Crystals of the Chlamydomonas reinhardtii cell wall: polymerization, depolymerization, and purification of glycoprotein monomers

Two of the three major outer layers of the Chlamydomonas reinhardtii cell wall (W6 and W4) can be solubilized from living cells with sodium perchlorate or other chaotropes and will repolymerize in vitro to form milligram amounts of wall crystals. Conditions for optimal crystalization are presented, and conditions that fail to induce polymerization are exploited to maintain monomers in aqueous solution for ion-exchange chromatography. The four major glycoproteins of the complex (GP1, 1.5, 2, and 3) have in this way been purified to apparent homogeneity and have been characterized morphologically by transmission electron microscopy using the quick-freeze, deep-etch technique and by amino acid composition. Three of the four are hydroxyproline-rich species that copolymerize to form the W6 layer. The fourth (GP1.5) is a glycine-rich species that binds to the interior of the in vitro crystal; it is apparently equivalent to the granules within the W4 layer in situ.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electron microscopy for the characterization of the vitelline coat glycoproteins of the polarized egg of Unio elongatulus

The vitelline coat (VC) glycoproteins of the Unio elongatulus egg, purified as previously described (Focarelli and Rosati, 1993: Mol Reprod Dev 35:44–51) and indicated as gp220 and gp180 by virtue of their apparent molecular weights in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The analysis confirmed the purity of our preparations and the mass of gp180, but gave a mass of 273,000 for gp220. Intact VCs and purified VC components were then visualized in stereo images of platinum replicas produced by the quick-freeze, deep-etch, and rotary shadowing techniques: gp180 revealed a c-like shape and gp273 a rosette-like shape. The intact VCs were found to consist of two layers, the internal one clearly fibrous and the external one compact. Since purified preparations of gp180 spontaneously formed fibrils of similar width to those present in the inner VC layer, this layer presumably consists mainly of this component. The prevalence of gp273 in the outer layer is also suggested and discussed. Mol. Reprod. Dev. 48:511–517, 1997. © 1997 Wiley-Liss, Inc.     

Egg jelly layers of Xenopus laevis are unique in ultrastructure and sugar distribution

Jelly coats surrounding the eggs of the South African clawed toad, Xenopus laevis, consist of three transparent, gelatinous layers: the innermost layer (J1), the middle layer (J2), and the outer layer (J3). The distribution of N-acetylglucosamine within these jelly coats, as probed with FITC-conjugated wheat germ agglutinin (WGA-FITC), and the matrix ultrastructure of each layer, as visualized in platinum replicas produced by the quick-freeze, deep-etch, and rotary-shadowing technique, suggests that each layer has a unique fiber and glycoprotein composition. J1 extends nearly 200 μm from the egg surface and exhibits no WGA-FITC staining. Stereo images of platinum replicas indicates that J1 consists of a tightly knit network of 5–10 nm fibers decorated with 10–20 nm particulate components. In contrast, J2 is a relatively thin layer, extending only 25–40 μm from the outer aspect of J1. When visualized by confocal microscopy, J2 displays a multilayered WGA-FITC staining pattern. The ultrastructure of J2 consists of sheets of fine fibers that run parallel to one another and that can be identified by their ability to bind WGA-colloidal gold. The fibers of each sheet run at an oblique angle to fibers in neighboring layers. J3 extends 100 μm or more from J2. The WGA-FITC staining pattern shows high intensity in its outer region and less intensity in regions closer to J2. Like J1, the J3 ultrastructure consists of a network of 5–10 nm fibers, decorated with 10–20 nm particulate components. The results of these studies add to a growing body of information that suggests the jelly coats surrounding the eggs of many animals consist of a fibrous glycoprotein superstructure that acts as a scaffold to which globular glycoproteins are bound. © 1996 Wiley-Liss, Inc.     

Nucleated assembly of Chlamydomonas and Volvox cell walls

The Chlamydomonas reinhardtii cell wall is made up of hydroxyproline-rich glycoproteins, arranged in five distinct layers. The W6 (crystalline) layer contains three major glycoproteins (GP1, GP2, GP3), selectively extractable with chaotropic agents, that self-assemble into crystals in vitro. A system to study W6 assembly in a quantitative fashion was developed that employs perchlorate-extracted Chlamydomonas cells as nucleating agents. Wall reconstitution by biotinylated W6 monomers was monitored by FITC-streptavidin fluorescence and quick-freeze/deep-etch electron microscopy. Optimal reconstitution was obtained at monomer concentrations (0.2-0.3 mg/ml) well below those required for nonnucleated assembly. Assembly occurred from multiple nucleation sites, and faithfully reflected the structure of the intact W6 layer. Specificity of nucleated assembly was demonstrated using two cell-wall mutants (cw-2 and cw-15); neither served as a substrate for assembly of wild-type monomers. In addition, W6 sublayers were assembled from purified components: GP2 and GP3 coassembled to form the inner (W6A) sublayer; this then served as a substrate for self-assembly of GP1 into the outer (W6B) sublayer. Finally, evolutionary relationships between C. reinhardtii and two additional members of the Volvocales (Chlamydomonas eugametos and Volvox carteri) were explored by performing interspecific reconstitutions. Hybrid walls were obtained between C. reinhardtii and Volvox but not with C. eugametos, confirming taxonomic assignments based on structural criteria.     

DEEP-ETCH ANALYSIS OF ADHESION COMPLEXES BETWEEN GAMETIC FLAGELLAR MEMBRANES OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)

The ultrastructure of adhesion complexes between gametic flagellar membranes of Chlamydomonas reinhardtii Dangeard was analyzed using the quick-freeze deep-etch technique. The sexual agglutinin fibrils interact by forming hybrid fibers that frequently branch, forming extensively cross-bridged meshworks. This pattern of interaction mimics a prominent mode of cell wall formation in Chlamydomonas, supporting the notion that the agglutinins evolved from cell wall proteins and that sexual adhesion and cell wall assembly are homologous events.     

The cell wall of the chlamydomonad flagellate,Gloeomonas kupfferi (Volvocales,Chlorophyta)

Summary The large unicellular flagellate,Gloeomonas kupfferi, has recently been used as an important tool in chlamydomonad cell biology research, especially in studies dealing with the structure and function of the endomembrane system. However, little is known about the main secretory product, the cell wall. This study presents structural, chemical and immunological information about this wall. This 850–900 nm thick matrix is highly elaborate and consists of three distinct layers: an inner stratum (325 nm thick) consisting of tightly interwoven fibers, a medial crystalline layer consisting of 22–23 nm subunits and an outer wall layer (500 nm thick) of outwardlyradiating fibrils. Rapid freeze-deep etch analysis reveals that the 35–40 nm fibers of the outer layer form a quasi-lattice of 160 nm subunits. The outer wall can be removed from whole pellets using the chelator, CDTA. The medial wall complex can be solubilized by perchlorate. SDS-gel electrophoresis reveals that the perchlorate soluble-material consists of five high molecular weight glycoproteins and five major low molecular weight glycoproteins. The electrophoretic profile is roughly similar to that ofChlamydomonas reinhardtii. Antibodies were successfully raised against the outer wall component and were shown to label the outer wall layer.     

Ultrastructure and Composition of the Nannochloropsis gaditana Cell Wall

Marine algae of the genus Nannochloropsis are promising producers of biofuel precursors and nutraceuticals and are also harvested commercially for aquaculture feed. We have used quick-freeze, deep-etch electron microscopy, Fourier transform infrared spectroscopy, and carbohydrate analyses to characterize the architecture of the Nannochloropsis gaditana (strain CCMP 526) cell wall, whose recalcitrance presents a significant barrier to biocommodity extraction. The data indicate a bilayer structure consisting of a cellulosic inner wall (∼75% of the mass balance) protected by an outer hydrophobic algaenan layer. Cellulase treatment of walls purified after cell lysis generates highly enriched algaenan preparations without using the harsh chemical treatments typically used in algaenan isolation and characterization. Nannochloropsis algaenan was determined to comprise long, straight-chain, saturated aliphatics with ether cross-links, which closely resembles the cutan of vascular plants. Chemical identification of >85% of the isolated cell wall mass is detailed, and genome analysis is used to identify candidate biosynthetic enzymes.     

The sea urchin egg jelly coat is a three-dimensional fibrous network as seen by intermediate voltage electron microscopy and deep etching analysis

The egg jelly (EJ) coat which surrounds the unfertilized sea urchin egg undergoes extensive swelling upon contact with sea water, forming a threedimensional network of interconnected fibers extending nearly 50 μm from the egg surface. Owing to its solubility, this coat has been difficult to visualize by light and electron microscopy. However, Lytechinus pictus EJ coats remain intact, if the fixation medium is maintained at pH 9. The addition of alcian blue during the final dehydration step of sample preparation stains the EJ for visualization of resin embedded eggs by both light and electron microscopy. Stereo pairs taken of thick sections prepared for intermediate voltage electron microscopy (IVEM) produce a threedimensional image of the EJ network, consisting of interconnected fibers decorated along their length by globular jelly components. Using scanning electron microscopy (SEM), we have shown that before swelling, EJ exists in a tightly bound network of jelly fibers, 50–60 nm in diameter. In contrast, swollen EJ consists of a greatly extended network whose fibrous components measure 10 to 30 nm in diameter. High resolution stereo images of hydrated jelly produced by the quick-freeze/deep-etch/rotary-shadowing technique (QF/DE/RS) show nearly identical EJ networks, suggesting that dehydration does not markedly alter the structure of this extracellular matrix. © 1993 Wiley-Liss, Inc.     

The substructure of isolated and in situ outer dynein arms of sea urchin sperm flagella

Outer-arm dynein from the sperm of the sea urchin S. purpuratus was adsorbed to mica flakes and visualized by the quick-freeze, deep-etch technique. Replicas reveal particles comprised of two globular heads joined by two irregularly shaped stems which make contact along their length. One head is pear-shaped (18.5 X 12.5 nm) and the other is spherical (14.5-nm diam). The stems are decorated by a complex of bead-like subunits. The same two-headed protein is found in the 21S dynein-1 fraction of sucrose gradients. The beta-heavy chain/intermediate chain 1 (beta/IC-1) dynein subfraction, produced by low-salt dialysis and zonal centrifugation of the high-salt-extracted dynein-1, contains only single-headed molecules with single stems. These heads are predominantly pear-shaped (18.5 X 12.5 nm). Since 21S dynein-1 contains two heavy chains (alpha and beta), and the beta/IC-1 subfraction is comprised of only the beta-heavy chain (Tang et al., 1982, J. Biol. Chem. 257: 508-515), we conclude that each head is formed by a heavy chain, that the pear-shaped head contains the beta-heavy chain, and that the spherical head contains the alpha-heavy chain. The in situ outer dynein arms of demembranated sperm were also studied by the quick-freeze, deep-etch method. When frozen in reactivation buffer devoid of ATP, each arm consists of a large globular head that attaches to the A-microtubule by distally skewed subunits and attaches to the B-microtubule by a slender stalk. In ATP, this head shifts its orientation such that it can be seen to be constructed from two globular domains. We offer possible correlates between the in situ and the in vitro images, and we compare the structure of sea-urchin dynein with dynein previously described from Chlamydomonas and Tetrahymena.     

In vitro formation of the "S" layer, a unique component of the fertilization envelope in Xenopus laevis eggs

The extracellular matrix (ECM) of unfertilized Xenopus laevis eggs consists of an elaborate filamentous network in the perivitelline space (PS) and a thick fibrillar vitelline envelope (VE), with a thin layer of horizontal filaments (HF) separating the two. At fertilization this ECM is converted into the fertilization envelope (comprised of the fertilization (F) layer and altered VE), and a third layer, the smooth (S) layer, is formed at the upper boundary of the PS (Larabell and Chandler, 1988). In this report, we use quick-freeze, deep-etch, rotary-shadow electron microscopy to show that an intact S layer can be formed in vitro by incubation of unfertilized eggs in an exudate obtained from cortical granules. Within 5 min numerous 36-nm-diameter particles assemble in a highly ordered array at the microvillar tips. These particles appear to "melt" and to form patches of smooth material and within 10 min one continuous sheet has formed. The presence of the VE is required for formation of the S layer, and we suggest that the HF layer is the site of assembly.     

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.     

Imaging of plant microtubules with high resolution scanning electron microscopy

Summary High resolution scanning electron microscopy was used to obtain images of cortical microtubules and associated structures in onion root tips. Specimens were prepared using a modified quick-freeze deep-etch technique utilising cytosolic extraction with saponin and conductive staining with osmium.Abbreviations DMSO dimethylsulfoxide - HRSEM high resolution scanning electron microscope/microscopy - MTSB microtubule stabilising buffer - TEM transmission electron microscope/microscopy     

Evidence for the existence of two assembly domains within the sea urchin fertilization envelope.

The sea urchin fertilization envelope (FE) is a complex, macromolecular aggregate assembled by the addition of cortical granule secretions to the vitelline layer. The completed, trilaminar structure has a dense layer sandwiched between surface coats of paracrystalline material. Two cortical granule enzymes, ovoperoxidase and protease, and a cell surface transglutaminase are required for the assembly process. We have examined, by quick-freeze, deep-etch, rotary-shadow electron microscopy, the effects of inhibiting each of these enzymes upon FE assembly. These experiments reveal two domains within the FE, distinguishable by their enzymatic requirements for proper maturation. The first domain consists of the microvillar casts which require both protease and transglutaminase activities to obtain a normal paracrystalline coat. The second domain comprises the regions between casts and appears to mature by ovoperoxidase-mediated cross-linking of paracrystalline material to the envelope.     

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.     

Colony Organization in the Green Alga Botryococcus braunii (Race B) Is Specified by a Complex Extracellular Matrix

Botryococcus braunii is a colonial green alga whose cells associate via a complex extracellular matrix (ECM) and produce prodigious amounts of liquid hydrocarbons that can be readily converted into conventional combustion engine fuels. We used quick-freeze deep-etch electron microscopy and biochemical/histochemical analysis to elucidate many new features of B. braunii cell/colony organization and composition. Intracellular lipid bodies associate with the chloroplast and endoplasmic reticulum (ER) but show no evidence of being secreted. The ER displays striking fenestrations and forms a continuous subcortical system in direct contact with the cell membrane. The ECM has three distinct components. (i) Each cell is surrounded by a fibrous β-1, 4- and/or β-1, 3-glucan-containing cell wall. (ii) The intracolonial ECM space is filled with a cross-linked hydrocarbon network permeated with liquid hydrocarbons. (iii) Colonies are enclosed in a retaining wall festooned with a fibrillar sheath dominated by arabinose-galactose polysaccharides, which sequesters ECM liquid hydrocarbons. Each cell apex associates with the retaining wall and contributes to its synthesis. Retaining-wall domains also form “drapes” between cells, with some folding in on themselves and penetrating the hydrocarbon interior of a mother colony, partitioning it into daughter colonies. We propose that retaining-wall components are synthesized in the apical Golgi apparatus, delivered to apical ER fenestrations, and assembled on the surfaces of apical cell walls, where a proteinaceous granular layer apparently participates in fibril morphogenesis. We further propose that hydrocarbons are produced by the nonapical ER, directly delivered to the contiguous cell membrane, and pass across the nonapical cell wall into the hydrocarbon-based ECM.     

Structure of the Chlamydomonas agglutinin and related flagellar surface proteins in vitro and in situ

Using the quick-freeze, deep-etch technique, we compare the structure of the cane-shaped plus and minus sexual agglutinin molecules purified from gametes of Chlamydomonas reinhardi. We also describe the structure of three additional gamete-specific fibrillar molecules, called short canes, loops, and crescents, which are structurally related to the agglutinins. Four non-agglutinating mutant strains are found to produce the three latter fibrils but not canes, supporting our identification of the cane-shaped molecule as the agglutinin. The heads of the plus and minus canes are shown to differ in morphology. Moreover, two treatments that inactivate the plus agglutinin in vitro--thermolysin digestion and disulfide reduction/alkylation--bring about detectable structural changes only in the head domain of the cane, suggesting that the head may play an indispensible role in affecting gametic recognition/adhesion. We also present quick-freeze, deep-etch images of the flagellar surfaces of gametic, vegetative, and mutant cells of Chlamydomonas reinhardi. The gametic flagella are shown to carry the canes, short canes, loops, and crescents present in in vitro preparations. The cane and crescent proteins self-associate on the flagellar surface into stout fibers of uniform caliber, and they align along the longitudinal axis of the flagellum. The short canes and loops co-purify with flagella but, in the presence of mica, dissociate so that they lie to the sides of the flagella. The agglutinin canes of both mating types are oriented with their hooks at the membrane surface and their heads directed outward, where they are positioned to participate in the initial events of sexual agglutination.     

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.     

The cytoskeletal architecture of Trypanosoma brucei

The cytoskeleton of Trypanosoma brucei has been analyzed by the high-resolution technique of quick-freeze deep-etch rotary-shadowing electron microscopy. The study provides detailed structural information on the subpellicular array of microtubules, the flagellum, and the interaction of these 2 major structures of the trypanosomal cytoskeleton with each other. The subpellicular microtubules closely interact both with the cell membrane and with each other. At the anterior tip of the cell they converge into a tightly closed structure, whereas at the posterior end the microtubular array remains open ended. The microtubular array is involved also in forming the opening of the flagellar pocket. The microtubular array interacts with the paraflagellar rod of the flagellum through a dense meshwork of fibers that are anchored on the microtubular surface with one end and within the paraflagellar rod structure with the other. The highly ordered, 3-dimensional network of the paraflagellar rod itself is connected tightly to the microtubular axoneme of the flagellum through a regular array of fleur-de-lis-shaped linking structures.     

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.     

3,4-Dehydroproline inhibits cell wall assembly and cell division in tobacco protoplasts.

We investigated the function of cell wall hydroxyproline-rich glycoproteins by observing the effects of a selective inhibitor of prolyl hydroxylase, 3,4-dehydro-L-proline (Dhp), on wall regeneration by Nicotiana tabacum mesophyll cell protoplasts. Protoplasts treated with micromolar concentrations of Dhp do not develop osmotic stability and do not initiate mitosis. The architecture of regenerated cell walls was examined using deep-etch, freeze-fracture electron microscopy of rapidly frozen tobacco cells. Untreated protoplasts assemble a dense fibrillar cell wall consisting of laterally associating subelementary fibrils. In contrast, treatment of protoplasts with Dhp alters the structure of the regenerated wall fibrils in several ways: first, the microfibrils are coated with globular knobs; second, some larger fiber bundles have an open ribbon-like appearance; and third, the smallest subelementary fibrils were not visible. Tobacco cells develop an abnormal morphology as a consequence of this abnormal cell wall structure. Thus, inhibition of prolyl hydroxylase results in the regeneration of a cell wall with abnormal structural and functional properties. These data provide experimental evidence that hydroxyproline-rich glycoproteins are important for the structural integrity of primary cell walls and for the correct assembly of other wall polymers, and that wall structure is an important regulator of cell division and cell morphology.