Chlamydomonas Reinhardtii Cells Research Articles

Inositol phospholipid metabolism may trigger flagellar excision in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii cells shed their flagella in response to environmental stress. Under favorable conditions, flagella are quickly regrown. To learn more about the signals that trigger flagellar excision and regrowth we have investigated inositol phospholipid metabolites, molecules implicated in signal transduction in several other systems. After deflagellation by low pH or mastoparan, a potent activator of G proteins, there was a rapid increase in levels of inositol 1,4,5-trisphosphate measured by use of receptor-binding assays and HPLC. This increase was concomitant with a decrease in levels of phosphatidylinositol 4,5-bisphosphate and was followed by an increase in phosphatidic acid, results consistent with activation of phospholipase C and diacylglycerol kinase. Additional experiments suggest that this activated phospholipase C is not important for flagellar regrowth but plays a role in informing the excision apparatus of the environmental stress. Addition of neomycin (an inhibitor of phospholipase C) before exposure of cells to low pH or mastoparan prevented the increase in inositol 1,4,5-trisphosphate and also prevented deflagellation. Addition of neomycin after deflagellation blocked increases in inositol 1,4,5-trisphosphate that normally followed deflagellation, but did not block flagellar assembly. Furthermore, a flagellar excision-defective mutant, fa-1, did not shed its flagella in response to low pH or mastoparan, yet both of these agents activated phospholipase C in these cells. The results suggest that activation of phospholipase C, possibly via a G protein, is a proximal step in the signal transduction pathway inducing deflagellation in Chlamydomonas.

Actin dynamics during the cell cycle in Chlamydomonas reinhardtii.

We have used two monoclonal antibodies to demonstrate the presence and localization of actin in interphase and mitotic vegetative cells of the green alga Chlamydomonas reinhardtii. Commercially available monoclonal antibodies raised against smooth muscle actin (Lessard: Cell Motil. Cytoskeleton 10:349-362, 1988; Lin: Proc. Natl. Acad. Sci. USA 78:2335-2339, 1981) identify Chlamydomonas actin as a approximately 43,000-M(r) protein by Western immunoblot procedures. In an earlier study, Detmers and coworkers (Cell Motil. 5:415-430, 1985) first identified Chlamydomonas actin using NBD-phallacidin and an antibody raised against Dictyostelium actin; they demonstrated that F-actin is localized in the fertilization tubule of mating gametes. Here, we show by immunofluorescence that vegetative Chlamydomonas cells have an array of actin that surrounds the nucleus in interphase cells and undergoes dramatic reorganization during mitosis and cytokinesis. This includes the following: reorganization of actin to the anterior of the cell during preprophase; the formation of a cruciate actin band in prophase; reorganization to a single anterior actin band in metaphase; rearrangement forming a focus of actin anterior to the metaphase plate; reextension of the actin band in anaphase; presence of actin in the forming cleavage furrow during telophase and cytokinesis; and finally reestablishment of the interphase actin array. The studies presented here do not allow us to discriminate between G and F-actin. None the less, our observations, demonstrating dynamic reorganization of actin during the cell cycle, suggest a role for actin that may include the movement of basal bodies toward the spindle poles in mitosis and the formation of the cleavage furrow during cytokinesis.

An equilibrium model for electron transfer in photosystem II acceptor complex: an application to Chlamydomonas reinhardtii cells of D1 mutants and those treated with formate

La decomposition des emissions de fluorescence de la chlorophylle a des systemes photosynthetiques apres un flash unique saturant est d'habitude analysee par trois ou quatre exponentielles, qui sont en relation avec les differents modes de reoxidation du [Q⁻A]. Ce modele est appele le modele independant de decomposition. Dans la presente publication nous avons teste ces decompositions des emissions de fluorescence chez 'Chlamydomonas reinhardtii' (type sauvage et cinq mutants) d'une maniere differente que nous appellerons le modele d'equilibre, car nous tenons compte des reactions d'equilibres apparents entre le Q⁻AQB et QAQ⁻B et, en plus, d'un flux d'electrons quittant le complexe QAQB. Ce modele suppose que les constantes de vitesse de liaison et de degagement de la plastoquinone s'aneantissent et n'influencent pas les resultats. Cette analyse nous permet la prediction du temps de vie moyen pour le transport d'electrons en avant (τab) du Q⁻A vers le QB et le Q⁻B, la constante d'equilibre apparente (kab/kba), le temps de vie moyen (τPQ) d'electrons quittant le PS II, essentiellement vers le pool de la plastoquinone (PQ) ainsi que le rapport entre les centres rapides et lents du photosysteme II. Nous presenterons en plus une comparaison de ces resultats avec ceux obtenus par le modele independant (Govindjee et al. 1992). Du fait que dans la presente publication les trois parametres independants (τab, τba et τPQ) sont obtenus a partir d'une seule mesure de decomposition des emissions de fluorescence de la chl a, nous presentons nos resultats pour qu'ils puissent etre verifies par d'autres mesures et methodes. Si l'on suppose que le parametre de transfert d'energie (p) entre les systemes est de 0.5, les mutants Dl Ar-207 (F255Y), Br-202 (L175F)et Dr-18 (V219I) montrent un transfert d'energie en avant (QAQB⁽⁻⁾ → Q⁻AQ⁻B⁽⁼⁾ pratiquement inchange (temps de vie moyen τab = 600 - 950 μs) et, en plus, un rapport des centres reactionnels du PS II rapides et lents pratiquement egale de 0.1 a 0.2 en comparaison avec le type sauvage. Par contre, chez les mutants Ar-204 (G256D) et DCMU-4 (S264A), on observe un temps de vie moyen du transport d'electrons en avant augmente plusieurs fois (τab ≈ 2'000 μs), des constantes d'equilibre apparentes du Q⁻AQB = QAQ⁻B modifiees (la plus faible chez S264A et G256D, intermediaire chez V219I et la plus elevee chez F255Y), et des rapports exceptionnellement eleves (0.6 - 0.8) de centres reactionnels rapides et lents du PS II. L'adjonction de formiate a 100 ou 200 mM provoque une augmentation du temps de vie du transport d'electrons en avant (Q⁻A vers QB) chez le type sauvage de 640 a 990 μs ainsi qu'aux cinq mutants testes. Le plus grand changement est observe chez S264A (de 1.4 a ~30 ms), le plus petit chez L275F (de 950 μs a 1.4 ms). Le traitement au formiate (appauvrissement en bicarbonate) conduit a une augmentation du temps de vie pour le transport d'electrons vers le pool de la PQ de 1.5 a 5 fois selon le mutant, une diminution des constantes d'equilibre apparentes de la reaction Q⁻AQB = QAQ⁻B d'un facteur de 2 a 6 ainsi qu'a une augmentation remarquable en centres lents. Tous ces effets sont reversibles par le bicarbonate.

Changes in Photorespiratory Enzyme Activity in Response to Limiting CO2 in Chlamydomonas reinhardtii

The activity of two photorespiratory enzymes, phosphoglycolate phosphatase (PGPase) and glycolate dehydrogenase (glycolate DH), changes when CO(2)-enriched wild-type (WT) Chlamydomonas reinhardtii cells are transferred to air levels of CO(2). Adaptation to air levels of CO(2) by Chlamydomonas involves induction of a CO(2)-concentrating mechanism (CCM) which increases the internal inorganic carbon concentration and suppresses oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. PGPase in cell extracts shows a transient increase in activity that reaches a maximum 3 to 5 hours after transfer and then declines to the original level within 48 hours. The decline in PGPase activity begins at about the time that physiological evidence indicates the CCM is approaching maximal activity. Glycolate DH activity in 24 hour air-adapted WT cells is double that seen in CO(2)-enriched cells. Unlike WT, the high-CO(2)-requiring mutant, cia-5, does not respond to limiting CO(2) conditions: it does not induce any known aspects of the CCM and it does not show changes in PGPase or glycolate DH activities. Other known mutants of the CCM show patterns of PGPase and glycolate DH activity after transfer to limiting CO(2) which are different from WT and cia-5 but which are consistent with changes in activity being initiated by the same factor that induces the CCM, although secondary regulation must also be involved.

Glycolate photoproduction by free and alginate-entrapped cells of Chlamydomonas reinhardtii

Chlamydomonas reinhardtii cells provide an effective system for glycolate photoproduction, operative during 30 h when they are growing under low CO2, in the presence of 1 mM aminooxyacetate and 50 μM acetazolamide. Glycolate excretion by the cells can proceed for about 4 days if every other 12 h a high CO2 level is restored in the culture in the absence of inhibitors. The immobilized system in alginate beads has about a twofold higher glycolate photoproduction rate (23 μmol·mg chlorophyll (Chl)−1·h−1) than free-living cells (12 μmol · mg Chl−1 · h−1).

Molecular studies of linkage group XIX of Chlamydomonas reinhardtii: evidence against a basal body location.

Linkage group XIX (also known as the UNI linkage group) in the green alga, Chlamydomonas reinhardtii, exhibits a number of unusual properties that have lead to the suggestion that it represents a basal body-associated chromosome. To begin a molecular analysis of this linkage group, we have identified DNA sequences from it and used them to determine the copy number of linkage group XIX within the cell. We find that linkage group XIX is present in the same copy number per cell as nuclear linkage groups in both haploid and diploid strains. We also find that the copy number of linkage group XIX is unchanged in mutants lacking basal bodies. We conclude that there is no convincing evidence that linkage group XIX localizes to the basal bodies of Chlamydomonas reinhardtii cells.

Regulation of molybdenum cofactor species in the green alga Chlamydomonas reinhardtii

Molybdenum cofactor (MoCo) of molybdoenzymes is constitutively produced in cells of the green alga Chlamydomonas reinhardtii grown in ammonium media, under which conditions certain molybdoenzymes are not synthesized. In soluble form, MoCo was found to be present in several forms: (i) as a low Mr free species; (ii) bound to a MoCo-carrier protein of about 50 kDa that could release MoCo to directly reconstitute in vitro nitrate reductase activity in the nit-1 mutant of Neurospora crassa, but not to Thiol-Sepharose which, in contrast, bonded free MoCo; and (iii) bound to other proteins, putatively constitutive molybdoenzymes, which only released MoCo after a denaturing treatment. The amount of total MoCo (free, carrier-bound and heat releasable forms) was dependent on the growth phase of cell cultures. Constitutive levels of total MoCo in ammonium-grown cells markedly increased when cells were transferred to media lacking ammonium (nitrate, urea or nitrogen-free media). This increase did not require de novo protein synthesis and was stimulated by light. Levels of both total MoCo and free plus carrier-bound MoCo seemed to be unrelated to either nitrate reductase synthesis or functioning of nit-1 and nit-2 genes responsible for nitrate reductase structure and regulation, respectively. Results suggest that MoCo is continuously synthesized in C. reinhardtii and that its levels are regulated by ammonium in a way independent of nitrate reductase synthesis.

Spectral sensitivity, structure and activation of eukaryotic rhodopsins: Activation spectroscopy of rhodopsin analogs in Chlamydomonas

Retinal normally binds opsin forming the chromophore of the visual pigment, rhodopsin. In this investigation synthetic analogs were bound by the opsin of living cells of the alga Chlamydomonas reinhardtii; the effect was assayed by phototaxis to give an activation spectrum for each rhodopsin analog. The results show the influence of different chromophores and the protein on the absorption of light. The maxima of the phototaxis action spectra shifted systematically with the number of double bonds conjugated with the imine (CN +H) bond of the chromophore. Chromophores lacking a β-ionone ring, methyl groups and all CC double bonds photoactivated the rhodopsin of Chlamydomonas with normal efficiency. On the basis of a simple model involving one-electron transitions between occupied and virtual molecular orbitals, we estimate the charge distribution along the chromophore in the binding site. With this restraint we define a unique structural model for eukaryotic rhodopsins and explain the spectral clustering of pigments, the spectral differences between red and green rhodopsins and the molecular basis of color blindness. Our results are consistent with the triggering of the activation of rhodopsin by the light-mediated change in electric dipole moment rather than the steric cis—trans isomerization of the chromophore.

Distinction between Hypoxanthine and Xanthine Transport in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii cells consumed hypoxanthine and xanthine by means of active systems which promoted purine intracellular accumulation against a high concentration gradient. Both uptake and accumulation were also observed in mutant strains lacking xanthine dehydrogenase activity. Xanthine and hypoxanthine uptake systems exhibited very similar Michaelis constants for transport and pH values, and both systems were induced by either hypoxanthine or xanthine. However, they differed greatly in the length of the lag phase before uptake induction, which was longer for hypoxanthine than for xanthine. Cells grown on ammonium and transferred to hypoxanthine media consumed xanthine before hypoxanthine, whereas cells transferred to xanthine media did not take up hypoxanthine until 2 hours after commencing xanthine consumption. Metabolic and photosynthetic inhibitors such as 2,4-dinitrophenol, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, and carbonylcyanide m-chlorophenylhydrazone inhibited to a different extent the hypoxanthine and xanthine uptake. Similarly, N-ethylmaleimide abolished xanthine uptake but slightly affected that of hypoxanthine. Hypoxanthine consumption was inhibited by adenine and guanine whereas that of xanthine was inhibited only by urate. We conclude that hypoxanthine and xanthine in C. reinhardtii are taken up by different active transport systems which work independently of the intracellular enzymatic oxidation of these purines.

A rapid procedure for the isolation of chloroplast DNA fromChlamydomonas using the TL-100 ultracentrifuge

We describe a rapid and efficient procedure for the isolation of chloroplast (and nuclear) DNA from walled cells ofChlamydomonas reinhardtii. Total nucleic acids are prepared and separated by equilibrium centrifugation in CsCl-bisbenzimide gradients using a high-speed table-top ultracentrifuge. Chloroplast DNA sufficient for several restriction analysis is obtained from 1 liter of cells in one to two days.

Absence of DNA in the basal body ofChlamydomonas reinhardtii by fluorimetry using a video-intensified microscope photon-counting system

A search was made for DNA in both the basal bodies (BBs) in situ and BBs isolated from cells ofChlamydomonas reinhardtii by high-resolution epifluorescence microscopy after staining with 4′-6-diamidino-2-phenylindole (DAPI), by fluorimetry using a video-intensified microscope photon-counting system (VIMPICS) and by immunofluorescence microscopy after staining with a monoclonal tubulin-specific antibody. The flagella and intracellular microtubules radiate from the BBs. The BBs in young vegetative cells, gametes and young zygotes do not emit fluorescence after staining with DAPI but the spherical cell nucleus, the ovoid chloroplast nuclei and the tiny mitochondrial nuclei emit bright, blue-white fluorescence. Thus, it appears that BBs do not contain larger amounts of DNA than do the other organelles. To avoid the halation effects of fluorescence from the cell debris and cytoplasm and to measure carefully any extremely low levels of DNA that might be present in the organelles, a complex, composed of two flagella, a pair of BBs and the cell nucleus, was isolated from the gametes by treatment with autolysin and 0.1% Triton X-100. After staining with DAPI, the BBs of such complexes exhibit faint fluorescence while the cell nucleus emits strong fluorescence. The point and total intensities of the fluorescence emitted from each portion of the complex were measured with the VIMPICS. When the fluorescence intensity “T” of T 4 phage is taken as a standard, the fluorescence intensities of the flagella, the pair of BBs, the cell nucleus and the nucleus ofEscherichia coli are respectively 0.2 T, 0.40 T, 1452.2 T and 20.4 T. The slight fluorescence emitted from the BB seems to be due to the halation of the fluorescence emitted from the cell nucleus. The intensity of the fluorescence from the BBs is reduced to the intensity of the fluorescence of the flagella when the cell nucleus is removed from the complex. From these results, we conclude that the BBs do not contain DNA. Discrepancies related to the reported presence of DNA in the BBs are discussed.

High-frequency nuclear transformation of Chlamydomonas reinhardtii.

By using a method in which cell-wall-deficient Chlamydomonas reinhardtii cells were agitated in the presence of DNA, glass beads, and polyethylene glycol, nuclear transformation rates of approximately 10(3) transformants per micrograms of plasmid DNA were achieved. The nitrate reductase gene from wild-type Chlamydomonas was used to complement a mutation in the corresponding gene of a strain containing nit1-305. Transformants were selected by growth with nitrate as sole source of nitrogen. The transforming DNA integrated into the genome at a low-copy number in nit+ transformants. When cells carrying nit1-305 were agitated in the presence of two plasmids, one with the gene for nitrate reductase and the second with an unselected gene, the unselected gene was present in 10-50% of nit+ transformants. This high frequency of cotransformation will allow any cloned gene to be introduced into Chlamydomonas. Moreover, the overall efficiency of transformation should be high enough to permit isolation of genes from genomic libraries by complementation of stable nuclear mutants. The availability of efficient nuclear and chloroplast transformation in Chlamydomonas provides specific advantages for the study of chloroplast biogenesis, photosynthesis, and nuclear-chloroplast genome interactions.

Photoproduction of ammonium by Chlamydomonas reinhardtii cells immobilized in barium alginate: a reactor feasibility study

Chlamydomonas reinhardtii cells immobilized in Ba-alginate beads provide a stable and effective system for photoproducing ammonium from nitrite in a culture medium containing l-methionine-d,l-sulphoximine. The process was studied in either air-lift, fluidized or packed-bed reactors, the last one providing the most suitable system with a volumetric activity of 2700 μmol NH inf+ sup4 ·1−1 per hour.

Fluorescence of the reaction centre of photosystem II in cells of green alga Chlamydomonas reinhardtii

Cells of green alga Chlamydomonas reinhardtii, mutant ASS66, which only exhibit the spectra and polypeptide composition of photosystem II, possess a weak fluorescence at about 673 nm along with two main emission bands at 686 and 698 nm, after cultivation in the dark. The 673 nm fluorescence exhibits an excitation spectrum similar to the absorbance of the isolated reaction centre of photosystem II (D1/D2 protein complex). It contains chlorophyll a, pheophytin a, and carotenoid bands with the chlorophyll:pheophytin ratio of about 3. Under illumination, the intensity of the 673 nm emission band decreases with concomitant increase in the emission band at 698 nm, which is thought to belong to the core antenna of photosystem II. Under illumination, in the presence of dithionite (strong reducing conditions), the relative intensity of 673 to 686 nm emission does not change, but the intensities of the pheophytin bands in its excitation spectrum were reduced two-fold. That the relative intensity of the 673 nm band in the emission spectrum under illumination in the presence of dithionite does not change indicates that it originates from an emitter other than pheophytin (possibly reaction centre chlorophyll a).

The nuclear‐coded chloroplast 22‐kDa heat‐shock protein of Chlamydomonas

A cDNA clone, pCHS62, was isolated using poly(A)-rich RNA from heat-shocked Chlamydomonas reinhardtii cells. The clone has a length of 1.1 kb and codes for the complete heat-shock protein which was reported to be associated with the grana region of the thylakoid membranes and ascribes protection against photoinhibition during heat-shock. An expression vector prepared in the pUC19 plasmid was used to obtain a fusion protein against which rabbit polyclonal antibodies have been raised. The antibodies react specifically with the heat-shock protein of 22 kDa synthesized in vivo during heat-shock, which is localized in the grana thylakoids, with the in vitro translated product using poly(A)-rich RNA from heat-treated cells as well as with the hybrid release translation product of the pCHS62 clone. The clone was sequenced. It contains a 5' region consisting of 85 nucleotides, an open reading frame of 471 nucleotides and a non-coding 3' region of 600 nucleotides. Northern hybridization indicates a length of 1.7 kb for the messenger RNA of heat-shock protein 22. Analysis of similarity between the derived amino acid sequence of this protein and other heat-shock proteins demonstrates that this protein belongs to the small-molecular-mass plant heat-shock protein family and also shows similarities with animal heat-shock proteins including the presence of a short region possessing similarity with bovine alpha-crystalline as reported for other heat-shock proteins. The molecular mass of the protein as determined from the sequence is 16.8 kDa. Despite its localization in the chloroplast membranes, it does not seem to include a transit peptide sequence, in agreement with previous data. The sequence contains only a short hydrophobic region compatible with its previously reported localization as a thylakoid extrinsic protein.

Ammonium photoproduction by free and immobilized cells of Chlamydomonas reinhardtii

Free-living or immobilized Chlamydomonas reinhardtii cells photoproduce ammonium from nitrite in a medium containing 1 mM of l-methionine-d,l-sulphoximine (MSX). Ammonium is accumulated in the medium to 8 mM final concentration, which inhibits nitrite uptake by the MSX-treated cells and consequently the excretion of ammonium is blocked. However, if ammonium was removed from the medium and nitrite and MSX periodically restored, the photoproduction process could be maintained over 96 h, with a final ammonium concentration of about 18 mM for free-living cells and 28 mM for immobilized ones. The MSX-treated cells showed a photoproduction productivity of 1300 μmol NH4+· mg chlorophyll (Chl)-1, with an average production rate of 14 μmol NH4+· mg Chl-1 per hour, for calcium alginate-entrapped cells, while the corresponding data for free-living ones was 650 μmol NH4+· mg Chl-1 and 6.7 μmol NH4+· mg Chl-1 per hour, respectively. Immobilized cells showed a significant increase in the nitrite uptake rate, probably due to a change in membrane permeability as a consequence of cell-matrix interactions.

Genetic analysis of long-flagella mutants of Chlamydomonas reinhardtii.

The length of the flagella of Chlamydomonas reinhardtii cells is tightly regulated; both short-flagella and long-flagella mutants have been described. This report characterizes ten long-flagella mutants, including five newly isolated mutants, to determine the number of different loci conferring this phenotype, and to study interactions of mutants at different loci. The mutants, each of which was recessive in heterozygous diploids with wild type, fall into three unlinked complementation groups. One of these defines a new gene, lf3, which maps near the centromere of linkage group I. The flagellar length distributions in populations of each mutant were broad, with the longest flagella measuring four times the length of the longest flagella seen on wild-type cells. Each of the ten mutants had defective flagellar regrowth after amputation. Some of the mutants showed no regrowth within the time required for wild-type cells to regenerate flagella completely. Other mutants had subpopulations with rapid regeneration kinetics, and subpopulations with no observable regeneration. The mutants were each crossed to wild type to form temporary quadriflagellate, dikaryon cells; in each case the long flagella were rapidly shortened in the presence of the wild-type cytoplasm, demonstrating that the mutants were recessive, and that length control could be exerted on already assembled flagella.

Tubulin gene expression in the Chlamydomonas reinhardtii cell cycle: elimination of environmentally induced artifacts and the measurement of tubulin mRNA levels.

To investigate the involvement of tubulin gene expression in controlling cell division events in Chlamydomonas reinhardtii we have measured tubulin mRNA levels during the cell cycle under different environmental conditions. In C. reinhardtii cells grown under the synchronizing conditions of 14 h of light followed by 10 h of darkness, mRNAs for tubulin and associated flagellar proteins were found to accumulate periodically with a peak just prior to cell division. This was not seen when previously synchronized cells were transferred to constant environmental conditions in a turbidostat, suggesting that dramatic changes in tubulin mRNA levels are not required for successful completion of the cell cycle. A hypothesis to explain the patterns of tubulin mRNA accumulation found under different environmental conditions is presented.

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O2 evolution (up to 120 μmol O2· (mg Chl)(-1)·h(-1) compared with 130 μmol O2· (mg Chl)(-1)·h(-1) of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO2 concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O2 uptake could be observed in the dark by high- and low-CO2 adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 μmol photons·m(-2)·s(-1) for high- and low-CO2 adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200-300 μM for low-CO2 adapted chloroplasts, whereas high-CO2 adapted chloroplasts were not saturated even at 700 μM DIC. The concentrations of DIC required to reach half-saturated rates of net O2 evolution (Km(DIC)) was 31.1 and 156 μM DIC for low- and high-CO2 adapted chloroplasts, respectively. These results demonstrate that the CO2 concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.

Nucleated assembly of Chlamydomonas and Volvox cell walls.

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