Molecular Organization of Cell-Wall Crystals from Chlamydomonas Reinhardtii and Volvox Carteri
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Molecular organization of cell-wall crystals from Chlamydomonas reinhardtii and Volvox carteri URSULA W. GOODENOUGH and JOHN E. HEUSER Departments of Biology and Cell Biology/Physiology, Washington University, St Louis MO 63130, USA Summary The extracellular matrices of Chlamydomonas layer of the crystal (W6A) is shown to be a co- reinhardtii and Volvox carteri contain homolo- polymer of GP2 and GP3. The bulky globular gous salt-extractable crystalline layers that will domains of these glycoproteins form the rows of self-assemble in vitro. The organization of these granules seen on the upper and lower surfaces of crystals is examined using the quick-freeze deep- W6A in both Chlamydomonas and Volvox, and etch technique. In C. reinhardtii, the outer layer their filamentous domains interact to form a of the crystal is an open polygonal weave (W6B); dense meshwork. this is shown to be constructed from regular overlapping associations between the fibrous hy- Key words: cell wall, Chlamydomonas reinhardtii, Volvox droxyproline-rich glycoprotein GP1. The inner carteri. Introduction In a previous article (Goodenough & Heuser, 1985), we showed that the crystalline layer, designated \V6 by The green algae of the Order Volvocales, the best Roberts et al. (1972), is in fact bilaminar, consisting of known genera of which are Chlamydomonas and Vol- a dense lattice (W6A) and a more open woven layer vox, are surrounded by cell walls constructed of (W6B), the latter apparently not visualized following hydroxyproline-rich glycoproteins (Miller et al. 1974). negative staining. Here we provide detailed images of The wall of the best studied species, Chlamydomonas each layer after crystal adsorption to mica flakes. Using reinhardlii, has been shown to possess two major purified crystal glycoproteins (Goodenough et al. domains: a chaotrope-insoluble inner trabcculum, and 1986), we show that none is able to crystallize on its a chaotrope-soluble outer layer with a crystalline struc- own, that the glycoproteins designated GP2 and GP3 ture (Goodenough & Heuser, 1985; Roberts et al. will co-assemble to form the W6A layer, and that GP1 1985). If the solubilized outer layer is dialysed against forms the W6B layer. We also report that the crystalline water, the glycoproteins will reassemble i?i vitro to layer of Volvox carteri can be solubilized in chaotropc form small crystals with a native substructure (Good- and reassembled /;/ vitro, yielding crystals that have the enough et al. 1986; Hills et al. 1975). Negatively same basic structure as those from C. reinhardtii. stained images of these crystals have been subjected to We conclude, for both organisms, that the globular optical filtering and computer reconstruction to pro- domains of glycoproteins GP2 and GP3 form the duce a detailed two-dimensional projection of the granular densities visualized on the upper and lower lattice (Roberts et al. 1982); however, the specimens surfaces of W6A, and that the filamentous domains of reportedly lack sufficient order for analysis in three these proteins form an intervening meshwork, which dimensions (Roberts et al. 1985). By contrast, native may serve important filtering functions. We also pro- wall fragments from several other genera (Chlorogo- pose a detailed model for the assembly of the W6B nium, ljobomonas) have more stable crystalline layers, weave from GP1 monomers, and we show that the GP2 which have yielded detailed three-dimensional maps monomer is highly conserved between C. reinhardtii (Shaw& Hills, 1982, 1984). and Volvox. A deep-etch analysis of the cell wall of C. In this article we analyse C. reinhardtii wall crystal eugamatos is presented in the accompanying paper structure using the quick-freeze deep-etch technique. (Goodenough & Heuser, 1988). Journal of Cell Science 90, 717-733 (1988) Printed in Great Britain © The Company of Biologists Limited 1988 717 Materials and methods zigzagging fibres, sloping slightly downwards from left to right in Fig. 2, that are interconnected by regularly All relevant methods for Chlamydomonas crystal formation, spaced crossbars. As shown diagrammatically in crystal protein purification, and quick-freeze deep-etch elec- Fig. 3A, the zigzags are not symmetrical — the 'zig' is tron microscopy have been presented (Goodenough et al. longer than the 'zag' - and the crossbars extend from 1986; Goodenough & Heuser, 1985). V. carteri spheroids the bottom of each zag to the top of each zig. The result were grown in synchronous liquid cultures as described (Kirk & Kirk, 1983). To prepare crystals, fifteen 300-ml cultures is a staggered array of asymmetric hexagons. Both the were harvested by filtration through a 30jum nylon screen, zigzag and the crossbar fibres are, in general, of were suspended in distilled water in two SO ml tubes and uniform diameter; however, in regions such as those pelleted at 27 000 g for 5 min. The pellets were put on ice for marked by large white arrows in Fig. 2, they appear to 15 min, during which time the spheroids quantitatively shed be unravelling into two strands. More informative their flagella. The pellets were then suspended in water, spun views of the unravelling process are presented in a later at 3000 £ for 3 min, and the flagella-containing supernatant section. was discarded. The pellets were again suspended in water, spun at 39 000 £ for 5 min, and the supernatants were discarded. The volume of the pellets was estimated, and for The W6A outer surface each 1 ml of pellet, 05 ml of 4 M-sodium perchlorate in water was added (two pellets, = 10ml each, are typically obtained). Where the W6B fibres have been fractured away in The pellets were suspended in the perchlorate for 15-30 min Fig. 2, the outer surface of W6A is exposed. This with occasional pipetting. The suspensions were then centri- surface carries parallel rows of granules, oriented fuged at 39 000if for 10 min and the clear supernatants vertically in this and subsequent figures along what will transferred into dialysis bags and dialysed for 24 h against hereafter be designated the A axis of the crystal (thick three changes of water. The dialysate was placed in a 100 ml vertical arrow). The granules also define a series of beaker, frozen at —80°C, and lyophilized to dryness. The parallelograms, one of which is highlighted, where each lyophilate was suspended with vigorous pipetting in a small parallelogram tilts upwards and to the left with respect volume (l-2ml) of 1 M-perchlorate; after 1 h, the suspension was ccntrifuged at 30000^ for 15 min to pellet insoluble to the A axis. The granules themselves tend to be oval material. The supernatant was dialysed overnight against rather than round, with their long axes oblique relative water to yield an abundant crop of crystals; by phase-contrast to the A axis. These relationships are illustrated in microscopy, these are similar in appearance to those obtained Fig. 3B. from C. reinhardtii (Goodenough et al. 1986) but are even To show that the W6A granules are not fractured-off more refractile. termini of W6B material, crystals were prepared that lack the W6B component. This was accomplished by mixing together the purified monomers GP2 and GP3 Results (Goodenough et al. 1986) in the presence of perchlor- ate, followed by dialysis against water. The abundant Overview crystals that form under these conditions are devoid of Fig. 1 shows a C. reinhardtii crystal 'sandwich', polym- W6B weave because the GP1 monomer, which forms erized in vitm, adsorbed to a mica flake, and quick- the weave (see below), is not present. The crystals frozen deep-etched. Each half of the sandwich is nonetheless carry prominent oval granules on their organized so that its 'woven' VV6B layer is on the W6A surfaces (Fig. 4), forming parallelograms (high- outside and its dense W6A layer is on the inside. As the lighted) with the same dimensions (24 nm X 27 nm) as fracture plane passes through such a sandwich, there- fore, different views of the crystal are obtained: the those in the native crystal. No crystals form if either outer surfaces of VV6B and W6A are exposed on the GP2 alone or GP3 alone is treated in an identical upper half, whereas the under surfaces of W6A and fashion, demonstrating that both monomers must in- W6B are exposed by fracturing through the sandwich teract to form the lattice. to the lower half. Both the upper and under surfaces of The material between the W6A granules consists of a W6A are seen to carry parallel rows of granules (thick dense fibrillar network (Figs 2 and 4). Some of the arrows). The lower W6B layer, making direct contact fibrils appear to interconnect the granules; others seem with the mica, has been induced to depolymerize into to course between them. Most conspicuous, perhaps, its component strands, as described in detail, below. are the fibrils that run through the centre of some of the The sections that follow present a stepwise analysis parallelograms in the direction of the A axis; several are of the structure of this crystal. indicated by thin white arrows in Figs 2 and 4. Although it is possible to recognize such patterns, it is The W6B weave also clear that the meshwork is readily distorted: the Fig. 2 shows the outer surface of a crystal sandwich. fibrils, for example, often appear coalescent, leaving Prominent is the VV6B weave, which consists of long concomitant holes. 718 U. IV. Goodenough and J. E. Heuser Fig. 1. Survey of C. reinhardtii crystal sandwich, showing the upper surfaces (up) of W6B and W6A in the upper layer followed by the under surfaces (un) of W6A and W6B in the lower layer. The VV4 domain, containing large spherical aggregates, occupies the centre of the sandwich. Thick arrows indicate parallel rows of granules defining the A axes, which are seen to be oriented in different directions in the two halves of the sandwich.