contribute to this differentiation process. cell--the non-innervated face--is specialized in the regeneration of the electrochemical gradient (40). The Ach-R is present exclusively on the innervated membrane, and Na ÷ K ÷ ATPase accumulates on the non-innervated one. These membranes thus constitute fully differentiated and stable domains of the plasma membrane. From this standpoint the diskshaped electrocyte shows a striking functional and structural polarity ( 18, 21).Postsynaptic membrane fractions purified from Torpedo comprise essentially the intrinsic Ach-R polypeptides plus few extrinsic components of apparent molecular mass 43 kD (63, 64) na...

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“…In the present experiment, a majority of the fodrin labeling was associated with amorphous materials. A likely explanation is that the material is derived from actin filaments which were not preserved through osmium fixation (12). In fact, some immunogolds were observed with short thin filaments, which may be remaining actin filaments.…”

Section: Discussionmentioning

confidence: 99%

Immunoelectron microscopy of fodrin in the unroofed speciment of the adherent human neutrophil.

Fujimoto

Ogawa

1992

Acta Histochem. Cytochem

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We reported previously that fodrin is concentrated in the posterior portion of the human neutrophil stimulated with the chemotactic peptide (Fujimoto and Ogawa, J. Cell Sci., 96: 477-486, 1990). In the present study, the mechanically unroofed specimen of glass-adherent neutrophils was examined to define finer details of fodrin distribution. By immunofluorescence microscopy, F-actin and fodrin were positively labeled; after stimulation with the chemotactic peptide, fodrin was found more densely in the posterior half of the cell as in the whole cell observation. In ultrathin sections of the unroofed and immunogold-labeled specimen, two dimensional distribution of fodrin could be observed. Some gold particles were observed with filaments of about 5 nm in diameter, but others were on amorphous materials. In the stimulated neutrophil, labeling for fodrin was mainly found between straight filaments of about 10 nm in diameter which were densely distributed in the posterior portion of the cell. The results indicate that the redistribution of fodrin in the stimulated neutrophil is concurrent with other cytoskeletal changes.Cell migration is enabled by a combination of multiple factors, but obviously cytoskeleton is the major player in the phenomenon (17). In moving cells, various cytoskeletal proteins show redistribution, and accumulation either in the anterior or in the posterior portion has been reported: F-actin (10, 14), myosin II and actin-binding protein (18) in neutrophils and myosin I in amoebae (9) are seen anteriorly, whereas myosin II in amoebae is in the posterior (9).In the previous study, we observed that fodrin is concentrated in the posterior portion of migrating neutrophils stimulated with the chemotactic peptide (8). Moreover, fodrin was not localized to the cell membrane itself, but in the peripheral cytoplasm appearing highly filamentous in thin sections. We therefore concluded that fodrin is a cytoskeletal rather than a peripheral membrane protein and may be related to the dynamic reorganization of the cytoskeleton in the neutrophil. A question raised by the result was where and how fodrin is localized in the macromolecular architecture of the cell. Presently we utilized a cytochemical technique which allows distinct visualization of the membrane associated cytoskeletal components, and localized fodrin in direct correlation with them.

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Actin filament destruction by osmium tetroxide

Cited by 345 publications

References 43 publications

The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections.

The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections.

Evidence for a polarity in the distribution of proteins from the cytoskeleton in Torpedo marmorata electrocytes.

Immunoelectron microscopy of fodrin in the unroofed speciment of the adherent human neutrophil.

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