J. Mol. Biol. (1975) 96, 431441

Self-assembly of a Plant Cell Wall in vitro-f G. J. HILLS, J. M. PHILLIPS, M. R. GAY AND K. ROBERTS John Innes Inetitute, Colney Lane, Norwich NR4 7UH, England (Received 19 December 1974) A method has been developed by which the cell wall of C?d&nydomonas reXuxdi may be dissociated into its components, and then reassembled in v&o into a product that is chemically and structurally identical to the original cell wall. Chaotropic agents, such as lithium chloride and sodium perchlorate, separate the wall into two fractions, an insoluble amorphous inner wall layer, which retains its integrity (7.5% by weight of the complete wall) and a salt-soluble fraction containing the homogeneous glycoproteins responsible for the outer crystalline layers of the cell wall. Removal of the salt from dissociated walls by dialysis leads to the rapid recovery of complete reassembled cell walls. The conditions necessary for successful reconstitution of the cell wall in vitro include the presence of a suitable surface, across which a decreasing salt gradient exists, and the presence of both the salt-insoluble and the salt-soluble components. The saltsoluble glycoproteins alone can self-assemble under various conditions to form fragments that have the crystalline structure characteristic of the outer layers of the complete cell wall. Both the inner wall layer and the salt-soluble glycoproteins have similar bulk amino acid and sugar (arabinose, galactose, mannose) compositions and both contain hydroxyproline. On the basis of the in v&o reconstitution of the cell wall we discuss certain aspects of in viwo cell wall morphogenesis. This communication describes the first case in which a plant cell wall has been reconstructed in. v&o, and indicates that components of very large cellular structures are capable of being built by a simple self-assembly process.

1. Introduction The study of the assembly processes by which the constituent parts of a cell are built is essential for an understanding of both cell morphogenesis and, ultimately, biological development in general. We have been investigating various aspects of these assembly processes using the cell wall of the green alga Chkz~~y&nno~ reinhurdi (Roberts et al., 1972; Hills, 1973; Hills et al., 1973; Roberts, 1974). There are three main ways in which the problem may be approached. The first uses genetics to analyse the properties of various mutants defective in cell wall assembly (Davies & Plaskitt, 1971; Davies, 1972a,b; Hyams & Davies, 1972). The second involves the analysis of the synthesis and assembly of the wall components in the living cell. In order to understand these processes, the third approach is necessary, the reassembly of the cell wall in vitro, and this forms the subject of this paper. Hopefully, a combination of these three approaches will eventually enable us to understand the factors that control wall formation in the living cell and the rules that govern the assembly. The advantages of the in vitro system that we have developed for the reassembly of t This is the third paper of a series entitled Structure, Composition and Morphogenesis of the Cell Wall of Chlamydvmonas reinhardi. The second paper in the series is Hills et al. (1973). 431

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the cell wall are numerous. Experiments may be done rapidly and under precisely controlled conditions. The effect of modifying various parameters may be quickly monitored, and the assay for the reassembled products is very simple. As a result of our previous studies on algal cell walls (Roberts et al., 1972 ; Hills et al., 1973; Roberts, 1974) we now have a considerable knowledge of the precise structure of the cell wall of C. reinhrdi. The wall comprises several species of highly ordered high molecular weight glycoprotein subunits, which form an essentially two-dimensional crystalline layer around the cell. Between this layer and the plasmalemma of the cell is a thin amorphous wall layer. The whole cell wall has been shown to contain a high proportion of the unusual amino acid hydroxyproline (Roberts et al., 1972 ; Roberts, 1974). Using various image analysis techniques we have now derived a detailed description of the fine structure of the crystalline layer to a resolution of approximately 2.5 nm (Hills et al., 1973), based on negatively stained cell walls and optical and electron diffraction methods. This structural information was considered essential in order to be able to assess the accuracy of the in vitro reassembly process. Previous studies on assembly processes have involved relatively small and simple structures, such as microtubules (reviewed by Borisy et aZ., 1974), viruses (e.g. Kellenberger & Edgar, 1971), bacterial flagella (reviewed by Smith & Koffler, 1971) and bacterial ribosomes (e.g. Fahnestock et al., 1973). The advantage of the algal cell wall system is that the entire assembly process, as well as the finished product, can be monitored in the ordinary light microscope (Hills, 1973). This paper describes the development of a method for the dissoci.ation and reassembly of the cell wall of C. reinhardi by changes in the ionic conditions. The reassembly product has been further characterized by electron microscopy and chemical analysis.

2. Materials and Methods Wild type cells of C. reinhard; were cultured in 500~ml batches of yeast/acetate/peptone medium (Davies & Plaskitt, 1971) placed 1 to 2 m from a continuously illuminated 360 W G.E.C. Solarcolour high pressure sodium lamp. After 2 weeks growth the cell culture was centrifuged at 9000 revs/min for 30 min in 260-ml capacity bottles in the JA14 head of a Beckman 521 centrifuge. The bottles were then carefully removed and the supernatant fluid sucked off, taking care not to disturb the white flocculent layer of cell walls over the pellet of green cells. The walls may be easily and cleanly removed in the remainder of the supernatant fluid, bulked with distilled water and recentrifuged to give a yield (from a 2 week old culture) of 8.1 x lo7 cell walls, or 14 mg dry weight of cell walls, per litre of culture. A cell wall suspension of 1 mg/ml contains 6.8 x lo6 cell walls and corresponds to 0.85 O.D. units of absorbance at 325 nm. Our particular strain of wild type C. rednhmdi may be unusual in the fact that the walls discarded during vegetative growth are not degraded to any appreciable extent. Wild types from other sources (e.g. 11/32a, c and 11/45 of the Culture Centre of Algae and Protozoa, Cambridge) appear to produce far less wall material in the culture medium. Negatively stained specimens were made on 400-mesh copper grids coated with evaporated carbon film stripped from mica. The stains used were either 5% aqueous ammonium molybdate, or 6% sodium tungstate adjusted to pH 6.8 with formic acid. When fixed walls were to be examined by thin sectioning, fixation was carried out according to the method of Franke et al. (1969). We used a JEOL JEMIOOB and an AEI EMBB electron microscope. The optical diffractometer used was of the type described by Home & Markham (1972). Light microscopy was carried out on a Zeiss Photomicroscope. Pre-formed caesium chloride gradients were prepared just before use by layering three 0*7-ml layers of aqueous caesium chloride (densities 1.88, 1.70 and 1.50 g/cm3). Then

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2.5 ml of salt-dissociated walls was floated on the top and centrifuged for 60 min at 24,000

revs/min at 4°C in either the SW39 or the SW41 head of the Beckman model L centrifuge. All solutions, and the centrifuge head, started at room temperature. If a sodium perchlorate dig& was to be centrifuged, 8 06ml layer of sucrose (1.4 g/om3) was positioned between the 1.6 g/cm3 caesium chloride band and the digest. A Beckman model E analytical ultracentrifuge was used. Polyaorylamide gel electrophoreais of the cell wall and its components was done using the procedures described by Roberts (1974). Antiserum to wild type cell walls was raised by 5 weekly injections of 3 mg each of pure cell walls into the marginal ear vein of a rabbit, in the absence of adjuvant. Amino acid analysis was done on a Beckman Spinco model 120 amino acid analyser (Spa&man et al., 1958), and sugars were essayed by gas chromatography of the trimethylsilated derivatives (Robinson & Monsey, 1971).

3. Results and Discussion (a) Di88ociahn

of the cell wall

In order to achieve reassembly of the crystalline component of the cell wall in vitro, it was necessary first to dissociate the wall into its respective components without denaturation. Various methods were tried (pH changes, temperature shifts, and ionic condition changes). Dissociation was followed by measuring the absorbance at 325 nm before and after treatment (Fig. l), and by monitoring the appearance of the walls with phase contrast light microscopy. It was found that reversible dissociation occurred with the following three salt solutions (optimum molarities are given), 5 M-caeaium chloride, 1 M-sodium perchlorate and 8 M-lithium chloride. Lower molarities in each case did not achieve complete dissociation and higher molar&a prevented maximum reassociation. These salts are well known as chaotropic agents and have been used in various systems for the solubilization of protein aggregates (Hetefi & Hanstein, 1974). Wall dissociation in the presence of high concentrations 2.0

I.6

-

275

300

325

320

Wavelengthinm)

FIG. 1. The absorption ml). Curve A, cell walls chloride solution. 29

spectra of a preparation

of wild type cell walls of C. ~einJaar& (04 mg/ of walls in 8 m-lithium

in distilled water: curve R, the same conaentrrttion

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of salt is never complete, an insoluble component remaining, which may be removed by centrifugation for 30 minutes at 12,000 g. Thie component can be just seen under optimal phase dontrast conditions as a wall-shaped structure, but extremely thin compared with the original wall (Hills, 1973). This layer, whioh corresponds to the inner amorphous wall component WI (Roberts et al., 1972) of the wall, is discussed in a later section. The supernatant fluid, after wall dissociation, contains the salt-soluble glycoproteins responsible for the crystalline component of the cell wall (Hills et aE., 1973 ; Roberta, 1974).

Analytical centrifugation of the supernatant fluid from 8 ~-lithium chloridedissociated walls shows the presence of one main sedimenting species at 810,w= 6.8. This sedimentation behaviour is very dependent however on both the concentration of wall material and the exact molarity of the salt used (Plate I(a)). We assume that the fact that two peaks are obtained (Pig. 2 in Hills, 1973) under certain conditions W8ao.w = 6.8 S and 9.3 S) indicates that they contain a common component and that different states of aggregation explain the variability. 5 ar-caesium chloride-dissociated walls, when run on a Spinw model E, revealed one main band floating at 1.379 g/cm3 and a minor component floating at I.391 g/cm3 (Plate I(b) and (c)). The supernatant fluid from caesium chloride-dissociated walls was also ruu in a preparative caesium chloride gradient (Plate I(d)) and revealed one main band (which showed some indication of splitting). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate of both the original supernatant fluid and this main band, compared with whole walls, showed that all the major electrophoretic species in the intact wall are present in the caesium chloride supernatant fluid and can be recovered as one main component on preparative caesium chloride flotation (Plate I(e) to (g)). On the basis of these hydrodynamic studies, we conclude that there is one major morphological component in the salt-soluble fraction of the cell wall. This major morphological component, it appears, may be further fractionated into several electrophoretic species in the presence of sodium dodecyl sulphate (Davies & Lyall, 1973; Hills, 1973; Roberts, 1974). The salt-soluble fraction of the walls contains about 30% (w/w) of protein, of which 18% is hydroxyproline. The amino acid composition of this fraction has already been reported by Roberts (1974). Further work on the chemistry of the salt-soluble glycoprotein is being undertaken. The supernatant fluid of salt-dissociated walls cannot reform intact cell walls after dialysis against water when examined in the light microscope. However, a white flocculent material does appear on dialysis and when this was examined in the electron microscope it was found to be composed of small irregular fragments that clearly showed the typical crystalline structure of intact cell walls (Plate I(h)). This was cordirmed by optical diffraction studies (Plate I(i)). Clearly, therefore, the saltsoluble fraction of the cell wall contains all the components necessary for the assembly of the crystalline layer of the original wall. These crystalline fragment,s, when concentrated by centrifugation at 15,000 g for 30 minutes and washed twice with water, showed identical electrophoretically mobile components on gels, in the presence of sodium dodecyl sulphate, to the control cell walls. An unexplained feature of fragment formation is that only 40% (w/w) of the saltsoluble material is recovered as fragments following dialysis. The remaining material however, is still electrophoretically identical to what has been incorporated into the

PLATES

I-III,

PLATE I. (a) The sedimentation Schlieren pattern of the salt-soluble component of the cell wall in 8 M-lithium chloride solution. The top trace is at a concentration of 1.07 mg/ml and the bottom trace is at one-third dilution, This represents two different concentrations of subunits in the same molarity salt. The picture was taken 40 min after the rotor had attained a speed of 44,770 revs/min in a Beckman model E ultracentrifuge. (b) The Schlieren pattern obtained by running 0.24 mg cell walls in caesium chloride (1.383 g/cm3). The rotor was run for 16 h at 44,770 revs/min. The salt-insoluble wall fraction is at the bottom of the cell and the salt-soluble component is floating as two components, a main band at 1.379 g/cm3 and a minor band at 1.391 g/ems. (c) An ultraviolet absorption trace of the equilibrium run shown in Plate (b), showing the two saltsoluble components. (d) A preparative caesium chloride gradient (36,000 revs/min for 22 h in the SW41 rotor of a Beckman L centrifuge). The salt-soluble fraction of the cell wall floated as one main band, with an indication of splitting. This band, when removed and dialysed in the presence of purified inner wall layers, gave rise to complete reconstituted cell walls. (e) to (g) Polyacrylamide, sodium dodeoyl sulphate gels stained with the periodic acid Schiff reaction. (e) Control wild type cell walls. (f) Reconstituted cell walls obtained from 6 ol-caesium chloride solution. (g) The band removed from the caesium chloride gradient shown in Plate (d). The inner wall layer is seen as material that does not enter the gel in (e) and (f). The salt-soluble fraction accounts for all the electrophoretically mobile components. (h) Fragments obtained by dialysis of the salt-soluble wall component alone, negatively stained in ammonium molybdate (magnification 100,000 x ). Two fragments showing the crystalline lattice structure are present. These are both “single” layer crystals (Hills et aE., 1973). (i) The optical diffraction pattern derived from the larger of the two fragments shown in (h). This is identical to the diffraction pattern obtained from the complete cell wall (Plate III(b)). (j) Phase contrast light micrograph of a very large fragment obtained by the rotary evaporation of the supernatant fluid from a dialysed and centrifuged solution of the salt-soluble wall components, i.e. in effect the glycoprotein subunits in water. Such fragments are many times larger than the original cell wall (magnification 260 x ). (k) Walls, dissociated in 8 M-lithium chloride, run on a preformed caesium chloride gradient (24,000 revs/min for 60 min in the SW39 rotor of a Beckman model L centrifuge). Band 1 represents the salt-soluble glycoprotein subunits. These do not sediment at this speed. Band 2 contains flagellar material. Band 3 contains the small spherical flagella-associated objects shown in II(e). Band 4 contains the inner wall layers. II. (a) Phase contrast light micrograph of purified cell walls (magnification 400 x ). (b) to (d) Time-course of wall reassembly (0, 5 and 10 min dialysis, respectively) taken on the dialysis slide shown in Fig. 3. It should be pointed out that the time zero picture was taken under normal optimal phase contrast conditions, as it was impossible to see the inner wall layers ( +) when using the apparatus shown in Fig. 3. (Magnification 400 x .) (e) Electron micrograph of a rotary shadowed purified inner wall layer (band 4 in Plate I(k)) showing the two attached flagellar collars ( +) and the circular amorphous object (c) mentioned in the text. (Magnification 2600 x .) (f) Phase contrast light micrograph of reconstituted cell walls prepared by dialysis of 8 Mlithium chloride-dissociated cell walls for comparison with the original wall material shown in (a). Magnification 400 x . PLATE

PLATE III. (a) Electron micrograph of the normal negatively stained cell wall. The crystalline lattice is readily visible. Magnification 180,000 x . (b) The optical diffraction pattern obtained from (a). (c) The comparable negatively stained image of the in vitro reconstituted cell wall. Magnification 180,000 x (d) The optical diffraction pattern of the micrograph shown in (c). A comparison with (b) confirms the identity of the reconstituted crystalline layer of the wall with that in the original cell wall. (e) Electron micrograph of a thin section of the purified wild type cell wall. I, Inner amorphous layer of the wall. 0, Outer crystalline layer of the wall. Magnification 100,000. (f) The in vitro reconstituted cell wall (magnification 100,000 x ). The same wall layers are visible. (g) Polyacrylamide gel electrophoresis of the control cell wall in the presence of sodium dodecyl sulphate. (The origin is on the right.) (h) A similar gel to (g) showing the identical bands obtainable from reconstituted cell walls. [facinsa. 434

PLATE

11.

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fragments. Furthermore, the material that remains in solution can be made to produce more crystalline fragments by simple rotq evaporation at room temperature (Plate I(j)). By this method, very large fragments, up to 160 @ &cross, can be assembled. It has been noticed that, although f&me&s from the supernatant fluid of sodium perchlorate-dissociated walls regularly displayed the typical crystalline structure, when using lithium chloride and Mium chloride this was not always the case ; fragments often being obtained with no discernible structure. By electron microscopy we have attempted to visualize both the subunits themselves and the intermediate aggregation states between them and the crystalline lattioe. Taking the supenmtant fluid from salt-dissociated walls after dialysis, we prepared negatively stained specimens (Plate I(h)). Structured fragments were never found that contained less than about 60 unit cells (approx. 093 pmz). Despite intensive searching, we have been unable to demonstrate convincingly the presence of discrete morphological monomeric subunits. No intermediate assembly stages have been visuslized. It would appear, therefore, that fragment formation is by the simple accretion of subunite. The fact that we do not see very small fragments may well be due to their instability in negative stein, resulting from the insu%cient number of crosslinks involved in such a structure. A possible approach to visu&ing structural intermediates in lattice assembly could be based on the observation that the supernatant fluid from salt-dissociated walls shows a characteristic jump in absorbance et 326 nm at a specific point when diluted with water (Pig. 2).

Sodium perchlorate concn (M) FIG. 2. The absorption at 326 run of the sodium perohloreta-solublewsll fraction clone when diluted with wetar. The points plotted repreeent eolutioua that oontain au identical oonoentration of material. The sudden incream in light scattering oomes when the salt ooncantration falls to about 0.4 M.

(c) The salt-insoluble wall component Purificstion of the salt-insoluble wall component (which was originally termed the nucleating agent) (Hills, 1973) has been accomplished by using preformed caesium chloride gradients. Two major bands and one minor band are found on such gradients (Plate I(k)). The densest band (1.74 g/cm3) when removed, added to the purified salt-soluble wall fraction and dialysed, produced intact cell walls as judged by light and electron microscopy. This band contains the inner layer of the wall (Wl) as

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ET AL.

judged by electron microscopy. (The top band contains flagellar remains and flagellar collars (Roberts et al., 1972).) The very high apparent density of the inner layer of the wall is probably due to selective caesium chloride binding as water-washed inner layers still retain bound salt. When examined by phase contrast light microscopy, the inner wall layers could just be detected under ideal conditions as sacs that were of the same size and shape as the complete cell walls, but considerably thinner. Pixed and sectioned inner wall layers revealed them to be identical to the inner amorphous layer (Wl) of the complete cell wall (Roberts et al., 1972), together with the two salt-insoluble flagellar collars that act as specific morphologiaal markers for this wall layer (Plate II(e)). Negative staining confirmed that there was no crystalline component in this layer of the wall. Surface replicas and rotary shadowing again revealed no periodic components (Plate II(e)). Treatment of complete walls with sodium dodecyl sulphate results in the removal of the crystalline outer layers of the wall and the remaining structure is morphologically identical to the inner layer (Wl) of the wall (Roberts et al., 1972). It cannot however be used successfully in wall reconstitution in vitro. The insolubility of the inner wall layer itself is further shown by the fact that sodium dodecyl sulphate digests of the salt-insoluble wall fraction contain no electrophoretically detectable components either by Coomassie blue or by periodic acid Schiff staining, even on 2.5% polyacrylamide gels. Purified inner wall layers were quantitatively recovered from salt-dissociated cell walls. They were washed, dried and shown to account for 7.5% (w/w) of the complete cell wall. They were assayed for both their amino acid and sugar composition (Tables 1 and 2). The assay revealed that the protein component contained 16.4% hydroxyproline, with aspartic acid and glutamic acid as the next most common residues. The inner wall layer is composed of at least 50% carbohydrate and at least 30% protein. The balance is made up by caesium chloride, which had become tightly bound during the isolation procedure. No change in dry weight of the complete wall or the inner wall layer was found following extraction with methanol and ether, suggesting that no lipid is present in the wall. It is both puzzling, and of considerable

TABLE 1 Amino acid composition of the inner layer of Chlamydomonas reinhardi cell walls Amino acid

Protein by m&88 (%)

Amino s&d

Protein by m&s8 (%)

LYE

4.74

Ala

5.06

His 47 AT Thr ser Glu Pro GUY

o-40

6.49 10~06 6.19 5a3 7.72 6.63 3.68

1.91 V&l Met Ile LCCl TV Phe WP

5.70 0.88 3.38 6*86 4.14 4.95 16.40

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TABLE 2

Sugar corpodbn

of the inner layer of Chlamydomonas reinhardi cell waEls

Sugar

Inner cell wall layer by mass

(%I Gal Ara Man

XYl

21.2 18.8

7.6 1.4

The total peroentage of sugars is higher than indicated, some tightly bound caesium chloride.

as the inner wall layer

sampleoont,ained

interest, that the inner wall layer and the salt-soluble wall fraction have very similar amino acid compositions (Roberts, 1974). This similarity also extends to the sugars found in the inner layer, identical sugars being found in approximately the same ratio (Roberts, 1974). We have no explanation for the apparent chemical similarity of the salt-soluble glycoproteins and the salt-insoluble inner wall layer. The third and minor component detected on the preformed caesium chloride gradient (Plate I(k)) was found by electron microscopy to consist of 0.5 pm diameter circular amorphous objects of unknown composition. It is of interest that similar objects have been found consistently in inner wall layer preparations (Plate II(e)) and in the complete cell wall, usually in close association with the basal end of the flagellar collars. Their possible role in the cell wall and its assembly remains obscure. (d) The in vitro reconstitution of the cmplete cell wall Following successful in vitro reassembly experiments (Hills, 1973), some more details of the precise conditions necessary are now discussed, together with the characterization of the reassembled product. Dialysis of salt-dissociated cell walls against water results rapidly in the formation of products which, under the light microscope, are similar to the original walls of the starting material (Plate II(a) and (f)). Neither the salt-soluble fraction on its own, nor the insoluble inner wall layers on their own (e.g. when removed by Millipore filtration, pore size l-2 pm) are capable of reforming complete cell walls. If both components are put back together again and dialysed, wall reassembly occurs. These reconstituted cell walls have been shown by various methods to be identical to the original walls. Negatively stained specimens of reconstituted walls reveal the same crystalline component (Plate III(c)) as is found in normal walls (Plate III(a)) and this was confirmed by a comparison of their optical diffraction patterns (Plates III(b) and (d)). Spun, washed, fixed (Franke et al., 1969) and sectioned reassembled walls (Plate III(f)) showed the same main wall layers (Plate III(e)) as the original walls (Roberts et al., 1972). The outer layer (W7) was barely present, however, and this fact coupled with a re-examination of walls from cells grown under a variety of conditions has suggested the possibility that this layer may not be a true constant component of the cell walls. The essential point is that the sectioned reassembled walls revealed an inner wall layer (Wl) together with an outer crystalline triplet layer (W2-W6), the arrangement found in normal walls.

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Spun and washed reassembled walls showed identical electrophoretic components following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and staining with Coomessie blue or periodic acid Schiff (Plate III(g) and ON. Preliminary experiments using antiserum raised against purified normal cell walls showed them to be antigenically similar to reassembled cell walls by agglutination tests. The antiserum showed no cross reactions with CAalamycEomona.s me-w&i or CMn-ogoniurn elonga&n cell walls, both closely related species but having a different crystalline lattice in their walls (Roberts, 1974). Not all of the salt-dissociated wall material is recovered in the form of cell walls. The dry weight recovery of reassembled cell walls following dialysis is about 35%. However, the subunits remaining in solution can be assembled onto purified additional inner wall layers to give a final wall recovery by number of about 9Oo/o.In other words, the reassembly process is being limited by the availability of intact inner wall layers. We attribute the low initial wall recovery to the fact that the original cell wall preparation has been obtained from a cell culture and it contains a mixture of whole walls and broken walls. Both of these will produce soluble subunits on salt dissociation but only the intact walls will produce complete inner wall layers capable of participating in the reassembly in vitro to give intact cell walls.

FIG. 3. A diagram showing the experimental arrangement used for the light miorosoope obser. vations of the sequenoe of in YiLro reassembly of cell walls during dialysis. 1, Cover&p; 2, saltdissoaiated 0011 walls; 3, square of dialysis membrane; 4, distilled water; 6, “well” miorosoope slide.

We have followed the time-course of wall reassembly under the phase contrast microscope. This was made possible by the simple dialysis arrangement shown in Pigure 3, essentially a sandwich of dialysis membrane between water and saltdissociated walls, Walls gradually appear in a matter of minutes (inner wall layers are too thin to detect with this technique). Wall reassembly does not appear to be a synchronous process, some walls being completed before others have appeared at all. Photographs of the reassembly process, after different times of dialysis, are shown in Plate II(b) to (d). An investigation into the conditions that are essential for the reassembly of the cell wall by dialysis was undertaken. Our initial idea was that the dialysis was equivalent simply to salt removal, i.e. dilution. However an 8 ns-lithium chloride digest of cell walls, when diluted to 2 Y with water, produces no walls, even though walls are normally not dissociated by 2 ~-lithium chloride itself. Dilution rate is also not significant, as an experiment set up using a motor driven Agla syringe to match the dilution rate exactly with that found experimentally during dialysis was also unsuccessful. We therefore turned our attention to the dialysis sac itself’. Modification of the dialysis sac (turning it inside out, modifying the pore size by acetylation)

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did not affect the ability to reconstitute cell walls. However, when chopped pieces of dialysis sac were added to salt-dissociated walls, even when accompanied by dilution, we observed no wall reassembly at all, thus ruling out surface effects associated with the didysis sac. As the dialysis membrane itself did not appear to be of importance in the reassembly prooess, we therefore asked whether we could bring about cell wall reassembly by any other method. An 8 r+lithium chloride digest of cell walls, either when placed in a cavity in a 2% agar block, or as a drop on a Mill&ore filter flo&ng on water, produced reconstituted walls. Again, it is not the nature of the surface itself that is of importance, as chopped agar or Millipore filters have no effect on a salt-dissociated wall digest at all. We can further conclude that hydrostatic pressure within the dialysis sac is unimportant, as dialysis in an open topped dialysis sac is successful. We also conclude that the rate of dialysis is significant, as a small piece of dialysis membrane tied over the end of a tube filled with salt-dissociated walls and immersed in water, does not allow wall resssembly, presumably because the dialysis rate across such a small area is too slow. It seems from these experiments that the essential requirements for the reassocitbtion of a salt-dissociated cell wall are (a) a decreasing salt gradient set up at an interface (b) a physical surface of the right kind. Unlike many other self-assembly systems, that described here does not seem to be dependent on the presence of ape&c cations such as magnesium or calcium. Cell wall reassembly takes place normally even in the presence of O-06 M-EDTA or O-05 MEGTA. The reaction also appears to be relatively insensitive to pH changes. Follow. ing dialysis against phosphate, Tris or acetate buffers between pH 46 and pH 8.5, we found no detectable differences in the final recovery of rmembled cell walls. The system we have described for cell wall assembly in vitro will be of great value in the understanding of the various cell wall mutants that are available (Davies & Plaskitt, 1971; Hyams & Davies, 1972). One mutant we have examined illustrates the sort of information obtaitinable. K3, a strain of CW18 (Davies & Lyall, 1973) produces the typical eleotrophoretic wall components of the wild type, but does not form a cell wall. Salt soluble subunits were pm&xl from the growth medium, taken up in 8 az-LiCl, and were dialysed in the presence of inner wall layers purified from wild type walls. Normal cell walls were produced, suggesting that K3 was deficient in some aspect of its inner wall layer. (e) The mammbly process On the basis of what we now know about the in vitro reassembly of the cell wall, coupled with the information we have about its detailed structure, we can make some suggestions about how the cell walls may be assembled in viva. Basically there are two ways in which the main layers of the cell wall could be laid down. The first of these involves the initial deposition of the crystalline triplet lrtyer, followed by the later accretion of the inner wall layer, a model proposed by Davies & Lyall (1973). The second possibility, essentially the reverse of the former suggestion, involves first laying down the inner wall layer around the daughter protoplasts, within the mother cell wall, followed by the attachment of the crystalline triplet layer onto the outer surface of this inner wall layer. Two main lines of evidence lead us to support the latter model of cell wall assembly in ,vivo. The main point is that, in the in v&o system, the production of a complete cell wall is completely contingent on the presence

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of an intact inner wall layer. In no circumstance can one build a wall-shaped reassembly product from the crystalline lattice components alone. The second line of evidence comes from our initial sectioning experiments. Sections of dividing cells indicate that the first layer of the wall to appear around the daughter protoplasts within the mother cell wall is the amorphous inner wall layer. The fact that sections very rarely reveal intermediate stages of wall assembly means that the in vivo process itself must be very fast. In this connection the evidence from the cell wall mutants is equivocal, as we have no firm clues as to which wall layer is defective in any one mutant. However, K3 (Davies & Lyall, 1973) appears to produce normal crystalline lattice components but is unable to form a cell wall unless a wild type inner wall layer is provided. What the in vitro experiments do not provide us with is any indication of whether the crystalline layer of the wall (W2-W6) is attached to the inner wall layer by the strict sequential addition of monomers, or whether fragments of lattice attach and then grow laterally to cover the surface. Two further points are of interest here. One is the presence in both the original cell walls, and the reconstituted walls, of dislocations within the crystalline lattice layer. It has been suggested that these act as the sites of addition of further subunits when the wall increases in area during cell growth (Roberts, 1974). The fact that during ila vitro cell wall reassembly the wall density becomes evenly and progressively more pronounced in the phase contrast microscope, and does not show localized increases in density, is strong evidence in favour of multiple sites of subunit attachment onto the inner wall layer (Plate II(b) to (d)). The second point is that during cell growth the cryst)alline layer of the wall can expand by simple addition of monomers at dislocations, but this must be accompanied by a corresponding growth of the amorphous inner wall layer. How the overall growth of the wall is programmed to match the growth of the cell, when the wall is not in contact with the plasmalemma, is difficult to understand. The inner wall layer as isolated, it must be realized, is not a simple closed sphere. Originating as it does from a discarded mother cell wall it inevitably has a split within it, and so consequently reassembled walls also have a split in them. Also present, attached to the inner wall layer, are the two salt-insoluble flagellar collars, described in detail by Roberts et al. (1975), which confer a bilateral symmetry to the wall. Whether or not the two new flagella’r collars that must’ be made for each daughter cell form the foci for the subsequent xssembly of the inner wall layer is not yet known. Considerable evidence now exists supporting the presence of glpcoproteins containing hydroxyproline in plant cell walls (Lamport, 1970; Miller et al.. 1974) and speculation exists concerning its structural and functiona. role. From our observations presented here, in which hydroxyproline is found in both the sa,ltinsoluble and the salt-soluble fractions of the cell wall, we would conclude that no direct common structural role can be attributed to the hydroxyproline-rich protein component. Although the amino acid composition is very similar for both fractions, the inner wall layer is composed of an insoluble amorphous fibrous matrix and the outer wa.11layer is composed of non-covalently bound glycoprotein subunits. It is important to point out here the fact that the method of a,ssembly of this algal cell wall appears to bea’r no similarity to the assembly of higher plant cell walls. being in fact more akin to that found in bacteria. The walls of several Gram negative bacteria. are particularly analogous. in possessing an ordered array of subunits on their external surface. However, the subunits in this case appear to be simple globucations are essential for their assembly (Thornr et r/l.. lar proteins, and divalent

SELF-ASSEMBLY

OF CHLAMYDOMUNAS

CELL

WALLS

441

1975). The case of Chblanaydonsonasis the first in which any plant cell wall has been reconstructed in vitro, and emphasizes that components of very large biological structures may be built by a simple self-assembly process. In order to understand the assembly of the Chla~ydon~~nas cell wall in greater detail, we need to be able to fractionate and characterize the individual components of the cell wall and to know more about its three-dimensional fine structure. Work is in progress on both of these fronts. We should like to thank M. W. Rees of this Institute for the amino acid analyses described in this paper, D. S. Robinson of the Food Research Institute, Norwich, for kindly performing the gas chromatography for the sugar analyses, and Professor D. R. Davies, also of this Institute, for advice and encouragement. REFERENCES Borisy, G. G., Olmsted, J. B., Marcum, J. M. & Allen, C. (1974). Fed. Proc. Fed. Amer. Sot. Exp. Biol. 33, 167-174. Davies, D. R. (1972a). Exp. Cell Res. 73, 512-516. Davies, D. R. (1972b). Mol. Gen. Genet. 115, 334-348. Davies, D. R. & Lyall, V. (1973). Mol. Gen. Genet. 124, 21-34. Davies, D. R. & Plsskitt, A. (1971). Gent. Rtx. 17, 33-43. Fahnestock, S., Held, W., & Nomura, M. (1973). John InnesSymp. 1, 179-217. Franke, W. W., Krien, S. & Brown, R. M. (1969). Hietochemie, 19, 162-164. Hate& Y. & Hanstein, W. G. (1974). Methods in EnzymoZogy, 31, 770-790. Hills, G. J. (1973). Planta, 115, 17-23. Hills, G. J., Gurney-Smith, M. & Roberts, K. (1973). J. UEtraatit. Res. 43, 179-192. Horne, R. W. & Markham, R. (1972). In Practical Methods in Electron Microscopy (Glauerb, A. M., ed.), vol. 1 (pt 2), pp. 327-434. North-Holland, Amsterdam. Hyams, J. & Davies, D. R. (1972). Mutat. Rea. 14, 381-389. Kellenberger, E. & Edgar, R. S. (1971). In The Phage Lambda (Hershey, A. D., ed.), pp. 271-295, Cold Spring Harbor Laboratory. Lamport, D. T. A. (1970). Annu. Rev. Plant. Physiol. 21, 235-270. Miller, D. H., Mellman, I. S., Lamport, D. T. A. & Miller, M. J. (1974). J. Cell BioZ. 63, 420-429. Roberts, K. (1974). PhiZ. Trans. Roy. Sot. Land. 368, 129-146. Rea. 40, 599-613. Roberts, K., Gurney-Smith, M. & Hills, G. J. (1972). J. UZkmtruct. Roberts, K., Phillips, J. M. & Hills, G. J. (1975). Micron, in the press. Robinson, D. S. & Monsey, J. B. (1971). B&hem. J. 121, 537-547. Smith, R. W. & Koffler, H. (1971). Advan. Microbial PhysioZ. 6, 219-339. Spackman, D. H., Stein, W. H. & Moore, S. (1958). Anal. Chem. 30, 1190-1206. Thorne, K. J. I., Thornley, M. J., Naisbitt, P. & Glauert, A. M. (1975). Biochim. Biophys. Acta, 389, 97-116.