Interaction of myosin.ADP.fluorometal complexes with fluorescent probes and direct observation using quick-freeze deep-etch electron microscopy

Myosin forms stable ternary complexes with ADP and phosphate analogues of fluorometals that mimic different ATPase reaction intermediates corresponding to each step of the cross-bridge cycle. In the present study, we monitored the formation of ternary complexes of myosin.ADP.fluorometal using the fluorescence probe prodan. It has been reported that the fluorescence changes of the probe reflect the formation of intermediates in the ATPase reaction [Hiratsuka (1998) Biochemistry 37, 7167-7176]. Prodan bound to skeletal muscle heavy-mero-myosin (HMM).ADP.fluorometal, with each complex showing different fluorescence spectra. Prodan bound to the HMM.ADP.BeFn complex showed a slightly smaller red-shift than other complexes in the presence of ATP, suggesting a difference in the localized conformation or a difference in the population of BeFn species of global shape. We also examined directly the global structure of the HMM.ADP.fluorometal complexes using quick-freeze deep-etch replica electron microscopy. The HMM heads in the absence of nucleotides were mostly straight and elongated. In contrast, the HMM heads of ternary complexes showed sharply kinked or rounded configurations as seen in the presence of ATP. This is the first report of the direct observation of myosin-ADP-fluorometal ternary complexes, and the results suggest that these complexes indeed mimic the shape of the myosin head during ATP hydrolysis.     

Fibers of RecA protein and complexes of RecA protein and single-stranded phi X174 DNA as visualized by negative-stain electron microscopy

Monomers of purified RecA protein polymerize into helical fibers whose pitch is 7.2 nm to 7.5 nm and whose diameter is 11 nm. Either short (approximately 0.2 micron), single fibers, or bundles of aligned, longer fibers, can be formed preferentially, by varying the Mg2+ concentration. When RecA protein is bound to circular, single-stranded phi X174 DNA it forms helical fibers of different classes of contour lengths, ranging from 0.98 micron, depending upon the conditions of assembly. Two different helical pitches are found, one of 9.3 nm when the incubation buffer contains, besides the obligatory Mg2+, either ATP gamma S or ATP accompanied by single-strand binding protein, and one of 5.5 nm when the latter additives are omitted. Preformed fibers of the compact type can be converted to open ones of 9.3 nm pitch upon addition of ATP gamma S, even after the removal of unbound RecA. All signs of helicity are obliterated upon glutaraldehyde cross-linking except in those fibers whose assembly has been mediated by ATP gamma S. RecA protein and single-strand binding protein are competitively bound to single-stranded DNA. Composite complexes, however, are not encountered unless ATP gamma S is present. Otherwise, segments of DNA that are coated by one or the other protein are seen as separate regions. When the assembly of complexes of single-stranded DNA and RecA is mediated by single-strand binding protein and ATP, the axial separation between successive bases is 0 X 42 nm, somewhat greater than the axial distance between bases in one strand of duplex DNA in the B form. It is proposed that the bases of the single-stranded DNA in the complex are located near its inner surface, and that base-pairing with double-stranded DNA takes place following invasion of the central cavity of the complex.     

Visualization of RecA filaments and DNA by fluorescence microscopy

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.     

Ultrastructural changes during fertilization envelope assembly in Lytechinus pictus eggs revealed by quick-freeze,deep-etch electron microscopy

Morphological features of fertilization envelope assembly in egges from the sea urchin Lytechinus pictus were examind in platinum replicas of samples quick-frozen, deep-etched, and rotary-shadowed at various times after insemination. Unfertilized eggs are surrounded by the vitelline layer, a glycocalyx, which faith-fully follows the contours of the microvillus-studded egg surface. The vitelline layer is secured to the plasma membrane below via a series of short projections called vitelline posts. The vitelline matrix itself is an elaborate meshwork of uniformly sized filaments, which are decorated in places with globular particles. At fertilization, the vitelline layer elevates off the egg surface and by 1 min after insemination appears as a thin, airy network of fibers. In contrast to Strongylocentrotus purpuratus, impressions of the underlying microvilli are not retained in this species. The vitelline template appears to become filled in by the deposition of amorphous secretory material between 1 and 5 min after fertilization. This smooth, amorphous layer is then coated with a thin sheet of paracrystalline material. Paracrystalline coating is incomplete at 5 min, but by 20 min after insemination the coat is complete, consisting of ordered parallel rows of roset-telike particles.     

DNA gyrase and its complexes with DNA: direct observation by electron microscopy

Electron microscopy of DNA gyrase holoenzyme, of gyrase A subunits, and of the complexes of both species with DNA enables us to deduce the relative locations of subunits in the holoenzyme and to indicate a plausible path for DNA complexed with gyrase. The structural results are discussed in terms of certain models for directional DNA strand transport.     

The effects of various nucleotides on the structure of actin-attached myosin subfragment-1 studied by quick-freeze deep-etch electron microscopy

Stereo electron microscopy of negatively stained images showed that myosin heads in acto-subfragment-1 (S1) covalently cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide were predominantly short and round when ATP was added, in contrast to their uniform tilted appearance in the rigor state. As an attempt to exclude molecules which were actually dissociated but still tethered to actin by artificial cross-links, quick-freeze deep-etch electron microscopy was coupled with the mica flake method to observe uncross-linked native acto-S1 in the presence of ATP. To maintain the low affinity S1 associated to actin in the presence of ATP, a high concentration of acto-S1 was applied to mica flakes whose absorption had been chemically modified. The image of acto-S1 with added ATP agreed well with the expected time-course of reversible dissociation and reassociation, confirming the applicability of this approach to examination of the structural changes of acto-S1. S1 molecules attached to F-actin under rigor conditions or in the presence of ADP were elongated, with the long axis tilted to F-actin. Actin-attached S1 became short and round upon addition of ATP or ADP-inorganic vanadate. Adenyl-5'-yl imidodiphosphate and inorganic pyrophosphate each partially dissociated S1 from actin, as expected.     

Conformational change and localization of calpactin I complex involved in exocytosis as revealed by quick-freeze, deep-etch electron microscopy and immunocytochemistry

Calpactin I complex, a calcium-dependent phospholipid-binding protein, promotes aggregation of chromaffin vesicles at physiological micromolar calcium ion levels. Calpactin I complex was found to be a globular molecule with a diameter of 10.7 +/- 1.7 (SD) nm on mica. When liposomes were aggregated by calpactin, quick-freeze, deep-etching revealed fine thin strands (6.5 +/- 1.9 [SD] nm long) cross-linking opposing membranes in addition to the globules on the surface of liposomes. Similar fine strands were also observed between aggregated chromaffin vesicles when they were mixed with calpactin in the presence of Ca2+ ion. In cultured chromaffin cells, similar cross-linking short strands (6-10 nm) were found between chromaffin vesicles and the plasma membrane after stimulation with acetylcholine. Plasma membranes also revealed numerous globular structures approximately 10 nm in diameter on their cytoplasmic surface. Immunoelectron microscopy on frozen ultrathin sections showed that calpactin I was closely associated with the inner face of the plasma membranes and was especially conspicuous between plasma membranes and adjacent vesicles in chromaffin cells. These in vivo and in vitro data strongly suggest that calpactin I complex changes its conformation to cross-link vesicles and the plasma membrane after stimulation of cultured chromaffin cells.     

Direct observation of the assembly of RecA/DNA complexes by atomic force microscopy

The formation of the RecA/DNA nucleofilament on nicked circular double stranded (ds) DNA in the presence of ATPgammaS was studied using the atomic force microscope (AFM) at nanometer resolution. The AFM allowed simultaneous observation of both dsDNA substrate and RecA protein-coated sections such that they are highly distinguishable. Using a time series of images, the complex formation was monitored. AFM imaging provided direct evidence that assembly of the nucleofilaments occurs via a nucleation and growth mechanism. The nucleation step is much slower than the growth phase, as demonstrated by the predominance of naked dsDNA at early and middle time points, followed by the rapid appearance of partially then fully formed complexes. Observation of the formation of nucleation sites without accompanying growth on unnicked dsDNA enabled an estimate of the nucleation rate, of 5 x 10(-5) RecA min(-1) bp(-1). The published model for the analysis of RecA assembly on dsDNA deduces a single kinetic parameter that prevents the separate determination of nucleation rate and growth rate. By directly measuring the nucleation rate with the AFM, this model is employed to determine a growth rate of 202 min(-1). These AFM results provide the first direct evidence of previous results on complex formation obtained only by indirect means.     

Visualization of DNA and RNA molecules, and protein-DNA complexes for electron microscopy

This article provides step-by step instructions for the preparation of double- and single-stranded DNA and RNA molecules and protein-DNA complexes for electron microscopy (EM). Absorption, spreading, staining, dark-field imaging, and metal shadowing techniques are described in detail. A number of examples are illustrated on analysis of DNA replication, DNA repair and DNA recombination to demonstrate the usefulness of the technique for EM visualisation. Application of immunogold labeling of specific protein in DNA-protein complexes is also covered.     

Specific hydraulic conductivity of corneal stroma as seen by quick-freeze/deep-etch

Previous studies of the hydraulic conductivity of connective tissues have failed to show a correspondence between ultrastructure and specific hydraulic conductivity. We used the technique of quick-freeze/deep-etch to examine the ultrastructure of the corneal stroma and then utilized morphometric studies to compute the specific hydraulic conductivity of the corneal stroma. Our studies demonstrated ultrastructural elements of the extracellular matrix of the corneal stroma that are not seen using conventional electron microscopic techniques. Furthermore, we found that these structures may be responsible for generating the high flow resistance characteristic of connective tissues. From analysis of micrographs corrected for depth-of-field effects, we used Carmen-Kozeny theory to bound a morphometrically determined specific hydraulic conductivity of the corneal stroma between 0.46 x 10(-14) and 10.3 x 10(-14) cm2. These bounds encompass experimentally measured values in the literature of 0.5 x 10(-14) to 2 x 10(-14) cm2. The largest source of uncertainty was due to the depth-of-field estimates that ranged from 15 to 51 nm; a better estimate would substantially reduce the uncertainty of these morphometrically determined values.     

The Trypanosoma brucei cytoskeleton: Ultrastructure and localization of microtubule-associated and spectrin-like proteins using quick-freeze, deep-etch, immunogold electron microscopy

We have used a combination of quick-freezing/deep-etching and colloidal gold immunocytochemistry (i) to analyze the molecular organization of the microtubular membrane skeleton and the flagellum of Trypanosoma brucei, and (ii) to localize two defined cytoskeletal proteins within these structures. The cell body of trypanosomatids is enveloped by a membrane skeleton consisting of a tightly packed array of microtubules which are closely associated with the cell membrane. The membrane-oriented face of these microtubules is richly decorated with microtubule-associated proteins, which form intermicrotubule and microtubule-membrane linkers. In contrast, the cytoplasmic faces of the microtubules have a smooth, nondecorated appearance. A previously identified, highly repetitive microtubule-associated protein is confined to the membrane-oriented face of the microtubular array, suggesting that the function of this protein may be that of a microtubule-membrane linker. Quickfreezing has also been used to reveal the geometric organization of the paraflagellar rod structure in the flagellum, its interaction with the cell body, and a unique series of fleur-de-lis-like molecules which link this organelle to axonemal microtubules. Immunohistochemistry using an antibody against human erythrocyte spectrin suggests that these linker structures may contain ancestral spectrin-like molecules.     

Native structure and arrangement of inositol-1,4,5-trisphosphate receptor molecules in bovine cerebellar Purkinje cells as studied by quick-freeze deep-etch electron microscopy.

We used quick-freeze deep-etch replica electron microscopy to visualize the native structure of inositol-1,4,5-trisphosphate receptor (IP3R) in the cell. In the dendrites of Purkinje neurons of bovine cerebellum there were many vesicular organelles whose surfaces were covered with a two-dimensional crystalline array of molecules. Detailed examination of the cytoplasmic true surface of such vesicles in replica revealed that the structural unit, identified as IP3R by immunocytochemistry and subsequent Fourier analysis, is a square-shaped assembly and is aligned so that the side of the square is inclined by approximately 20 degrees from the row-line of the lattice. Comparison with the ryanodine receptor (RyaR), another intracellular Ca2+ channel on the endoplasmic reticulum, suggested that IP3R, unlike RyaR, has a very compact structure, potentially reflecting the crucial difference in the function of the cytoplasmic portion of the molecule.     

Visualization of the reconstituted FRGY2-mRNA complexes by electron microscopy

Xenopus oocytes store large quantities of translationally dormant mRNA in the cytoplasm as storage messenger ribonucleoprotein particles (mRNPs). The Y-box proteins, mRNP3 and FRGY2/mRNP4, are major RNA binding components of maternal storage mRNPs in oocytes. In this study, we show that the FRGY2 proteins form complexes with mRNA, which leads to mRNA stabilization and translational repression. Visualization of the FRGY2-mRNA complexes by electron microscopy reveals that FRGY2 packages mRNA into a compact RNP. Our results are consistent with a model that the Y-box proteins function in packaging of mRNAs to store them stably for a long time in the oocyte cytoplasm.     

Visualization of cAMP receptor protein-induced DNA kinking by electron microscopy

The effect of specific DNA binding of the cAMP . cAMP receptor protein complex to two DNA fragments (301 and 2685 base-pairs in length) containing the lac operon has been investigated by electron microscopy. It is shown that specific DNA binding of the cAMP . cAMP receptor protein complex induces a kink of 30 to 45 degrees in the DNA with the apex of the kink located at the site of protein attachment. These findings lend direct visual support for the kinking hypothesis based on the observation of anomalous electrophoretic mobility of DNA fragments containing specifically bound cAMP receptor protein.     

The Chlamydomonas cell wall and its constituent glycoproteins analyzed by the quick-freeze, deep-etch technique

Using the quick-freeze, deep-etch technique, we have analyzed the structure of the intact cell wall of Chlamydomonas reinhardi, and have visualized its component glycoproteins after mechanical shearing and after depolymerization induced by perchlorate or by the wall-disrupting agent, autolysin. The intact wall has previously been shown in a thin-section study (Roberts, K., M. Gurney-Smith, and G. J. Hills, 1972, J. Ultrastruct. Res. 40:599-613) to consist of a discrete central triplet bisecting a meshwork of fibrils. The deep-etch technique provides additional information about the architecture of each of these layers under several different experimental conditions, and demonstrates that each layer is constructed from a distinct set of components. The innermost layer of the central triplet proves to be a fibrous network which is stable to perchlorate but destabilized by autolysin, disassembling into fibrillar units we designate as "fishbones." The medial layer of the triplet is a loose assemblage of large granules. The outer layer is a thin, crystalline assembly that is relatively unaffected by autolysin. It depolymerizes into two glycoprotein species, one fibrous and one globular. The wall glycoproteins prove to be structurally similar to two fibrous proteins that associate with the flagellar membrane, namely, the sexual agglutinins and the protomers of a structure we designate a "hammock." They are also homologous to some of the fibrous components found in the extracellular matrices of multicellular plants and animals. The quick-freeze, deep-etch technique is demonstrated to be a highly informative way to dissect the structure of a fibrous matrix and visualize its component macromolecules.     

Visualization of the bent helix in kinetoplast DNA by electron microscopy

Kinetoplast DNA minicircles from the trypanosomatid Crithidia fasciculata contain a segment of approximately 200 bp which is probably more highly bent than any other DNA previously studied. Electron microscopy (EM) of relaxed minicircles (2.5 kb) revealed 200-300 bp loops within the larger circles, and the loops could also be detected on full-length linear molecules. Examination by EM of a 219 bp cloned fragment which contains the bent helix revealed that up to 70% of the molecules appeared circular whether or not the ends were cohesive. In contrast, a 207 bp fragment from pBR322 showed no circles and the fragments in general appeared much straighter than the kinetoplast fragments. Treatment of the 219 bp bent kinetoplast fragment with the drug distamycin caused a striking reduction in curvature.     

Visualization of the nuclear lamina in mouse anterior pituitary cells and immunocytochemical detection of lamin A/C by quick-freeze freeze-substitution electron microscopy.

We examined the nuclear lamina in the quickly frozen anterior pituitary cells by electron microscopic techniques combined with freeze substitution, deep etching, and immunocytochemistry and compared it with that in the chemically fixed cells. By quick-freeze freeze-substitution electron microscopy, an electron-lucent layer, as thick as 20 nm, was revealed just inside the inner nuclear membrane, whereas in the conventionally glutaraldehyde-fixed cells the layer was not seen. By quick-freeze deep-etch electron microscopy, we could not distinguish definitively the layer corresponding to the nuclear lamina in either fresh unfixed or glutaraldehyde-fixed cells. Immunofluorescence microscopy showed that lamin A/C in the nucleus was detected in the acetone-fixed cells and briefly in paraformaldehyde-fixed cells but not in the cells with prolonged paraformaldehyde fixation. Nuclear localization of lamin A/C was revealed by immunogold electron microscopy also in the quickly frozen and freeze-substituted cells, but not in the paraformaldehyde-fixed cells. Lamin A/C was localized mainly in the peripheral nucleoplasm within 60 nm from the inner nuclear membrane, which corresponded to the nuclear lamina. These results suggest that the nuclear lamina can be preserved both ultrastructurally and immunocytochemically by quick-freezing fixation, rather than by conventional chemical fixation.     

Mechanism of brush border contractility studied by the quick-freeze, deep-etch method

We have analyzed terminal web contraction in sheets of glycerinated chicken small intestine epithelium and in isolated intestinal brush borders using a quick-freeze, deep-etch, rotary shadow replication technique. In the presence of Mg-ATP at 37 degrees C, the terminal web region of each cell in the glycerinated sheet and of each isolated brush border became severely constricted at the level of its zonula adherens (ZA). Consequently, the individual brush borders rounded up, splaying out their microvilli in fanlike patterns. The most prominent ultrastructural changes that occurred during terminal web contraction were a dramatic decrease in the diameter of the circumferential ring composed of a bundle of 8-9-nm filaments adjacent to the zonula adherens and a decrease in the number of cross-linkers between the microvillus rootlets. Microvilli were not retracted into the terminal web. We have used myosin S1 decoration to demonstrate that most of the circumferential bundle filaments are actin and that the actin filaments are arranged in the bundle with mixed polarity. Some filaments within the bundle did not decorate with myosin S1 and had tiny projections that appeared to be attached to adjacent actin filaments. Because of their morphology and immunofluorescent localization of myosin within this region of the terminal web, we propose that these undecorated filaments are myosin. From these results, we conclude that brush border contraction is caused primarily by an active sliding of actin and myosin filaments within the circumferential bundle of filaments associated with the ZA.     

Visualization of macromolecular complexes using cryo-electron microscopy with FEI Tecnai transmission electron microscopes

This protocol details the steps used for visualizing the frozen-hydrated grids as prepared following the accompanying protocol entitled 'Preparation of macromolecular complexes for visualization using cryo-electron microscopy.' This protocol describes how to transfer the grid to the microscope using a standard cryo-transfer holder or, alternatively, using a cryo-cartridge loading system, and how to collect low-dose data using an FEI Tecnai transmission electron microscope. This protocol also summarizes and compares the various options that are available in data collection for three-dimensional (3D) single-particle reconstruction. These options include microscope settings, choice of detectors and data collection strategies both in situations where a 3D reference is available and in the absence of such a reference (random-conical and common lines).