Chlamydomonas Cell Wall Research Articles

Autolysin Production from Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a model organism for various processes, from photosynthesis to cilia biogenesis, and a great chassis to learn more about biofuel production. This is due to the width of molecular tools available, which have recently expanded with the development of a modular cloning system but, most importantly, with CRISPR/Cas9 editing now being possible. This technique has proven to be more efficient in the absence of a cell wall by using specific mutants or by digesting Chlamydomonas cell wall using the mating-specific metalloprotease autolysin (also called gametolysin). Multiple protocols have been used and shared for autolysin production from Chlamydomonas cells; however, they provide very inconsistent results, which hinders the capacity to routinely perform CRISPR mutagenesis. Here, we propose a simple protocol for autolysin production requiring transfer of cells from plates into a dense liquid suspension, gametogenesis by overnight incubation before mixing of gametes, and enzyme harvesting after 2 h. This protocol has shown to be highly efficient for autolysin production regardless of precise control over cell density at any step. Requiring a minimal amount of labor, it will provide a simple, ready-to-go approach to produce an enzyme critical for the generation of targeted mutants.

The Chaotrope-soluble Glycoprotein GP2 Is a Precursor of the Insoluble Glycoprotein Framework of the Chlamydomonas Cell Wall

The cell wall of the unicellular green alga Chlamydomonas reinhardtii consists of an insoluble, hydroxyproline-rich glycoprotein framework and several chaotrope-soluble, hydroxyproline-containing glycoproteins. Up to now, there have been no data concerning the amino acid sequences of the hydroxyproline-containing polypeptides of the insoluble wall fraction. Matrix-assisted laser desorption ionization time-of-flight analyses of peptides released from the insoluble cell wall fraction by trypsin treatment revealed the presence of 14 peptide fragments that could be attributed to non-glycosylated domains of the chaotrope-soluble cell wall glycoprotein GP2. However, these peptides cover only 15% of the GP2 polypeptide backbone. Considerably more information concerning the presence of GP2 in the insoluble cell wall fraction was obtained by an immunochemical approach. For this purpose, 407 overlapping pentadecapeptides covering the whole known amino acid sequence of GP2 were chemically synthesized and probed with a polyclonal antibody raised against the deglycosylated, insoluble cell wall fraction. This particular antibody reacted with 297 of the 407 GP2-derived peptides. The peptides that were recognized by this antibody are distributed over the whole known GP2 sequence. The epitopes recognized by polyclonal antibodies raised against the 64- and 45-kDa constituents purified from the deglycosylation products of the insoluble cell wall fraction are also distributed over the whole GP2 backbone, although the corresponding antigens are considerably smaller than GP2. The significance of the latter results for the structure of the insoluble cell wall fraction is discussed.

14-3-3 proteins are constituents of the insoluble glycoprotein framework of the chlamydomonas cell wall.

The cell wall of the unicellular green alga Chlamydomonas reinhardtii consists predominantly of Hyp-rich glycoproteins, which also occur in the extracellular matrix of multicellular green algae and higher plants. In addition to the Hyp-rich polypeptides, the insoluble glycoprotein framework of the Chlamydomonas cell wall contains minor amounts of 14-3-3 proteins, as revealed by immunochemical studies and mass spectroscopic analysis of tryptic peptides. Polypeptides immunologically related to the 14-3-3 proteins also were found in the culture medium of Chlamydomonas. The levels of two of these 14-3-3-related polypeptides were decreased in the culture medium of the wall-deficient mutant cw-15. These findings indicate that 14-3-3 proteins are involved in the cross-linking of Hyp-rich glycoproteins in the Chlamydomonas cell wall.

Volvox as a Model System for Studying the Ontogeny and Phylogeny of Multicellularity and Cellular Differentiation

Volvox carteri, a spherical alga with a complete division of labor between approximately 2000 biflagellate somatic cells and 16 asexual reproductive cells called gonidia, provides a very attractive system for analyzing how a molecular-genetic program for cell-autonomous cellular differentiation may be encoded within a genome. Then, when considered in combination with a group of closely related volvocine algae that includes unicellular Chlamydomonas plus a series of colonial forms of increasing cell number and complexity, it also provides an attractive model system for analyzing how such a program for multicellularity and cytodifferentiation may have evolved. It is proposed that the following were some of the key steps in this evolutionary pathway: (1) The Chlamydomonas cell wall was transformed into an extracellular matrix (ECM) that joined sister cells into a colonial unit. (2) Larger organisms with more abundant ECM were favored because of the role the ECM plays in storing limiting nutrients. (3) In the V. carteri lineage the ancestral biphasic first biflagellate and then reproductive pathway of development became converted to a dichotomous pathway by introduction of two kinds of cell-type-specific negative regulators: one that blocked growth and reproduction in presumptive somatic cells and one that blocked somatic development in presumptive gonidia. Progress has been made in cloning and characterizing genes that are involved in setting apart the two cell lineages of V. carteri and in subsequently controlling their dichotomous differentiation. The strengths and weaknesses of V. carteri and its relatives as a model system for studying the evolution of multicellularity are discussed.

Production of cell wall polypeptides by different cell wall mutants of the unicellular green alga Chlamydomonas reinhardtii

Three classes of cell wall-defective mutants of the unicellular green alga Chlamydomonas reinhardtii have been described in the literature differing with respect to the amounts of cell wall material and its attachment to the plasma membrane, respectively. We have compared the production of the chaotrope-soluble cell wall polypeptides by the different mutants. These experiments have been performed by comparative Western-blot analyses using antibodies raised (1) against the deglycosylation products of the insoluble wall fraction of wild-type cells, (2) against the deglycosylation product of the ‘150 kDa’ chaotrope-soluble cell wall polypeptide and (3) against the carbohydrate side chains of the Chlamydomonas cell wall glycoproteins, respectively. Considerably different levels of cell wall polypeptides were found in the LiCl-extracts from intact cells of the various mutant strains containing the apoplastic, chaotrope-soluble cell wall glycoproteins. No correlation was found between the amounts and the patterns of cell wall glycoproteins present in the LiCl-extracts and the electron microscopical classification of the mutant strains. All the mutant strains were shown to contain the same amounts and patterns of intracellular cell wall precursors as wild-type cells as revealed by Western-blot analyses of urea-SDS lysates of LiCl-pretreated cells. These findings indicate that the different mutant strains produce the same set of cell wall polypeptides at the same relative amounts as wild-type cells. However, in the case of some strains belonging to different classes of cell wall mutants and showing differential seggregation patterns in crosses, alterations were observed for the pattern of extracellular cell wall polypeptides present in the LiCl-extracts from intact cells and in the culture medium, respectively.

Immunological Identification of a Putative Precursor of the Insoluble Glycoprotein Framework of the Chlamydomonas Cell Wall

To identify precursors of the insoluble glycoprotein framework of the Chlamydomonas cell wall, a polyclonal antibody was raised against the mixture of polypeptides released from the insoluble wall fraction by chemical deglycosylation. This antibody preferentially cross-reacted with a '150 kDa' salt-soluble cell wall glycoprotein. The conclusion that this '150 kDa' glycoprotein is a putative precursor of the insoluble cell wall fraction was corroborated by the results of pulse-chase experiments and by experiments with antibodies raised against the '150 kDa' salt-soluble glycoprotein and against its 100 kDa deglycosylation product, respectively. Whereas the antibody against the '150 kDa' glycoprotein preferentially recognized carbohydrate side chains, the antibody against its 100 kDa deglycosylation product was found to have essentially the same specificity towards glycosylated and deglycosylated cell wall components as the antibody against the deglycosylation products of the insoluble wall fraction. Furthermore, the antibody against the deglycosylated, insoluble wall fraction recognized almost the same set of peptide fragments derived by V8 protease treatment from the '150 kDa' salt-soluble cell wall glycoprotein and its 100 kDa deglycosylation product, respectively, as the antibody against the 100 kDa deglycosylated cell wall polypeptide.

Chaotrope-soluble cell wall glycoproteins of Volvox and some members of the Zygnematophyceae immunologically related to the ‘150 kDa’ cell wall glycoprotein of Chlamydomonas reinhardtii

As previously described, the cell wall of Chlamydomonas reinhardtii contains several chaotrope-soluble glycoproteins with similar sugar compositions, but considerably different proportions of hydroxyproline. All these hydroxyproline-containing cell wall glycoproteins are recognized by a polyclonal antibody raised against the purified ‘150 kDa’ cell wall glycoprotein of Chlamydomonas reinhardtii. This antibody also cross-reacts with some polypeptides present in the LiCl-extracts from intact cells of Spirotaenia erythrocephala, Spirotaenia obscura, Volvox aureus, and Volvox globator as revealed by Western blot analyses. Therefore, an IgG fraction of this particular antibody coupled to CNBr-activated Sepharose was used to isolate the immunologically related polypeptides extracted by aqueous LiCl from intact cells or cell wall preparations of different green algae belonging to the Volvocales or to the Zygnematophyceae. Different and more or less complex polypeptide patterns were observed by comparative SDS-PAGE analyses of the polypeptide fractions isolated by immunoaffinity chromatography from extracts of Chlamydomonas reinhardtii, Volvox aureus, Micrasterias rotata, Gonatozygon brebissonii and Netrium digitus, respectively, whereas the corresponding fraction prepared from Spirotaenia erythrocephala contained a predominant ‘160 kDa’ component. Amino acid and sugar analyses revealed that all these polypeptide fractions contained hydroxyproline (1.0–4.5 mol%) and the same sugars as the Chlamydomonas cell wall glycoproteins. However, the relative amounts of these sugars (arabinose, galactose, glucose, mannose and xylose) were found to be rather different. The highest proportion of hydroxyproline (4.5 mol%) and the highest ratio of arabinose: galactose (4.75:1) were determined for the glycoprotein fraction isolated from Spirotaenia erythrocephala indicating that at least the predominant ‘160 kDa’ component is an intrinsic cell wall glycoprotein containing hydroxyproline and oligo-arabinosyl side chains. All the polypeptide fractions isolated from the other green algae obviously also contained chaotropesoluble cell wall glycoproteins as revealed by the presence of arabinose and hydroxyproline. These findings show that all the investigated green algae contain chaotrope-soluble cell wall glycoproteins immunologically related to the ‘150 kDa’ cell wall glycoprotein of Chlamydomonas reinhardtii.

Polypeptide pattern of the insoluble wall component of Chlamydomonas reinhardii and its variation during the vegetative cell cycle

The cell wall of the unicellular green alga Chlamydomonas reinhardii consists of several salt-soluble glycoproteins and a framework of insoluble glycoproteins (K. Roberts, Phil. Trans. R. Soc. London B268 (1974) 129–146). This insoluble framework of the Chlamydomonas cell wall was completely solubilized by chemical deglycosylation with either trifluoromethanesulfonic acid or hydrogen fluoride in pyridine. Mild treatments with alkali or acid, however, did not destroy the insoluble wall component. Our findings suggest that the polypeptide subunits of the insoluble glycoprotein framework may be cross-linked through carbohydrate side chains. We demonstrate here that the pattern of polypeptides released from the insoluble wall component during chemical deglycosylation varies in a cell-cycle dependent manner. The insoluble wall component of all the analyzed cell cycle stages contained relatively constant amounts of polypeptides subunits with apparent molecular weights of >300, 150 and 64 kDa. Polypeptides with apparent molecular weights of 47 kDa and 22 kDa accumulated during cell growth, but were not present in the insoluble wall component of released zoospores. After apparent cytokinesis, increasing amounts of a 60-kDa and a 43-kDa polypeptide were detected. These polypeptides disappeared during liberation of zoospores.

The Chlamydomonas cell wall degrading enzyme, lysin, acts on two substrates within the framework of the wall.

The Chlamydomonas cell wall is a multilayered, extracellular matrix containing 20-25 proteins and glycoproteins, many of which are highly enriched in hydroxyproline. 80-90% of the wall protein is located in a crystalline portion of the wall that is soluble in sarkosyl-urea solutions as well as in chaotropic salts. Although the wall has no cellulose it contains a noncrystalline, highly insoluble framework portion that is responsible for the integrity and overall shape of the wall. In the present report we show that the framework of the wall is composed of two components that are acted upon by lysin, a wall degrading enzyme released by mating gametes. One, which makes up the major portion of the framework, is insoluble upon boiling in SDS-PAGE sample buffer. Lysin treatment of this portion leads to its physical degradation and the concomitant appearance of several SDS-dithiothreitol-soluble polypeptides ranging in relative molecular mass from greater than 400,000 to less than 60,000. The second component is the flagellar collar. This hollow cylinder composed of striated fibers aligned in parallel array serves as the tunnel in the wall through which the flagella protrude. Our evidence indicates that the primary collar polypeptide is a 225,000-Mr molecule that itself has at least two functional domains. One domain, contained in a 185,000-Mr fragment, permits the self-association of the molecules to form the main body of the collar. The second part of the molecule anchors the collar to the wall framework via sarkosyl-urea-insensitive, SDS-dithiothreitol-sensitive linkages.

Degradation of the framework of the Chlamydomonas cell wall by proteases present in a commercially available alpha-amylase preparation.

A commercially available alpha-amylase derived from Bacillus licheniformis contained an enzymatic activity able to degrade the inner portion or framework of the cell wall of Chlamydomonas reinhardtii. Both the wall-degrading activity and the contaminating protease were destroyed by heating the alpha-amylase preparation at 90 degrees C for 30 min. Since the alpha-amylase activity was uneffected by heat treatment, we conclude that it was not the alpha-amylase but the contaminating protease in the preparation that was responsible for the cell wall-degrading activity.

The Chlamydomonas cell wall and its constituent glycoproteins analyzed by the quick-freeze, deep-etch technique.

Using the quick-freeze, deep-etch technique, we have analyzed the structure of the intact cell wall of Chlamydomonas reinhardi, and have visualized its component glycoproteins after mechanical shearing and after depolymerization induced by perchlorate or by the wall-disrupting agent, autolysin. The intact wall has previously been shown in a thin-section study (Roberts, K., M. Gurney-Smith, and G. J. Hills, 1972, J. Ultrastruct. Res. 40:599-613) to consist of a discrete central triplet bisecting a meshwork of fibrils. The deep-etch technique provides additional information about the architecture of each of these layers under several different experimental conditions, and demonstrates that each layer is constructed from a distinct set of components. The innermost layer of the central triplet proves to be a fibrous network which is stable to perchlorate but destabilized by autolysin, disassembling into fibrillar units we designate as "fishbones." The medial layer of the triplet is a loose assemblage of large granules. The outer layer is a thin, crystalline assembly that is relatively unaffected by autolysin. It depolymerizes into two glycoprotein species, one fibrous and one globular. The wall glycoproteins prove to be structurally similar to two fibrous proteins that associate with the flagellar membrane, namely, the sexual agglutinins and the protomers of a structure we designate a "hammock." They are also homologous to some of the fibrous components found in the extracellular matrices of multicellular plants and animals. The quick-freeze, deep-etch technique is demonstrated to be a highly informative way to dissect the structure of a fibrous matrix and visualize its component macromolecules.

The Chlamydomonas cell wall: characterization of the wall framework.

The cell wall of the biflagellate alga Chlamydomonas reinhardtii is a multilayered, extracellular matrix composed of carbohydrates and 20-25 polypeptides. To learn more about the forces responsible for the integrity of this cellulose-deficient cell wall, we have begun studies to identify and characterize the framework of the wall and to determine the effects of the cell wall-degrading enzyme, lysin, on framework structure and protein composition. In these studies we used walls released into the medium by mating gametes. When isolated shed walls are degraded by exogenously added lysin, no changes are detected in the charge or molecular weight of the 20-25 wall proteins and glycoproteins when analyzed on one- and two-dimensional polyacrylamide gels, which suggests that degradation of these shed walls is due either to cleavage of peptide bonds very near the ends of polypeptides or that degradation occurs via a mechanism other than proteolysis. Incubation of walls with Sarkosyl-urea solutions removes most of the proteins and yields thin structures that appear to be the frameworks of the walls. Analysis by polyacrylamide gel electrophoresis shows that the frameworks are highly enriched in a polypeptide of Mr 100,000. Treatment of frameworks with lysin leads to their degradation, which indicates that this part of the wall is a substrate for the enzyme. Although lysin converts the Mr 100,000 polypeptide from an insoluble to a soluble form, there is no detectable change in Mr of the framework protein. Solubilization in the absence of lysin requires treatment with SDS and dithiothreitol at 100 degrees C. These results suggest that the Chlamydomonas cell wall is composed of two separate domains: one containing approximately 20 proteins held together by noncovalent interactions and a second domain, containing only a few proteins, which constitutes the framework of the wall. The result that shed walls can be solubilized by boiling in SDS-dithiothreitol indicates that disulfide linkages are critical for wall integrity. Using an alternative method for isolating walls from mechanically disrupted gametes, we have also shown that a wall-shaped portion of these unshed walls is insoluble under the same conditions in which shed walls are soluble. One interpretation of these results is that wall release during mating and the wall degradation that follows may involve distinct biochemical events.

Monoclonal antibodies to the major structural glycoprotein of the Chlamydomonas cell wall

A family of monoclonal antibodies (McAb) has been raised to the major cell-wall structural glycoprotein of the green alga Chlamydomonas reinhardii. The work represents an approach using McAb to the structure and function of plant cell wall-components. On the basis of cross-competition assays, the McAb have been subdivided into six groups. Various lines of evidence indicate that some of these group's e.g. group III, contain McAb which recognise specific oligosaccharide side chains of the glycoprotein. Heterogeneity of the oligosaccharide side chains is demonstrated, by probing 'Western' blots of separated glycoproteins and glycopeptides with the McAb. The results are discussed in relation to the possibility of raising McAb as probes for the structure and function of higher-plant cell-wall oligosaccharides, which are increasingly being shown to be important in cell-cell recognition phenomena and host-pathogen interactions.