Filamentous structure of the external surface of abalone eggs revealed by quick-freeze deep-etch technique

The external surface of abalone eggs was examined by thin section and quick-freeze, deep-etch electron microscopy. In thin sections, networks of fine filaments were found interconnecting the adjacent microvilli on the surface of unfertilized eggs. Quick-freeze, deep-etch electron microscopy revealed the three-dimensional structure of these networks of filaments on the external surface of the egg. Mainly two networks of filaments were identified; one was composed of thicker (14–19 nm) filaments interconnecting with the neighboring microvilli nearly horizontally, and the other was composed of thinner (8–14 nm) branched filaments closely surrounding the microvilli surface as well as highly interconnecting neighboring microvilli in a polygonal pattern. The overall structure of the filamentous network on the egg surface showed no distinct alteration after fertilization. These networks of filaments observed on the egg surface may play a key role in sperm–egg interaction.     

Cytoskeletal reorganization of human platelets after stimulation revealed by the quick-freeze deep-etch technique

We studied the cytoskeletal reorganization of saponized human platelets after stimulation by using the quick-freeze deep-etch technique, and examined the localization of myosin in thrombin-treated platelets by immunocytochemistry at the electron microscopic level. In unstimulated saponized platelets we observed cross-bridges between: adjoining microtubules, adjoining actin filaments, microtubules and actin filaments, and actin filaments and plasma membranes. After activation with 1 U/ml thrombin for 3 min, massive arrays of actin filaments with mixed polarity were found in the cytoplasm. Two types of cross-bridges between actin filaments were observed: short cross-bridges (11 +/- 2 nm), just like those observed in the resting platelets, and longer ones (22 +/- 3 nm). Actin filaments were linked with the plasma membrane via fine short filaments and sometimes ended on the membrane. Actin filaments and microtubules frequently ran close to the membrane organelles. We also found that actin filaments were associated by end-on attachments with some organelles. Decoration with subfragment 1 of myosin revealed that all the actin filaments associated end-on with the membrane pointed away in their polarity. Immunocytochemical study revealed that myosin was present in the saponin-extracted cytoskeleton after activation and that myosin was localized on the filamentous network. The results suggest that myosin forms a gel with actin filaments in activated platelets. Close associations between actin filaments and organelles in activated platelets suggests that contraction of this actomyosin gel could bring about the observed centralization of organelles.     

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 RecA protein and its complexes with DNA by quick-freeze/deep-etch electron microscopy

Freeze-etch electron microscopy of pure RecA protein aggregates, as well as of RecA protein complexes on single-stranded and double-stranded DNA formed with various nucleotides, has permitted a clearer discrimination between the two different helical polymers that this protein forms. Both are continuous, single-start, right-handed helices; however, the form observed when ATP or non-hydrolyzable ATP analogs are present has a pitch of 9.5 nm and a diameter of 10 nm, while the other form, observed in the absence of ATP or its analogs, or in the presence of ADP, has a pitch of 6 nm and a diameter of 12 nm. The former "long pitch" helix is found only when RecA protein is bound to DNA. The latter "short pitch" helix is also observed in pure RecA protein polymers (also termed rods) and in the needle-like paracrystals of RecA protein that form in the presence of magnesium or spermidine ions, representing bundles of rods closely packed in register. Addition of ATP or non-hydrolyzable ATP analogs in the absence of DNA dissociates the pure RecA protein crystals, as well as individual helical rods, into short curvilinear chains of attached monomers. These chains typically form closed, circular rings of 7(+/- 1) protein monomers, similar in construction to a single turn of the RecA protein helix, but significantly broader in diameter. The role of ATP in interconverting the various polymeric forms of RecA protein is discussed within the context that ATP functions as a reversible allosteric effector of RecA protein, much as it mediates reversible conformational changes in other vectoral motor proteins such as myosin, dynein, kinesin and the 70,000 Mr "heat shock" ATPases. We discuss how cyclic conversions back and forth between the short- and long-pitch conformations of RecA protein could mediate in reversible single-stranded and double-stranded DNA interactions during the search for homology.     

The coelomic envelope of Xenopus laevis eggs: a quick-freeze, deep-etch analysis

The extracellular matrix of Xenopus laevis oocytes was analyzed before and after meiotic maturation using quick-freeze, deep-etch, rotary-shadow electron microscopy. The perivitelline space (PS) of the meiotically immature oocyte contains a filamentous network which connects microvilli (MV) and follicle cell macrovilli to the folded oocyte surface below. The envelope overlying the PS is composed of bundles of large fibers which course between the tips of the MV. Spaces between these bundles contain smaller fibrils which secure the egg envelope to the microvillar tips. Meiotic maturation is accompanied by flattening of the oocyte plasma membrane, formation of an orderly array of MV, and elevation of the egg envelope. In the coelomic eggs, the reorganized envelope is composed of loosely bundled large fibers which course above the microvillar tips rather than between them. The spaces between these bundles contain small fibers similar to those seen in the meiotically immature oocyte. This reorganized envelope, however, will not bind sperm; further modifications must transpire during passage through the oviduct to render it sperm receptive.     

Cell wall glycoproteins from Chlamydomonas reinhardii,and their self-assembly

We have investigated the in vitro reassembly of the salt soluble, hydroxyproline rich, glycoproteins from the cell wall of Chlamydomonas reinhardii, into structured cell wall fragments. We have devised an assay which has been used to follow the reassembly of the unfractionated and fractionated (2BI and 2BII) cell wall glycoproteins. Reassembly has a pH optimum of 5, a temperature optimum of 20°C, and the final size of the reassembled fragments appears to be promoted by the minor component 2BI. Periodate oxidation experiments show that sugar residues, in particular mannose, are important for accurate reassembly. Using electron microscopy, the structure of the reassembled products has been elucidated, as have intermediate stages in the reassembly process.Abbreviations TRIS Tris(hydroxymethyl)-methylamine - SDS Sodium dodecyl sulphate - PAGE Polyacrylamide gel electrophoresis This is the fifth paper in a series entitled Structure composition and morphogenesis of the cell wall of Chlamydomonas reinhardii. The last paper in this series was Catt et al. (1976)     

Ultrastructure of detergent-resistant cytoskeletons in the noncortical domain of sea urchin eggs as revealed by the quick-freeze deep-etch technique.

The ultrastructure of detergent-resistant cytoskeletons in the noncortical cytoplasm of sea urchin eggs was studied by quick-freeze, deep-etch electron microscopy. Two different cytoskeletal organizations were identified in the detergent-treated sea urchin eggs. They were distinguished by the presence or the absence of long actin filaments and probably correspond to the cortex and the noncortical cytoplasm, respectively. The non-cortical cytoplasm was composed of a complex network (designated here as the ground network) of filaments 6 to 13 nm in diameter, that interconnected aggregates of small globular materials, yolk granules and a meshwork of uniform filaments (8-9 nm in diameter). The 6 to 13 nm filaments comprising the ground network were branched and associated with filaments of the same or other sizes, resulting in the formation of an extremely complex network. The meshwork of 8-9 nm filaments was homogeneous in composition and constitutes a novel structure which has not been previously described. The 8-9 nm filaments were connected to one another at their ends, forming a meshwork of polygons. Meshworks, ranging up to 3 microns in diameter, were distributed throughout the non-cortical cytoplasm of the egg. Similar cytoplasmic structures were also observed in fertilized eggs.     

The chaotrope-soluble glycoprotein GP1 is a constituent of the insoluble glycoprotein framework of the Chlamydomonas cell wall

Chlamydomonas reinhardtii wild-type cells are surrounded by the insoluble cell wall component, a sac-like framework of cross-linked glycoproteins containing 22% hydroxyproline. The chaotrope-soluble cell wall glycoprotein GP1 is the only polypeptide with an even higher proportion of hydroxyproline (35%) occurring in vegetative C. reinhardtii cells. Mass spectrometric analyses of peptides released from the purified insoluble cell wall fraction by trypsin treatment and epitope analyses of polyclonal antibodies raised against different deglycosylation products of this particular wall fraction using 181 chemically synthesized GP1-derived pentadecapeptides revealed evidence that GP1 is indeed a constituent of the insoluble wall component.     

The Chlamydomonas cell wall: characterization of the wall framework

The cell wall of the biflagellate alga Chlamydomonas reinhardtii is a multilayered, extracellular matrix composed of carbohydrates and 20-25 polypeptides. To learn more about the forces responsible for the integrity of this cellulose-deficient cell wall, we have begun studies to identify and characterize the framework of the wall and to determine the effects of the cell wall-degrading enzyme, lysin, on framework structure and protein composition. In these studies we used walls released into the medium by mating gametes. When isolated shed walls are degraded by exogenously added lysin, no changes are detected in the charge or molecular weight of the 20-25 wall proteins and glycoproteins when analyzed on one- and two-dimensional polyacrylamide gels, which suggests that degradation of these shed walls is due either to cleavage of peptide bonds very near the ends of polypeptides or that degradation occurs via a mechanism other than proteolysis. Incubation of walls with Sarkosyl-urea solutions removes most of the proteins and yields thin structures that appear to be the frameworks of the walls. Analysis by polyacrylamide gel electrophoresis shows that the frameworks are highly enriched in a polypeptide of Mr 100,000. Treatment of frameworks with lysin leads to their degradation, which indicates that this part of the wall is a substrate for the enzyme. Although lysin converts the Mr 100,000 polypeptide from an insoluble to a soluble form, there is no detectable change in Mr of the framework protein. Solubilization in the absence of lysin requires treatment with SDS and dithiothreitol at 100 degrees C. These results suggest that the Chlamydomonas cell wall is composed of two separate domains: one containing approximately 20 proteins held together by noncovalent interactions and a second domain, containing only a few proteins, which constitutes the framework of the wall. The result that shed walls can be solubilized by boiling in SDS-dithiothreitol indicates that disulfide linkages are critical for wall integrity. Using an alternative method for isolating walls from mechanically disrupted gametes, we have also shown that a wall-shaped portion of these unshed walls is insoluble under the same conditions in which shed walls are soluble. One interpretation of these results is that wall release during mating and the wall degradation that follows may involve distinct biochemical events.     

Between-species analysis of short-repeat modules in cell wall and sex-related hydroxyproline-rich glycoproteins of Chlamydomonas

Protein diversification is commonly driven by single amino acid changes at random positions followed by selection, but, in some cases, the structure of the gene itself favors the occurrence of particular kinds of mutations. Genes encoding hydroxyproline-rich glycoproteins (HRGPs) in green organisms, key protein constituents of the cell wall, carry short-repeat modules that are posited to specify proline hydroxylation and/or glycosylation events. We show here, in a comparison of two closely related Chlamydomonas species-Chlamydomonas reinhardtii (CC-621) and Chlamydomonas incerta (CC-1870/3871)-that these modules are prone to misalignment and hence to both insertion/deletion and endoduplication events, and that the dynamics of the rearrangements are constrained by purifying selection on the repeat patterns themselves, considered either as helical or as longitudinal face modules. We suggest that such dynamics may contribute to evolutionary diversification in cell wall architecture and physiology. Two of the HRGP genes analyzed (SAG1 and SAD1) encode the mating-type plus and minus sexual agglutinins, displayed only by gametes, and we document that these have undergone far more extensive divergence than two HRGP genes (GP1 and VSP3) that encode cell wall components-an example of the rapid evolution that characterizes sex-related proteins in numerous lineages. Strikingly, the central regions of the agglutinins of both mating types have diverged completely, by selective endoduplication of repeated motifs, since the two species last shared a common ancestor, suggesting that these events may have participated in the speciation process.     

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.     

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.     

Cytoskeletal filaments in embryonic chick myocardial cells as revealed by the quick-freeze deep-etch method combined with immunocytochemistry

Summary The three-dimensional organization of cytoskeletal filaments associated with the myofibrils and sarcolemma of the myocardial cells of early chick embryos was studied by the rapid-freeze deep-etch method combined with immunocytochemistry. In the endoplasmic region of saponin-treated myocardial cells, 12–14 nm filaments formed a loose network surrounding nascent myofibrils. These 12–14 nm filaments attached to the myofibrils and some of them converged into Z disc regions. In the non-junctional cytocortical region thinner 8–11 nm filaments composed a dense network just beneath the sarcolemma. In myofibril terminating regions at the sarcolemma, i.e., the fascia adherens, 3–5 nm cross-bridges were observed among the thin filaments. In Triton-permeabilized and myosin subfragment 1 (S1)-treated samples, subsarcolemmal 8–11 nm filaments proved to be S1-decorated actin filaments under which there was a loose network of S1-undecorated filaments. Subsarcolemmal S1-decorated actin filaments had mixed polarity and attached to the sarcolemma at one end. A loose network of S1-undecorated filaments among myofibrils in the endoplasmic region was revealed to consist of desmin-containing intermediate filaments after immuno-gold staining for desmin. These networks connecting myofibrils with sarcolemma were assumed to play an important role in integrating and transmitting the contractile force of individual myofibrils within early embryonic myocardial cells.     

A technique for labelling the sample surface for quick-freeze,deep-etch,rotary replication electron microscopy: application to the study of geletin gel structure

A method using magnesium oxide crystals to label the surface of physical gels, such as gelatin gel before quick-freezing is described and discussed. The quick-freeze, deep-etch, rotary replication technique is most adapted to 3-D visualization of physical gel structure. However, it is known that the depth which ultrarapid freezing may reach is limited by the growth of ice crystals as the distance from the surface of the specimen (rapidly cooled by smashing against a cooled metal plate) increases. Consequently, intact preservation of structures occurs only in superficial zones of the specimen. The MgO surface labelling technique provides a simple means for surface recognition. It enables the estimation of a given replicated area depth, taking into account the angle of specimen scraping before etching and replicating. By comparison of views of the same replica at different depths, freezing artifacts may be recognized even when they cause only slight deformations in the structure. This is particularly necessary for interpretation of gel network geometry: interpretation can be made with certainty only if a reliable surface reference marker exists. For gelatin gels, the depth of best freezing can be estimated to be around 5 μm from the frozen sample surface.     

Stepwise transformation of the vitelline envelope of Xenopus eggs at activation: a quick-freeze, deep-etch analysis

The extracellular matrix of Xenopus laevis eggs was analyzed at fixed intervals after prick-activation using quick-freeze, deep-etch, rotary-shadow electron microscopy. This technique revealed that the modifications of the matrix seen at fertilization do not occur simultaneously, but that instead there is an orderly progression of alterations at activation. The first modification, conversion of the vitelline envelope (VE) to the altered vitelline envelope (VE), occurs within 2 to 3 min after activation. Intermediate stages of the VE to VE transformation can be visualized traveling around the egg in a wave-like fashion. Upon completion of the wave, the loosely woven outer surface of the VE, believed to be the prefertilization layer, remains unaltered. Subsequent formation of the fertilization (F) layer at this VE-jelly interface occurs between 4 and 8 min postactivation. Finally, between 10 and 15 min postactivation, the smooth (S) layer forms on the tips of the microvilli and surrounds the entire egg.     

Substructure of cisternal organelles of neuronal perikarya in immature rat brains revealed by quick-freeze and deep-etch techniques

Summary Membrane-bounded organelles possessing cisternae, i.e., rough endoplasmic reticulum and Golgi apparatus, in immature rat central neurons were examined by quick-freeze and deep-etch techniques to see how their intracisternal structures are organized and how ribosomes are associated with the membrane of the endoplasmic reticulum. Cisternae of endoplasmic reticulum, 60–100 nm wide, were bridged with randomly-distributed strands (trabecular strands, 12.5 nm in mean diameter). Luminal surfaces of cisternae of the endoplasmic reticulum were decorated with various-sized globular particles, some as small as intramembrane particles, and others as large as granules formed by soluble proteins seen in the cytoplasm. A closer examination revealed much thinner strands (3.3. nm in mean diameter). Such thin strands were short, usually winding toward the luminal surface, and sometimes touching the luminal surface with one end. Ribosomes appeared to be embedded into the entire thickness of cross-fractured membranes of endoplasmic reticulum, that is, their internal portions appeared to be situated at almost the same level as the cisternal luminal surface. From the internal portion of ribosomes, single thin strands occasionally protruded into the lumen, suggesting that these thin strands were newly synthesized polypeptides. A horizontal separation within ribosomes appeared to occur at the same level as the hydrophobic middle of the membrane of the endoplasmic reticulum. Interiors of the Golgi apparatus cisternae, which were much narrower than cisternae of endoplasmic reticulum, were similarly bridged with trabecular strands, but the Golgi trabecular strands were thinner and more frequent. Their cisternal lumina were also dotted with globular particles. No identifiable profiles corresponding to the thin strands in the endoplasmic reticulum were observed. Golgi cisternae showed a heterogeneous distribution of membrane granularity; the membrane in narrow cisternal space was granule-rich, while that in expanded space was granule-poor, suggesting a functional compartmentalization of the Golgi cisternae.     

The Chlamydomonas cell wall degrading enzyme, lysin, acts on two substrates within the framework of the wall

The Chlamydomonas cell wall is a multilayered, extracellular matrix containing 20-25 proteins and glycoproteins, many of which are highly enriched in hydroxyproline. 80-90% of the wall protein is located in a crystalline portion of the wall that is soluble in sarkosyl-urea solutions as well as in chaotropic salts. Although the wall has no cellulose it contains a noncrystalline, highly insoluble framework portion that is responsible for the integrity and overall shape of the wall. In the present report we show that the framework of the wall is composed of two components that are acted upon by lysin, a wall degrading enzyme released by mating gametes. One, which makes up the major portion of the framework, is insoluble upon boiling in SDS-PAGE sample buffer. Lysin treatment of this portion leads to its physical degradation and the concomitant appearance of several SDS-dithiothreitol-soluble polypeptides ranging in relative molecular mass from greater than 400,000 to less than 60,000. The second component is the flagellar collar. This hollow cylinder composed of striated fibers aligned in parallel array serves as the tunnel in the wall through which the flagella protrude. Our evidence indicates that the primary collar polypeptide is a 225,000-Mr molecule that itself has at least two functional domains. One domain, contained in a 185,000-Mr fragment, permits the self-association of the molecules to form the main body of the collar. The second part of the molecule anchors the collar to the wall framework via sarkosyl-urea-insensitive, SDS-dithiothreitol-sensitive linkages.     

The cell wall of Chlamydomonas eugametos. Immunological aspects

An antiserum was raised against the major cell wall glycoprotein of Chlamydomonas eugametos which after purification reacted specifically with all individual wall components but not with intact cell walls. The antigenic sites in intact walls appear to be cryptic but become exposed on partial enzymatic degradation or in situ during daughter-cell release when the insoluble component is digested. Using the antiserum as a specific label for cell walls in various stages of disintegration, cell wall digestion during asexual and sexual reproduction was studied. It is also shown that while cell wall material is associated with isolated flagella, it is not normally associated with the flagella of intact cells.     

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.