Faculty Opinions recommendation of Formation of Hirano bodies after inducible expression of a modified form of an actin-cross-linking protein.

ABSTRACT Hirano bodies are cytoplasmic inclusions composed mainly of actin and actin-associated proteins. The formation of Hirano bodies during various neurodegenerative disorders, including Alzheimer's disease and amyotrophic lateral sclerosis, has been reported. Although the underlying molecular mechanisms that lead to the formation of these inclusions in the brain are not known, expression of the C-terminal fragment (CT) (amino acids 124 to 295) from the endogenous 34-kDa actin-binding protein of Dictyostelium discoideum leads to the formation of actin inclusions in vivo. In the current study, we report the development of an inducible expression system to study the early phases of Hirano body formation using an inducible promoter system (rnrB). By fusing the CT to a green fluorescent protein (CT-GFP), we monitored protein expression and localization by fluorescence microscopy, flow cytometry, and Western blot analysis. We observed an increase in the number and size of inclusions formed following induction of the CT-GFP vector system. Time-lapse microscopy studies revealed that the CT-GFP foci associated with the cell cortex and fused to form a single large aggregate. Transmission electron microscopy further demonstrates that these inclusions have a highly ordered ultrastructure, a pathological hallmark of Hirano bodies observed in postmortem brain samples from patients with various neurodegenerative disorders. Collectively, this system provides a method to visualize and characterize the events that surround early actin inclusion formation in a eukaryotic model.

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Formation of Hirano Bodies Induced by Expression of an Actin Cross-Linking Protein with a Gain-of-Function Mutation

Eukaryotic Cell ◽

10.1128/ec.2.4.778-787.2003 ◽

2003 ◽

Vol 2 (4) ◽

pp. 778-787 ◽

Cited By ~ 25

Author(s):

Andrew Maselli ◽

Ruth Furukawa ◽

Susanne A. M. Thomson ◽

Richard C. Davis ◽

Marcus Fechheimer

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Cellular Response ◽

Biological Function ◽

Point Mutations ◽

Actin Binding ◽

Cross Linking ◽

Hirano Bodies ◽

Transmission Electron ◽

Ef Hand

ABSTRACT Hirano bodies are paracrystalline actin filament-containing structures reported to be associated with a variety of neurodegenerative diseases. However, the biological function of Hirano bodies remains poorly understood, since nearly all prior studies of these structures were done with postmortem samples of tissue. In the present study, we generated a full-length form of a Dictyostelium 34-kDa actin cross-linking protein with point mutations in the first putative EF hand, termed 34-kDa ΔEF1. The 34-kDa ΔEF1 protein binds calcium normally but has activated actin binding that is unregulated by calcium. The expression of the 34-kDa ΔEF1 protein in Dictyostelium induces the formation of Hirano bodies, as assessed by both fluorescence microscopy and transmission electron microscopy. Dictyostelium cells bearing Hirano bodies grow normally, indicating that Hirano bodies are not associated with cell death and are not deleterious to cell growth. Moreover, the expression of the 34-kDa ΔEF1 protein rescues the phenotypes of cells lacking the 34-kDa protein and cells lacking both the 34-kDa protein and α-actinin. Finally, the expression of the 34-kDa ΔEF1 protein also initiates the formation of Hirano bodies in cultured mouse fibroblasts. These results show that the failure to regulate the activity and/or affinity of an actin cross-linking protein can provide a signal for the formation of Hirano bodies. More generally, the formation of Hirano bodies is a cellular response to or a consequence of aberrant function of the actin cytoskeleton.

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Influence of Calcium Ions on the Plant Cell Surface: Membrane Fusions and Conformational Changes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050780 ◽

1974 ◽

Vol 32 ◽

pp. 154-155

Author(s):

D. James Morré ◽

Charles E. Bracker ◽

William J. VanDerWoude

Keyword(s):

Cell Wall ◽

Cell Surface ◽

Conformational Changes ◽

Calcium Ions ◽

Surface Membrane ◽

Carboxyl Groups ◽

Cross Linking ◽

Ultrastructural Evidence ◽

Glycine Max L ◽

Wall Extensibility

Calcium ions in the concentration range 5-100 mM inhibit auxin-induced cell elongation and wall extensibility of plant stems. Inhibition of wall extensibility requires that the tissue be living; growth inhibition cannot be explained on the basis of cross-linking of carboxyl groups of cell wall uronides by calcium ions. In this study, ultrastructural evidence was sought for an interaction of calcium ions with some component other than the wall at the cell surface of soybean (Glycine max (L.) Merr.) hypocotyls.

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Embedding Procedure for Water Swollen Samples

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010006951x ◽

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.

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The Current Situation in Chemical Stabilization of Biological Materials

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100093912 ◽

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.

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Parathyroid gland before and after cisplatin treatment

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128584 ◽

1987 ◽

Vol 45 ◽

pp. 860-861

Author(s):

S.K. Aggarwal ◽

J.M. Fadool

Keyword(s):

Parathyroid Gland ◽

Wistar Rats ◽

Antitumor Agent ◽

The Body ◽

Cross Linking ◽

Limiting Factor ◽

Hormonal Changes ◽

Primary Mechanism ◽

Before And After ◽

Dna Strands

Cisplatin (CDDP) a potent antitumor agent suffers from severe toxic side effects with nephrotoxicity being the major dose-limiting factor, The primary mechanism of its action has been proposed to be through its cross-linking DNA strands. It has also been shown to inactivate various transport enzymes and induce hypocalcemia and hypomagnesemia that may be the underlying cause for some of its toxicities. The present is an effort to study its influence on the parathyroid gland for any hormonal changes that control calcium levels in the body.Male Swiss Wistar rats (Crl: (WI) BR) weighing 200-300 g and of 60 days in age were injected (ip) with cisplatin (7mg/kg in normal saline). The controls received saline injections only. The animals were injected (iv) with calcium (0.5 ml of 10% calcium gluconate/day) and were killed by decapitation on day 1 through 5. Trunk blood was collected in heparinized tubes.

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Large-cluster and combined fluorescent and gold immunoprobes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166920 ◽

1996 ◽

Vol 54 ◽

pp. 892-893

Author(s):

Richard D. Powell ◽

James F. Hainfeld ◽

Carol M. R. Halsey ◽

David L. Spector ◽

Shelley Kaurin ◽

...

Keyword(s):

Metal Cluster ◽

Sulfonyl Chloride ◽

Gel Filtration ◽

Cross Linking ◽

Site Specific ◽

Fab Fragments ◽

Cluster Label ◽

Covalently Linked ◽

Texas Red ◽

Fold Excess

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100179993 ◽

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.

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