Molecular Organization of Cell-Wall Crystals from Chlamydomonas Reinhardtii and Volvox Carteri

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 perchlorate, 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, therefore, 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. X184 000.

Crystal organization in algal cell walls 719 Fig. 2. W6B weave and, where the weave is fractured off, VV6A surface of C. reinhardtii crystal. A thick vertical arrow designates the A axis of W6A. A unit parallelogram of W6A is highlighted. The thin white arrow indicates central fibrils in the meshwork, which bisect the parallelograms. Large white arrows designate areas where the W6B weave unravels into its two component strands. X250 000.

W6A/W6B relationships The W6A inner siuface The topological relationship between the W6B weave Fig. 5 shows the W6A under surface. Parallel rows of and the W6A upper granules is well illustrated by granules again define the A axis of the crystal (thick Fig. 2. If, starting from the upper left, one follows the arrow), with fibrillar material in between. The granules also define 24nmX27nm parallelograms (high- long zigzagging elements of the W6B weave until one lighted), but on this surface each unit tilts upwards and reaches the place where they have been fractured off to the right with respect to the A axis (Fig. 3D), the and the W6A granules arc exposed, the extension of flip side of the orientation seen from above (Fig. 3B). each zigzag is seen to pass between sets of major The fibrillar meshwork seen from the inner surface is granules, and the major granules are seen to lie directly again complex, and again highly variable. One can beneath the centre of each crossbar. These relation- define, for example, a C axis running diagonally across ships are shown diagrammatically in Fig. 3C. the parallelogram (Fig. 3D). Fibrous elements with

720 U. W. Goodenough and J. E. Heuser t A axis 27nm

A axis

Fig. 3. Diagrams of W6B and W6A construction. See text Fig. 4. Upper surface of a crystal formed by mixing for details. purified C. reinhardtii GP2 and GP3 monomers in 1 M- sodium perchlorate and dialysing overnight against water. Thick vertical arrow and thin white arrows, as in Fig. 2. this orientation are detected in reconstructions of Xs are placed to the right of small granular elements lying negatively stained images (Roberts et al. 1982), and between the large granules on the A axis. X250000. they are also prominent in some deep-etch images, an example being Fig. 6 (arrows). In contrast, very few C diagonals arc evident in Fig. 5. This variability is white arrows); with this image in mind, they can be highlighted in Fig. 7, where in a single W6A layer the identified as well in Fig. 5 and on the left side of Fig. 7 rows on the right display many strong C diagonals (thin white arrows). These central elements are suf- (arrows), whereas those on the left display only a few ficiently similar to those pointed out earlier in Figs 2 weak ones. and 4 (thin white arrows) to suggest that we arc viewing Rows that do not carry C diagonals are usually the same structures from above and below. The central instead bisected by central elements. Such elements are elements apparently correspond to the medial com- very conspicuous in the crystal shown as Fig. 8 (thin ponents labelled B in optical reconstructions of nega-

Crystal organization in algal cell walls 721 Fig. 5. Under surface of the W6A layer of a C. reinhardtii crystal. Thick vertical arrow and thin white arrows, as in Fig. 2; Xs as in Fig. 4. Monomers of GPI, GP2 and GP3 lying free on the mica are labelled. X250000.

Fig. 6. Under surface of the W6A layer of a C. reinhardtii crystal. Thick vertical arrow, as in Fig. 2. Arrows indicate fibrillar C diagonals running across the parallelograms from upper left to lower right. X250000. tively stained crystals (Roberts et al. 1982), com- surface, the two sides of W6A are in general difficult to ponents that are particularly striking in fig. 2 of Davies distinguish morphologically (unless, of course, W6B is & Lyall (1973). present). The homologous crystals of V. carteri, by Although the granules of the inner surface are contrast, display much sharper differences between generally rounder than those seen from the outer front and back; their fibrillar components, moreover,

722 U. W. Goodenough and J. E. Heuser Fig. 7. Under surface of the W6A layer of a C. reinhardtii crystal. Thick vertical arrow and thin white arrows, as in Fig. 2. Large white arrows indicate fibrillar C diagonals. X250 000. Fig. 8. Under surface of the W6A layer of a C. reinhardtii crystal. Thick vertical arrow and thin white arrows, as in Fig. 2. x 250 000. are less prone to destabilization. In the next section, On the under surface, by contrast, the major gran- therefore, we analyse the Volvox crystal. ules are very prominent (Fig. 12). A rightward-sloping parallelogram is indicated, as usual, by the black-on- The W6 layer of Volvox crystals white highlighting. In fact, however, this unit is not the Fig. 9 shows a survey view of the surface of a Volvox first to catch the eye: strong diagonals along the C axis spheroid, with structural details provided in the legend (arrow) (cf. Fig. 3D) accentuate parallelograms that and by Kirk et al. (1986). As with C. reinhardtii cells slope steeply down to the right (one of these is (Goodenough et al. 1986), Volvox spheroids can be indicated by 4 white bars in Fig. 12). With low-angle stripped of their W6 crystalline layer by immersion in shadowing (Fig. 13), the C diagonals arc particularly sodium perchlorate, and the released monomers will prominent (arrow). In addition, a second system of recrystallize upon dialysis against water. Fig. 10 shows diagonals becomes apparent (Fig. 13, large white ar- a survey view of a Volvox crystal, which, like its C. rowheads). Each fibril in this system begins near a reinhardtii counterpart, usually forms a 'sandwich' (cf. major granule and then curves downwards and towards Fig. 1) so that one sees both the outside of the upper the midline of the parallelogram, an axis designated E lattice and, upon fracturing through the sandwich, the in Fig. 3D. In an unusual instance in which a Volvox under side of the lower lattice. Since Volvox carries no crystal has stretched during mica adsorption W6B weave, either in situ (Fig. 9) or in vitro (Fig. 10), (Fig. 14A), both the C and the E diagonals are replaced the outside of its W6A lattice can be viewed directly. It by sets of central elements (arrows), with fibrils is clear even at low magnification (Fig. 10) that this entering into them from either side. Fig. 14B shows a surface displays closely packed chains of small par- comparable image from C. reinhardtii, indicating that ticles, whereas the under surface displays more widely the fibrillar interiors of the two crystals can appear separated arrays of larger granules. remarkably similar. Fig. 11 shows the outer surface of a Volvox crystal at In areas where the fibrillar meshwork displays any higher magnification. The thick arrow indicates the A sort of uniform patterning, one half of the meshwork axis. One of the unit parallelograms is highlighted; its generally looks different from the other. Thus, in the dimensions (21nmx26nm) are somewhat different region under consideration in Fig. 14A, distinct indi- from those of C. reinhardtii (24 nm X 27 nm), but the vidual fibrils extend from the right row of granules to same structural plan is clearly being followed. The the midline, whereas random platinum grains decorate granules forming such parallelograms are in most cases whatever lies to the left of the midline. In Fig. 13, the difficult to make out; instead, the upper surface fibrils along the E axis (cf. Fig. 3D) run from the upper appears to consist of closely packed particles along the left to the midline, whereas a second set of fibrils runs A axis, interconnected by fibrillar material. from the upper right granule towards the left half of the

Crystal organization in algal cell walls 723 Fig. 9. V. carteri spheroid showing the constituent layers of its outer wall. A loosely anastomosing layer (Wl) extends from the cell surface (located at lower right, not included in field) to a denser meshwork designated W2. A narrow palisade layer, designated W4, is devoid of the granules present in the homologous W4 layer of C. reinhardtii (Fig. 1). The dense crystalline W6 layer carries no W6B weave. Streaming radial fibres on the outermost surface are topologically equivalent to the W7 domain of the C. reinhardtii wall (Goodenough et al. 1986), but they are much less densely packed and apparently not cross-connected. X37 000.

724 U. W. Goodenough and J. E. Heuser Fig. 11. Upper surface of the W6 layers of a V. carteri crystal. Thick vertical arrow, as in Fig. 2; Xs as in Fig. 4. X 250 000.

unit (black-on-white arrowhead). And finally, the C diagonals in both Chlamydomonas and Volvox are usually much stronger in one half of each parallelogram than in the other (Figs 6 and 12). We conclude, therefore, that in both crystal types, the fibrillar domains are not symmetrically organized. Dissociation of the W6B weave on mica When the W6B weave approaches the mica surface, it is invariably induced to depolymerize. Even after ex- posure to glutaraldehyde, arrays of hexagohs arc never encountered on mica, suggesting that some of the interactions that stabilize the hexagons are very sensi- tive to the strong field exerted by the negative charge on the mica surface. Mica adsorption instead yields the kinds of images shown in Figs 15 and 16. The downward vertical arrows in Fig. 15 indicate parallel rows of fibres that, in places, can be seen to be double- stranded. Connecting these rows are double-stranded rungs (opposed small white arrows). In the leftward part of Fig. 15 these rungs slope gently upward with Fig. 10. Survey of V. carteri crystal sandwich showing the respect to the parallel fibres; moving rightwards, they upper (up) and under (un) surfaces of its W6 layers. Thick tend to tilt more acutely, adopting a distinct lenticular vertical arrows, as in Fig. 2. As with C. reinhardtii (Fig. 1), the A axes of the two halves of the sandwich are shape. In Fig. 16A, parallel rows of vertical fibres are oriented in different directions. X 145 000. again interconnected by lenticular pairs of horizontal

Crystal organization in algal cell walls 725 Fig. 12. Under surface of the W6 layer of a V. carteri crystal, stereo pair. Thick vertical arrow, as in Fig. 2; Xs as in Fig. 4. Four white lines indicate a second parallelogram unit accentuated by strong C diagonals (arrow). X250000. rungs (arrows). In the upper part of the field, however, Dissociation of the W6A lattice on mica the disassembly process has progressed much farther, The GP2 and GP3 glycoproteins that form the W6A and long S-shaped units predominate. Such units are crystal possess large globular 'bodies' and fibrous also seen in the depolymerized W6B region of Fig. 1. A 'necks', which terminate in small globular 'heads' model relating these states of disassembly to the native (Goodenough et al. 1986; see also the monomers hexagonal weave of W6B is presented in the Dis- indicated in Figs 5 and 17). It is logical to suppose that cussion. the granules of the crystal are formed by the bodies and The W6B layer is formed of the glycoprotein GP1: that the necks interact to form the fibrous network, and during crystallization of purified monomer mixtures deep-etch replicas are fully consistent with this notion. (cf. Fig. 4), a weave will form only if GP1 is present; For example, in Fig. 17 the edges of a depolymerizing moreover, we find that if GP1 monomer is added to a crystal show head-plus-neck domains (arrows) emanat- GP2/GP3 assemblage such as that illustrated in Fig. 4, ing from A-axis rows of granules. Unfortunately, we a weave will form on its outer surface (Adair et al. have been unable to detect any informative patterns in 1987). Individual GP1 monomers are indicated in such depolymerizing images; that is, we have found Fig. 5 (see also Goodenough et al. 1986). Interestingly, nothing equivalent to the regular images displayed by whereas GP1 monomers invariably carry distinct the depolymerizing W6B. Instead, as is evident in globular domains, designated 'heads' (Goodenough et Figs 5-8, 12-14 and 17, the frayed edges of W6A al. 1986), at one end, such heads are not evident in the crystals either curl up, as if avoiding the mica surface, unravelling waves. It could be argued that the heads in or else splay out into an indecipherable jumble of the unravelling images are not adequately shadowed molecules. We have attempted to improve the situation because the proteins are so close to one another. by pretreating the mica with such agents as polylysine However, fig. 10 of Goodenough et al. (1986) shows a or cytochrome c, but without success. Therefore, we field of very concentrated GP1 monomers where the are unable to offer any detailed molecular model for the heads are nonetheless shadowed normally. Possibly, placement of GP2 and GP3 in the W6A lattice, and therefore, the head configuration is lost during the indeed, we do not know whether the conformations course of weave polymerization. More specifically, adopted by the monomers bear any resemblance to the binding sites important for polymerization may be conformations they adopt in polymerized state. buried in the globular heads of the monomers and become exposed once the correct initial contacts are made with other monomers and/or with sites on the Molecular components of Volvox aystals W6A lattice. Fig. 18 compares the SDS/PAGE profile of Chlamy-

726 U. W. Goodenough andjf. E. Heuser domonas and Volvox crystal monomers. It is evident that Volvox lacks any GP1 species; since it also lacks a W6B weave (Figs 10-11), this offers further support for the conclusion that GP1 creates the W6B layer. Three major and two minor Volvox glycopolypeptide (GP) species enter the running gel having apparent molecular weights of 350, 270, 230, 200 and 135 (X 103), respectively. The GP270 species is of particular interest because it migrates just slightly behind the GP2 species of C. reinhardtii. Moreover, when Volvox crystals are dissolved in perchlorate and the monomers are adsorbed to mica and washed free of salt, one of the proteins strongly resembles the C. reinhardtii GP2 (Fig. 19; upper two rows are Volvox GP270, bottom row is C. reinhardtii GP2), the only difference being that its neck is somewhat longer. We conclude, therefore, that the GP2 and GP270 species are homologous and that they are in part responsible for the homology between the two crystal types.

Discussion

Molecular organization of the W6B weave Fig. 20 presents a model of the molecular organization of the W6B weave, which indicates how a glycoprotein with the dimensions (100 nm) and configuration (Figs 5 and 17) of GP1 could assemble into the hexagons seen in situ (Fig. 2) and disassemble into the double- stranded ladders and S-shaped units seen after adsorption to mica (Figs 15 and 16). The model is based on the following premises and lines of reasoning. First, we assumed that each strand of the weave is double, formed of two GP1 proteins. This is indicated by the unravelling regions in Fig. 2 (arrows), and by the double-stranded nature of the depolymerizing units on mica (Figs 15 and 16). Second, we reasoned that a fibrous protein like GP1, with a globular head at one terminus, a blunt end at the other terminus (hereafter called the tail), and two kinks (nos 1 and 2) in its shaft (Goodenough et al. 1986), might well follow the same kinds of rules as those that govern the assembly of the 'hammock' described by Goodenough & Heuser (1985). The hammock monomer also bears a head, a single kink and a tail, and the monomers form a network of rectangles by head-to-tail and head-to-kink interactions. In the Fig. 13. Under surface of the W6 layer of a V. carteri model shown in Fig. 20A, three of the six vertices in crystal. Thick vertical arrow, as in Fig. 2. Arrow indicates each hexagon (boxed) are formed by interactions C diagonals; white arrowheads indicate E diagonals; black- between a head, a tail, a kink no. 1, and a kink no. 2, on-white-arrowhead indicates diagonals extending from each domain deriving from a different GP1 monomer. upper right granule in some of the parallelograms. X 250 000. We designate these as tetranodal vertices. As noted in Fig. 14. A,B. Under surface of the W6 layers of V. carteri Results, the heads of GP1 may in fact straighten out (A) and C. reinhardtii (B) crystals. Thick vertical arrows during the course of polymerization, but for orientation and thin white arrows, as in Fig. 2. X250 000. purposes they are drawn as globular in Fig. 20.

Crystal organization in algal cell walls 1T1 Fig. 15. W6B weave undergoing depolymerization upon adsorption to a mica surface. Thick arrow, as in Fig. 2 (note the shift in orientation from previous figures). Large white arrows indicate parallel rows of double-stranded fibres; pairs of small white arrows indicate double-stranded rungs that interconnect the parallel rows. The pair of opposing large white arrows in centre of field indicates a parallel row of fibres and the position it occupies with respect to the unit parallelogram of W6A. x250 000,

Third, it was important to identify units in the ation with W6A, they might well tend to migrate underlying W6A lattice that might dictate the pattern towards the tetranodes, creating the upwardly sloping of the hexagons, since GP1 is incapable of assembling lenticular units seen in Fig. 15. into hexagons on its own (Adair et al. 1987). The Fig. 20A shows obviously only one of many possible Fig. 20A model shows the three tetranodal vertices ways that GP1 monomers can be arranged to form a interacting with the major granules exposed on the W6B weave. We have experimented with numerous upper surface. As considered in detail in the next alternative arrangements, but none was capable of section, minor granules apparently lie between the generating the ladder-with-lenticular-rung pattern that major granules, as shown diagrammatically in Fig. 3F, is observed on mica. Therefore, while the model and we propose that the three non-tetranodal vertices illustrated in Fig. 20 requires independent verification, are stabilized by interactions with these minor gran- it is capable of explaining all our observations to date. ules. A noteworthy feature of Fig. 20A is its proposal that Fourth, we postulated that the non-tetranodal inter- each GP1 forms hexagons by interacting with six other actions with W6A are less stable than the tetranodal GP1 proteins in a staggered fashion. Thus, if the six interactions, so that as the VV6B weave approaches the hexagon-forming sides of a given protein are labelled mica surface, these are the first to be released. This 1—>6 from head to tail, then domain 1 interacts with release causes the zigzags to straighten out into the domain 5 of one protein; domain 2 interacts with do- parallel rows marked by arrows in Fig. 15, a conversion main 4 of a second protein; domain 3 interacts with illustrated in Fig. 20B. The crossbars, by this model, domain 6 of a third protein; domain 4 interacts with do- convert into the 'rungs' and, indeed, careful inspection main 2 of a fourth protein; domain 5 interacts with of the leftward area of Fig. 15 shows that each pair of domain 1 of a fifth protein; and domain 6 interacts with rungs is separated from adjacent pairs by the same domain 3 of a sixth protein. Such staggering is, of staggered spacing as the native crossbars (cf. Fig. 3A). course, reminiscent of the strategy followed by collagen Fifth, we reasoned that once the non-tetranodal molecules to form fibres (Bornstein & Sage, 1980) and portions of the strands were released from their associ- clathrin trimers to form hexagonal coats (Crowther &

728 U. W. Goodenough and J. E. Heuser Fig. 16. A. W6B weave undergoing depolymerization upon adsorption to a mica surface. Thick arrow, as in Fig. 2 (note that the same altered orientation is adopted here as in Fig. 15). Arrows indicate lenticular units. B. Stereo pair of A where the parallel white lines lie over the sets of parallel fibres in the W6B weave, and the circles indicate the position of identifiable large granules in the W6A crystal. X250000.

Pierce, 1981; Heuser & Kirchhausen, 1985). surface (Fig. 1; and Goodenough et al. 1986). On the Interestingly, sexual mating between Chlamydomonas other hand, each surface carries parallelograms with gametes involves membrane-bound fibrillar proteins the same dimensions. One question, then, is how these that are morphologically very similar to GPl (Good- two sets of surface features relate to each other in three enough et al. 1985), and we have proposed (Cooper et dimensions. One possibility is that the granules seen al. 1983; Goodenough et al. 1985) that the adhesion from above and below are directly superimposed. A between gametes may entail a complex interaction of second possibility is that the upper granules arc stag- such fibrils akin to the assembly of the cell-wall gered in a 'north-south' sense with respect to the lower networks being discussed here. granules, in such a way that the two sets alternate along the A axis. A third possibility is that the granules are Molecular organization of the W6A lattice staggered in an 'east-west' sense, with the A axes seen The two surfaces of W6A are clearly not identical. This from the top overlying the central elements seen from is evident from direct inspection in the case of Volvox below and vice versa. (Fig. 10); in the case of C. reinhardtii, it is made The east-west stagger model is not supported by our evident by the fact that GPl binds only to the upper replicas; for example, in Figs 5-8, the frayed edges of surface of W6A and GPl.5 binds only to the lower the crystals give no indication that large granules lie

Crystal organization in algal cell walls 729 Fig. 17. Under surface of a depolymerizing W6A layer of a C. reinhardtii crystal. Thick vertical arrow, as in Fig. 2. Arrows show head-plus-neck domains emerging from the unravelling crystal. Monomers of GP1, GP2 and GP3 lying free on the mica are labelled. X 250 000. beneath the central meridians. It is also refuted by image could, of course, also arise if the granules seen cross-sectional thin-section images (Catt et al. 1978): from above and below are indeed superimposed, creat- these show regular, =25 nm-spaced accumulations of ing the A densities, while a second set of 'minor stain spanning the full thickness of W6, indicating that granules', not readily visible in deep etchings, creates each major axis of mass on the upper surface lies above the D densities. a major axis of mass on the under surface. The model proposing a direct superposition of granules would at first sight seem to be countered by computer-reconstructed images of negatively stained crystals (Roberts et al. 1982). Such images, which should indicate the position of mass on both surfaces, show two types of granules along the A axis, a large species designated A alternating with a smaller species designated D, as might be expected from a north- south stagger of upper and lower granules. Such an

Fig. 18. SDS/PAGE of crystal glycopolypeptides from C. reinhardtii (C) and V. carteri (V), with samples prepared and PAS-stained as described by Goodenough et al. (1986). The C. reinhardtii glycopolypeptides migrate as >400 (GP1), 260 (GP2), 165 (GP3A) and ISO (GP3B) 3 (XlO )A/r species in this particular gel. (We find some gel- to-gel variation with these glycopolypeptides; for example, 3 values of 260-280 (xlO )Mr have been obtained for GP2 in different preparations.) The three major V. carteri bands 3 migrate as 270, 200 and 135X W Mr species.

730 U. W. Goodenough and jf. E. Heuser Fig. 19. Top two rows: GP2-like proteins from V. carter/. Bottom row: GP2 proteins from C. reinhardtii. X250000.

Crossbar

Fig. 20. Proposed molecular organization of the W6B weave. A. Organization of GP1 proteins is in the native weave. Open ovals represent the heads of GP1; filled rectangles represent the tails; #1 and #2 indicate the two major kinks in the protein. The A axis, zig, zag and crossbar are as in Fig. 3. Broken ovals represent the major granules of the W6A lattice; the crystal is being viewed from its under surface. B. Depolymenzation of W6B on mica. A represents a GP1 head; B and C are the two major kinks; and D is the tail. Broken ovals as in A. The crystal is again being viewed from its under surface.

Fig. 16B presents direct evidence that the major the W6A crystal, and circles were drawn over the major granules seen on the top and bottom of the W6A layer granules in this crystal. The lines are seen to pass are in fact superimposed. Lines were placed over the between the major granules. Since the parallel lines are vertical 'parallel-line' components of the unravelling thought to correspond to the W6B zigzags (Fig. 20), W6B weave on the mica; they were then extended into and since the zigzags also pass between the major

Crystal organization in algal cell walls 731 granules seen on the upper surface (Figs 2 and 3C), the be designed to absorb such impacts by undergoing major granules on the two surfaces are apparently local, and perhaps reversible, dislocations. superimposed. This same relationship can also be seen The higher plants also carry abundant hydroxypro- in Fig. 15, where an opposing set of large white arrows line-rich glycoproteins in their cell walls (Showalter & in the centre of the field indicates a parallel-line unit Varner, 1987). These lack bulky globular domains and passing between the large granules of the under surface consist only of head-plus-neck units (Strafstrom & along the same plane as that on which the zigzags would Staehelin, 1986), and their disposition in the wall has lie on passing between the large granules of the upper yet to be elucidated. If, as has been postulated (Lam- surface. port, 1986), the higher plant glycoproteins carry bind- If the major granules are indeed superimposed, then ing sites for such structural polymers as cellulose a second set of minor granules must lie on the A axis and/or the pectins, then there would be no 'structural between the major granules to account for the minor D need' for the globular domains found in the algal densities seen in image reconstructions. Although not glycoproteins. Retained by higher plants, however, is obvious in every parallelogram, possible candidates are an apparent 'need' for the necks and heads of these in fact visible in many instances. Thus in Fig. 4 Xs glycoproteins, perhaps for their ability to assemble into point to small granular elements that lie between the meshworks with useful biophysical properties. large granules of the upper surface, and in Fig. 5 Xs point to small granular elements that lie between the Members of Dr David Kirk's laboratory are thanked for the large granules of the lower surface. Similar Xs in Figs cultures of Volvox carten. Carol Hwang and Valerie Mermall 11 and 12 identify 'staggered' minor granules on the provided Chlamydomonas cells and purified GP proteins; upper and lower surfaces of the Volvox crystal. Robyn Roth produced the replicas and Comfree Coleman the final plates; Ann Dillon made the drawings. Supported by Granted this basic organization and the body-neck- grants GM-261S0 to U.W.G., GM-27215 to D. L. Kirk, head configuration of the GP2 and GP3 monomers that USDA 85-CRCR-1-18S8 to U.W.G. and D. L. Kirk, and form the lattice (Figs 5 and 18), it seems plausible that GM-29657 to J.E.H. most of the body mass of each protein forms the major granules, that portions of the body mass of each protein form the minor granules, and that the necks and heads References form the fibrillar meshwork. The major disappoint- ment of this study is that despite exhaustive scrutiny of ADAIR, W. S., STEINMETZ, S. A., MATTSON, D. M., some 500 stereo-pair images, we cannot offer any GOODENOUGH, U. W. & HEUSER, J. E. (1987). Nucleated assembly of Chlamydomonas and Volvox cell specific suggestions as to how the GP2 and GP3 walls. J CellBiol. 105, 2373-2382. monomers are positioned in the lattice. It is clear that BORNSTEIN, P. & SAGE, H. (1980). Structurally distinct the fibrils in the meshwork often lie along specific axes; collagen types. A. Rev. Biochem. 49, 967-1003. however, they are apparently capable of shifting pos- CATT, J. W., HILLS, G. J. & ROBERTS, K. (1976). A itions, perhaps as they are subjected to different structural glycoprotein, containing hydroxyproline, degrees of stretch, so that precise assignments are isolated from the cell wall of Chlamydomonas reinhardi. difficult. Moreover, it has proved impossible to assign a Planta 131, 154-171. particular neck to a particular body in the crystal and CATT, J. W., HILLS, G. J. & ROBERTS, K. (1978). Cell wall thereby to understand how the two proteins co-polym- glycoproteins from Chlamydomonas reinhardi, and their erize. self-assembly. Planta 138, 91-98. COOPER, J. B., ADAIR, W. S., MECHAM, R. P., HEUSER, J. We can, nevertheless, offer the general concept that E. & GOODENOUGH, U. W. (1983). Chlamydomonas the crystal has a substantial thickness, created by the agglutinin is a hydroxyproline-rich glycoprotein. Proc. superposition of GP2 and GP3 bodies along the natn.Acad. Sci. U.SA. 80, 5898-5901. granular A axes, and that this thickness is filled with a CROWTHER, R. A. & PEARSE, B. M. F. (1981). Assembly three-dimensional meshwork of fibrils that criss-cross and packing of clathrin into coats. J. Cell Biol. 91, one another. The bulky globular domains, particularly 790-797. if stabilized by side-to-side interactions, would be DAVIES, D. R. & LYALL, V. (1973). The assembly of a expected to maintain the integrity and thickness of the highly ordered component of the cell wall: The role of layer, while the fibrillar meshwork could perform such heritable factors and of physical structure. Molec. gen. functions as retaining water and solutes and deterring Genet. 124, 21-34. potential pathogens from access to the cell. Although GOODENOUGH, U. W., ADAIR, W. S., COLLIN-OSDOBY, P. & HEUSER, J. E. (1985). Structure of the the distortions visible in replicas of the fibrous mesh- Chlamydomonas agglutinin and related flagellar surface work (e.g. see Figs 2 and 4) may well occur during proteins in vitro and in situ. J. Cell Biol. 101, 924-941. sample preparation, similar distortions may occur GOODENOUGH, U. W., GEBHART, B., MECHAM, R. P. & when Chlamydomonas and Volvox encounter small HEUSER, J. E. (1986). Crystals of the Chlamydomonas particles in their environment; that is, the matrix may reinhardtn cell wall: Polymerization, depolymerization,

732 U. W. Goodenough and J. E. Heuser and purification of glycoprotein monomers. J. Cell Biol. cell wall of Chlamydomonas gymnogama and the concept 103, 405-417. of a plant cell wall protein. J. Cell Biol. 63, 420-429. GOODENOUGH, U. W. & HEUSER, J. E. (1985). The ROBERTS, K., GRIEF, C, HILLS, F. J. & SHAW, P. J. Chlamydomonas cell wall and its constituent (1985). Cell wall glycoproteins: structure and function. glycoproteins analyzed by the quick-freeze deep-etch J. Cell Sci. Suppl. 2, 105-127. techniques. J. Cell Biol. 101, 1550-1568. ROBERTS, K., GURNEY-SMITH, M. & HILLS, G. J. (1972). GOODENOUGH, U. W. & HEUSER, J. E. (1988). Molecular Structure, composition, and morphogenesis of the cell organization of the cell wall and cell-wall crystals from wall of Chlamydomonas reinhardi. I. Ultrastructure and Chlamydomonas eugamatos. J. Cell Sci. 90, 735-750. preliminary chemical analysis. .7. Ultrastruct. Res. 40, HEUSER, J. E. & KIRCHHAUSEN, T. (1985). Deep-etch 599-613. views of clathrin assemblies. J. Ullrastruct. Res. 92, ROBERTS, K., HILLS, G. J. & SHAW, P. J. (1982). The 1-27. structure of algal cell walls. In Electmn Microscopy of HILLS, G. J., PHILLIPS, J. M., GAY, M. R. & ROBERTS, K. Proteins (ed. J. R. Harris), vol. 3, pp. 1-40. London: (1975). Self-assembly of a plant cell wall in vitro. J. Academic Press. molec.Biol. 96, 431-441. SHAW, P. J. & HILLS, G. J. (1982). Three-dimensional KIRK, D. L., BIRCHEM, R. & KING, N. (1986). The structure of a cell wall glycoprotein. jf. molec. Biol. 162, extracellular matrix of Volvox: A comparative study and 459-471. proposed system of nomenclature. J. Cell Sci. 80, SHAW, P. J. & HILLS, G. J. (1984). The three-dimensional 207-231. structure of the cell wall glycoprotein of Chlorogonitnn KIRK, D. L. & KIRK, M. M. (1983). Protein synthesis elongatum.J. Cell Sci. 68, 271-284. patterns during the asexual life cycle of Volvox carteri. SHOWALTER, A. M. & VARNER, J. E. (1987). Biology and molecular biology of plant hydroxyproline-rich Devi Biol. 96, 493-506. glycoproteins. In The Biochemistry of Plants (ed. A. LAMPORT, D. T. A. (1986). The primary cell wall: A new Marcus), vol. 11. NY: Academic Press (in press). model. In Cellulose: Structure, Modification, and STAFSTROM, J. P. & STAEHELIN, L. A. (1986). Cross- Hydrolysis (ed. R. A. Young & R. Rowell), pp. 77-90. linking patterns in salt-extractable extensin from carrot New York: Wiley & Sons. cell walls. PI. Physiol. 81, 234-241. MILLER, D. H., MELLMAN, I. S., LAMPORT, D. T. A. & MILLER, M. (1974). The chemical composition of the (Received 23 February 1988-Accepted 10 May 1988)

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