Nucleated assembly of Chlamydomonas and Volvox cell walls.

Abstract

Abstract. 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 FITCstreptavidin fluorescence and quick-freeze/deep-etch electron microscopy. Optimal reconstitution was obtained at monomer concentrations (0.2-0.3 mg/mi) 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 GPI into the outer (W6B) sublayer. Finally, evolutionary relationships between C. reinhardtii and two additional members of the Volvocales (Ch/amydomonas 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.

A Transglutaminase Immunologically Related to Tissue Transglutaminase Catalyzes Cross-Linking of Cell Wall Proteins inChlamydomonas reinhardtii

“…The assembly of outer wall glycoproteins onto the cells can be studied in vitro as well (Hills et al, 1975;Adair et al, 1987). When "shells" (vegetative cells that have been perchlorate extracted and concomitantly killed) are dialyzed together with the solubilized glycoproteins, the glycoproteins assemble onto the shells, reforming a normal V-wall (Adair et al, 1987).…”

“…This selfassembly has been well documented in vitro for the saltextractable outer V-wall HRGPs: when the salt is dialyzed away from these proteins, they form a crystalline structure similar to that found in muro (Hills et al, 1975;Goodenough et al, 1986). In addition, salt-extracted vegetative cells added to the dialysis bag can nucleate the assembly of the soluble HRGPs to reform a crystalline layer (Adair et al, 1987).…”

Influence of the Cell Wall on Intracellular Delivery to Algal Cells by Electroporation and Sonication

“…The walls of C. reinhardtii are made of glycoproteins containing an extensin-like protein rich in hydroxyproline and sugar residues including arabinose, galactose and mannose (Adair et al 1987). Wall structure contains a central framework of polygonal plates bounded externally and internally by fibrous layers.…”

Molecular Characterization of a Zygote Wall Protein: An Extensin-Like Molecule in Chlamydomonas reinhardtii

“…The walls of the vegetative and gametic cells are highly ordered structures that carry a chaotrope-soluble crystalline layer composed of well-characterized HRGPs (Catt, Hills, and Roberts, 1978;Goodenough andHeuser, 1985, 1988;Roberts et al, 1985;Goodenough et al, 1986;Adair et al, 1987). When gametes mate, their walls are shed, and the zygotes proceed to elaborate new walls that are denser, thicker, lack the crystalline layer, and are insoluble in chaotropes and SDS (Minami and Goodenough, 1978;Grief, O'Neill, and Shaw, 1987).…”