Tectorins crosslink type II collagen fibrils and connect the tectorial membrane to the spiral limbus.

All inner ear organs possess extracellular matrix appendices over the sensory epithelia that are crucial for their proper function. The tectorial membrane (TM) is a gelatinous acellular membrane located above the hearing sensory epithelium and is composed mostly of type II collagen, and α and ß tectorins. TM molecules self-assemble in the endolymph fluid environment, interacting medially with the spiral limbus and distally with the outer hair cell stereocilia. Here, we used immunogold labeling in freeze-substituted mouse cochleae to assess the fine localization of both tectorins in distinct TM regions. We observed that the TM adheres to the spiral limbus through a dense thin matrix enriched in α- and ß-tectorin, both likely bound to the membranes of interdental cells. Freeze-etching images revealed that type II collagen fibrils were crosslinked by short thin filaments (4±1.5nm, width), resembling another collagen type protein, or chains of globular elements (15±3.2nm, diameter). Gold-particles for both tectorins also localized adjacent to the type II collagen fibrils, suggesting that these globules might be composed essentially of α- and ß-tectorins. Finally, the presence of gold-particles at the TM lower side suggests that the outer hair cell stereocilia membrane has a molecular partner to tectorins, probably stereocilin, allowing the physical connection between the TM and the organ of Corti.

Novel configuration of a myosin II transient intermediate analogue revealed by quick-freeze deep-etch replica electron microscopy.

In the present paper, we described our attempt to characterize the rough three-dimensional features of the structural analogue of the key intermediate of myosin's cross-bridge cycle. Using quick-freeze deep-etch replica electron microscopy, we observed that actin-attached myosin during in vitro sliding was bent superficially as postulated by the conventional hypothesis, but in the opposite direction of the putative pre-power-stroke configuration, as for ADP·Vi (inorganic vanadate)-bound myosin. We searched for the conformational species with a similar appearance and found that SH1-SH2 (thiols 1 and 2)-cross-linked myosin is a good candidate. To characterize such small asymmetric structures, we employed a new pattern-recognition procedure that accommodates the metal-replicated samples. In this method, the best-matched views of the target microscopic images were selected from a comprehensive set of images simulated from known atomic co-ordinates of relevant proteins. Together with effective morphological filtering, we could define the conformational species and the view angles of the catalytic domain and the lever arm cropped from averaged images of disulfide-cross-linked myosin. Whereas the catalytic domain of the new conformer closely resembled the pPDM (N,N'-p-phenylenedimaleimide)-treated, but SH2 Lys705-cross-linked, structure (PDB code 1L2O), a minor product of the same cross-linking reaction, the lever arm projected differently. Using separately determined view angles of the catalytic domain and the lever arm, we built a model of disulfide-cross-linked myosin. Further combination with the 'displacement-mapping' procedure enabled us to reconstruct the global three-dimensional envelope of the unusual structure whose lever arm orientation is compatible with our reports on the actin-sliding cross-bridge structure. Assuming this conformer as the structural analogue of the transient intermediate during actin sliding, the power stroke of the lever arm might accompany the reversal of the disorganized SH1 helix.

Ultrastructure of the denitrifying methanotroph "Candidatus Methylomirabilis oxyfera," a novel polygon-shaped bacterium.

"Candidatus Methylomirabilis oxyfera" is a newly discovered denitrifying methanotroph that is unrelated to previously known methanotrophs. This bacterium is a member of the NC10 phylum and couples methane oxidation to denitrification through a newly discovered intra-aerobic pathway. In the present study, we report the first ultrastructural study of "Ca. Methylomirabilis oxyfera" using scanning electron microscopy, transmission electron microscopy, and electron tomography in combination with different sample preparation methods. We observed that "Ca. Methylomirabilis oxyfera" cells possess an atypical polygonal shape that is distinct from other bacterial shapes described so far. Also, an additional layer was observed as the outermost sheath, which might represent a (glyco)protein surface layer. Further, intracytoplasmic membranes, which are a common feature among proteobacterial methanotrophs, were never observed under the current growth conditions. Our results indicate that "Ca. Methylomirabilis oxyfera" is ultrastructurally distinct from other bacteria by its atypical cell shape and from the classical proteobacterial methanotrophs by its apparent lack of intracytoplasmic membranes.

Use of the unroofing technique for atomic force microscopic imaging of the intra-cellular cytoskeleton under aqueous conditions.

Atomic force microscopy (AFM) combined with unroofing techniques enabled clear imaging of the intracellular cytoskeleton and the cytoplasmic surface of the cell membrane under aqueous condition. Many actin filaments were found to form a complex meshwork on the cytoplasmic surface of the membrane, as observed in freeze-etching electron microscopy. Characteristic periodic striations of about 5 nm formed by the assembly of G-actin were detected along actin filaments at higher magnification. Actin filaments aggregated and dispersed at several points, thereby dividing the cytoplasmic surface of the membrane into several large domains. Microtubules were also easily detected and were often tethered to the membrane surface by fine filaments. Furthermore, clathrin coats on the membrane were clearly visualized for the first time in water by AFM. Although the resolution of these images is lower than electron micrographs of freeze-etched samples processed similarly, the measurement capabilities of the AFM in a more biologically relevant conditions demonstrate that it is an important tool for imaging intracellular structures and cell surfaces in the native, aqueous state.

The origins and evolution of freeze-etch electron microscopy.

The introduction of the Balzers freeze-fracture machine by Moor in 1961 had a much greater impact on the advancement of electron microscopy than he could have imagined. Devised originally to circumvent the dangers of classical thin-section techniques, as well as to provide unique en face views of cell membranes, freeze-fracturing proved to be crucial for developing modern concepts of how biological membranes are organized and proved that membranes are bilayers of lipids within which proteins float and self-assemble. Later, when freeze-fracturing was combined with methods for freezing cells that avoided the fixation and cryoprotection steps that Moor still had to use to prepare the samples for his original invention, it became a means for capturing membrane dynamics on the millisecond time-scale, thus allowing a deeper understanding of the functions of biological membranes in living cells as well as their static ultrastructure. Finally, the realization that unfixed, non-cryoprotected samples could be deeply vacuum-etched or even freeze-dried after freeze-fracturing opened up a whole new way to image all the other molecular components of cells besides their membranes and also provided a powerful means to image the interactions of all the cytoplasmic components with the various membranes of the cell. The purpose of this review is to outline the history of these technical developments, to describe how they are being used in electron microscopy today and to suggest how they can be improved in order to further their utility for biological electron microscopy in the future.

Studies on gonococcus infection. II. Freeze-fracture, freeze-etch studies on gonocci.

Gonococci have been studied by electron microscopy after freeze-cleavage, freeze-etching and the findings correlated with those obtainable through thin sectioning and negative staining. The outer membrane of the cell wall is composed of round to hexagonal subunits 80 A in diameter. This membrane is also punctuated by 80-A holes visible on the exterior of the organism and extending into the substance or through the outer membrane. Pili coursing over the surface of the organisms appear to maintain a close anatomical relationship with the cell wall. In some instances, the surfaces of the organisms are virtually covered by a layer of pili.

The structure of erythrocyte membranes studied by freeze-etching. II. Localization of receptors for phytohemagglutinin and influenza virus to the intramembranous particles.

The distribution of specific glycoprotein receptors on the external surfaces of red cells was mapped, by the freeze-etching technique, to determine if the receptors coincided with the underlying 75-A intramembranous particles. Phytohemagglutinin, ferritin-conjugated phytohemagglutinin, and influenza virus were used as labeling agents since they can be seen by freeze-etching techniques and each reacts with a different site on the same glycoprotein molecule. The distribution of these labels was studied on intact human red cells, isolated ghost membranes, and trypsin-treated ghost membranes. The results show that the receptors for these labels are distributed uniformly over the surfaces of normal red cell membranes in the same apparent distribution as that of the 75-A particles within the membrane. The association between the external receptors and the underlying particles is especially evident when trypsin-treated ghost membranes are labeled: the labeled receptor sites and the intramembranous particles both form sharply defined, reticulated networks, which overlap. These results provide further support for the idea that membrane-bound glycoproteins are oriented so that their carbohydrate-rich segments, which bear the antigenic sites and receptors, are exposed to the external medium, while hydrophobic segments of the same molecules interact with lipids, and possibly other proteins, to form the intramembranous particles.

Ligand-induced movement of lymphocyte membrane macromolecules. II. Mapping of surface moieties.

Anti-immunoglobulin (Ig) coupled to ferritin or hemocyanin was used to map the distribution of Ig molecules on lymphocytes derived from bone marrow (B lymphocytes) by freeze-etching. The labeled anti-Ig was distributed all over the membrane in the form of random interconnected patches forming a lacy, continuous network. This was the pattern of lymphocytes labeled at 4 degrees C with the anti-Ig. After warming at 37 degrees C, the labeled molecules concentrated into a single area of the cell (forming the cap) and were rapidly internalized in small vesicles Freeze-etching showed close packing of the labeled molecules in the cap area. There was evidence that in the cap area the Ig molecules were exfoliated from the plane of the membrane, suggesting that the Ig may be superficial to the bilipid layer, or weakly anchored to the membrane. Similar studies were made using antibodies to histocompatibility antigens. Thymocytes were labeled with anti-H-2 and ferritin anti-Ig at 4 degrees C. Freeze-etching showed large patches scattered over the membrane and separated from each other by several thousand angstroms. This distribution may, in part, explain why H-2 antigens do not readily form a cap; the large patches are beyond the reach of even a double ligand (sandwich) reaction. The antigens that reacted with heterologous anti-lymphocyte globulin (ALG) were found in small noninterconnected clusters a few hundred angstroms apart. Such clusters presumably cannot be linked by a single antibody but can by a sandwich (ligand to ligand-antigen) reaction. In previous studies it was found that ALG antigens form a cap only after a sandwich reaction. Finally, the receptors for concanavalin A (Con A) were found in a lacy, irregular interconnected, random network. The spatial distribution of these moieties on the membrane may, in great part, determine their movement after reaction with one or two ligands.

Ligand-induced movement of lymphocyte membrane macromolecules. 3. Relationship between the formation and fate of anti-Ig-surface Ig complexes and cell metabolism.

Spleen lymphocytes were studied for the movement and interiorization of complexes of anti-Ig-surface Ig. The movement of the complex into a small, compact zone of the cell membrane (forming a cap) was inhibited by drugs that inhibited glycolysis and oxidative phosphorylation, but not by drugs that affected protein synthesis. Dead lymphocytes did not form caps. Freeze-etching techniques revealed that inhibited lymphocytes showed formation of multiple small complexes over the entire cell surface. Inhibitors of glycolysis and of oxidative phosphorylation also inhibited the interiorization and catabolism of radioiodinated anti-Ig. We hypothesize that cross-linking of all the surface Ig triggers the membrane movements that are required to pull the lattice into one zone of the cell.

Deep-etch EM reveals that the early poxvirus envelope is a single membrane bilayer stabilized by a geodetic "honeycomb" surface coat.

Three-dimensional "deep-etch" electron microscopy (DEEM) resolves a longstanding controversy concerning poxvirus morphogenesis. By avoiding fixative-induced membrane distortions that confounded earlier studies, DEEM shows that the primary poxvirus envelope is a single membrane bilayer coated on its external surface by a continuous honeycomb lattice. Freeze fracture of quick-frozen poxvirus-infected cells further shows that there is only one fracture plane through this primary envelope, confirming that it consists of a single lipid bilayer. DEEM also illustrates that the honeycomb coating on this envelope is completely replaced by a different paracrystalline coat as the poxvirus matures. Correlative thin section images of infected cells freeze substituted after quick-freezing, plus DEEM imaging of Tokuyasu-type cryo-thin sections of infected cells (a new application introduced here) all indicate that the honeycomb network on immature poxvirus virions is sufficiently continuous and organized, and tightly associated with the envelope throughout development, to explain how its single lipid bilayer could remain stable in the cytoplasm even before it closes into a complete sphere.

External scaffold of spherical immature poxvirus particles is made of protein trimers, forming a honeycomb lattice.

During morphogenesis, poxviruses undergo a remarkable transition from spherical immature forms to brick-shaped infectious particles lacking helical or icosahedral symmetry. In this study, we show that the transitory honeycomb lattice coating the lipoprotein membrane of immature vaccinia virus particles is formed from trimers of a 62-kD protein encoded by the viral D13L gene. Deep-etch electron microscopy demonstrated that anti-D13 antibodies bound to the external protein coat and that lattice fragments were in affinity-purified D13 preparations. Soluble D13 appeared mostly trimeric by gel electrophoresis and ultracentrifugation, which is consistent with structural requirements for a honeycomb. In the presence or absence of other virion proteins, a mutated D13 with one amino acid substitution formed stacks of membrane-unassociated flat sheets that closely resembled the curved honeycombs of immature virions except for the absence of pentagonal facets. A homologous domain that is present in D13 and capsid proteins of certain other lipid-containing viruses support the idea that the developmental stages of poxviruses reflect their evolution from an icosahedral ancestor.

Improved unroofing protocols for cryo-electron microscopy, atomic force microscopy and freeze-etching electron microscopy and the associated mechanisms.

Unroofing, which is the mechanical shearing of a cell to expose the cytoplasmic surface of the cell membrane, is a unique preparation method that allows membrane cytoskeletons to be observed by cryo-electron microscopy, atomic force microscopy, freeze-etching electron microscopy and other methods. Ultrasound and adhesion have been known to mechanically unroof cells. In this study, unroofing using these two means was denoted sonication unroofing and adhesion unroofing, respectively. We clarified the mechanisms by which cell membranes are removed in these unroofing procedures and established efficient protocols for each based on the mechanisms. In sonication unroofing, fine bubbles generated by sonication adhered electrostatically to apical cell surfaces and then removed the apical (dorsal) cell membrane with the assistance of buoyancy and water flow. The cytoplasmic surface of the ventral cell membrane remaining on the grids became observable by this method. In adhesion unroofing, grids charged positively by coating with Alcian blue were pressed onto the cells, thereby tightly adsorbing the dorsal cell membrane. Subsequently, a part of the cell membrane strongly adhered to the grids was peeled from the cells and transferred onto the grids when the grids were lifted. This method thus allowed the visualization of the cytoplasmic surface of the dorsal cell membrane. This paper describes robust, improved protocols for the two unroofing methods in detail. In addition, micro-unroofing (perforation) likely due to nanobubbles is introduced as a new method to make cells transparent to electron beams.

Freeze-fracture of microtubules and bridges in motile axostyles.

A freeze-fracture study of the motile axostyles of the flagellate protozoa Saccinobaculus and Pyrsonympha has been undertaken in order to obtain a view of the relationships of microtubules and their cross bridges not dependent on conventional preparative procedures. Reactivation studies using isolated axostyles prepared for freeze-fracture and then thawed demonstrate that we are observing the structure of a potentially functional axostyle. Cross fractures through the axostyle demonstrate more extensive interrow bridging than expected on the basis of observations of thin-sectioned material. Each microtubule has approximately sixfold bridge-binding sites with connections to as many as four interrow bridges. Measurements of microtubule diameter and spacing are significantly larger than those made from sectioned material and may indicate that conventional processing for electron microscopy results in the loss of structurally important water within the microtubule in addition to loss of intertubule material. Longitudinal fractures through the axostyle at various orientations demonstrate a minimum longitudinal periodicity of 160 A for both the spacing of the globular subunits within the microtubule wall and the spacing of the intrarow bridges. While intrarow bridges are strictly periodic and always oriented in parallel, interrow bridges are not strictly periodic and can be oriented at varying angles to the microtubule axis.

Substructure of the glomerular slit diaphragm in freeze-fractured normal rat kidney.

In the renal glomerulus, the narrow slits between adjacent epithelial podocytes are bridged by a diaphragm (2, 8, 11). In rat and mouse kidneys fixed by perfusion with tannic acid and glutaraldehyde (TAG), it has recently been discovered that this diaphragm has a highly ordered, isoporous substructure (9). It consists of a regular array of alternating cross bridges extending from the podocyte plasma membranes to a centrally running filament. This zipperlike pattern results in two rows of rectangular pores, approximately 40 X 140 A in cross section, dimensions consistent with the proposed role of the diaphragm as an important filtration barrier to plasma proteins (6). In the present study, we found in freeze-cleaved and in freeze-etched normal rat glomeruli that the surface of the slit diaphragm has an appearance conforming to the pattern found in sectioned material.

Ultrastructural organization of chloroplast thylakoids of the green alga Oocystis marssonii.

Intact cells of "Oocystis marssonii" were thin sectioned and freeze-etched, using conventional and double-recovery techniques. Thylakoids extend the length of the single chloroplast and occur in stacks of three to five. The peripheral thylakoids in a stack often alternate between adjacent stacks. Interpretation of double-recovery results suggests that membranes in unstacked regions are asymmetrical, with one face smooth and the matching face covered with closely packed 85-90 A diameter particles. Adjacent membranes in stacked regions evidently share 170 A diameter particles, and either membrane in a stacked region may fracture. The two fracture planes thus made possible may expose nearly entire 170 A particles or only the upper portion of such particles, creating in the latter case images of 125-135 A diameter particles. Fracture planes in all cases appear to occur through the interior of the membrane, in the plane between the hydrophobic ends of the lipid bilayer proposed in numerous membrane models.

Identification of chloroplast coupling factor by freeze-etching and negative-staining techniques.

Identification of chloroplast coupling factor particles, by the freeze-etching and negative-staining techniques, was made utilizing chloroplast thylakoids isolated from spinach leaves. Complete removal of particles, comparable in diameter to purified coupling factor particles, from the outer surface of freeze-etched thylakoids was achieved by treatment with 0.8% silicotungstate. Reappearance of particles, comparable in diameter to purified coupling factor particles, on the outer surface of freeze-etched thylakoids was demonstrated by combining silicotungstate-treated thylakoids with purified chloroplast coupling factor. Negative-staining results were in agreement with the freeze-etch data. The results demonstrate that the chloroplast coupling factor particles are exposed on the outer surface.

Resting myosin cross-bridge configuration in frog muscle thick filaments.

Clear images of myosin filaments have been seen in shadowed freeze-fracture replicas of single fibers of relaxed frog semitendinosus muscles rapidly frozen using a dual propane jet freezing device. These images have been analyzed by optical diffraction and computer averaging and have been modelled to reveal details of the myosin head configuration on the right-handed, three-stranded helix of cross-bridges. Both the characteristic 430-A and 140-150-A repeats of the myosin cross-bridge array could be seen. The measured filament backbone diameter was 140-160 A, and the outer diameter of the cross-bridge array was 300 A. Evidence is presented that suggests that the observed images are consistent with a model in which both of the heads of one myosin molecule tilt in the same direction at an angle of approximately 50-70 degrees to the normal to the filament long axis and are slewed so that they lie alongside each other and their radially projected density lies along the three right-handed helical tracks. Any perturbation of the myosin heads away from their ideal lattice sites needed to account for x-ray reflections not predicted for a perfect helix must be essentially along the three helical tracks of cross-bridges. Little trace of the presence of non-myosin proteins could be seen.

The crystal lattice of Paramecium trichocysts before and after exocytosis by X-ray diffraction and freeze-fracture electron microscopy.

Paramecium trichocysts are unusual secretory organelles in that: (a) their crystalline contents are built up from a family of low molecular mass acidic proteins; (b) they have a precise, genetically determined shape; and (c) the crystalline trichocyst contents expand rapidly upon exocytosis to give a second, extracellular form which is also an ordered array. We report here the first step of our study of trichocyst structure. We have used a combination of x-ray powder diffraction, freeze-etching, and freeze-fracture electron microscopy of isolated, untreated trichocysts, and density measurements to show that trichocyst contents are indeed protein crystals and to determine the elementary unit cell of both the compact intracellular and the extended extracellular form.

The extracellular matrix of Xenopus laevis eggs: a quick-freeze, deep-etch analysis of its modification at fertilization.

Eggs of the amphibian, Xenopus laevis, were quick-frozen, deep-etched, and rotary-shadowed. The structure of the extracellular matrix surrounding these eggs, including the perivitelline space and the vitelline envelope (VE), was visualized in platinum replicas by electron microscopy. The perivitelline space contains an elaborate filamentous glycocalyx which connects microvillar tips to the plasma membrane, to adjacent microvilli, and to the overlying VE. The VE is comprised of two layers, the innermost of which is a thin network of horizontal fibrils lying on the tips of the microvilli. The outermost is a thicker layer of large, cable-like fibers which twist and turn throughout the envelope. Upon fertilization, three dramatic modifications of the matrix occur. A thin sheet of smooth material, termed the smooth layer, is deposited on the tips of the microvilli and separates the egg from the overlying envelopes. The VE above is transformed from a thick band of cable-like fibers to concentric fibrous sheets, the altered VE. Finally, an ornate band of particles, corresponding to the fertilization layer in previous studies, is deposited at the altered VE/jelly interface. The altered VE and the fertilization layer comprise the fertilization envelope, which effects the structural block to polyspermy.

Analysis of the thylakoid outer surface. Coupling factor is limited to unstacked membrane regions.

The structure of the spinach thylakoid outer surface has been examined by deepetching, a technique which exposes the true surfaces of biological membranes by sublimination of frozen dilute buffer. The membrane surface is covered with large (150 A average diameter) and small (90 A average diameter) particles. Approximately 30% of the large particles can be removed under conditions reported to selectively remove carboxydismutase from the membrane surface. The remaining large particles can be removed only under conditions which cause a loss of coupling factor activity. When purified coupling factor is readded to membranes from which all coupling factor activity has been removed, large particles reappear, indicating that they represent coupling factor molecules. Since the number of particles and the amount of ATPase activity in the reconstituted and control membranes were the same, coupling factor molecules may be attached to specific binding sites. Analysis of antibody labeling experiments, enzyme assays, and experiments involving the unstacking and restacking of thylakoid membranes indicate that coupling factor is excluded from regions of membrane stacking (grana) and is present only in unstacked membrane regions. The exclusion of coupling factor from grana, which are known to be centers of intense photosynthetic activity, strongly suggests that the mechanism coupling electron transport to photophosphorylation is indirect. In addition to the large and small particles, in some cases regularly spaced ridges are visible on the outer surface after unstacking. Coupling factor binding sites seem to be excluded from regions where these structures occur.