Nucleated assembly of Chlamydomonas and Volvox cell walls

• Published in: The Journal of cell biology Vol. 105; no. 5; pp. 2373 - 2382
• Main Authors: Adair, W.S, Steinmetz, S.A, Mattson, D.M, Goodenough, U.W, Heuser, J.E
• Format: Journal Article
• Language: English
• Published: New York, NY Rockefeller University Press 01.11.1987; The Rockefeller University Press
• Subjects: ALGAE; ALGUE; Biological and medical sciences; Cell coat. Cell surface; Cell extracts; Cell Nucleus - ultrastructure; Cell structures and functions; Cell Wall - ultrastructure; CELL WALLS; Cells; Chlamydomonas - ultrastructure; Chlamydomonas reinhardtii; Chlorophyta - ultrastructure; Crystals; Electron microscopy; Freeze Etching; Fundamental and applied biological sciences. Psychology; GLICOPROTEINAS; GLYCOPROTEINE; GLYCOPROTEINS; Glycoproteins - isolation & purification; Glycoproteins - physiology; Microscopy, Electron; Molecular and cellular biology; Molecular Weight; Monomers; MUTANT; MUTANTES; MUTANTS; PARED CELULAR; PAROI CELLULAIRE; Perchlorates; Sodium; Species Specificity; Spheroids; Molecular assembly; Algae; Chlorophycophyta; Molecular hybrid; Glycoproteins; Comparative study; Cell wall; Thallophyta
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.105.5.2373

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.