The Chlamydomonas Cell Wall Glycoproteins Analyzed by the Technique and Its Constituent Quick-Freeze, Deep-Etch

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. Ultrastmct. 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. It is becoming increasingly clear that interactions between cells often involve the molecular merger of their extracellular matrices and/or the establishment of a common extracellular matrix. During the formation of a bilayered epithelium in animals, for example, an essential event is the formation of a common basal lamina, rich in such fibrous glycoproteins as type IV collagens and laminin (for review see reference 1), which is produced and secrected by both types of epithelial cells. In the case of higher plants, the initial event in cell wall formation appears to be the secretion of a fibrous hydroxyproline-rich glycoprotein, named extensin, accompanied by the deposition of such polysaccharides as pectins and cellulose (for review see reference 2). The unicellular alga Chlamydomonas reinhardi is surrounded by an extracellular coat composed of hydroxyproline-rich glycoproteins (3-5). An extracellular matrix of similar material holds together the individual Chlamydomonastype cells that form such colonial genera as Pandorina and 1550 Volvox (6-8). It is therefore reasonable to imagine that there may be evolutionary relationships between the matrix of Chlamydomonas and of the true metazoa. This article presents an analysis of how this matrix is constructed, using the quick-freeze, deep-etch technique developed in one of our laboratories (9, 10). The central region of the Chlamydomonas wall has previously been shown to be trilaminar and highly ordered ( 1116), analogous to the layered construction of the glomerular basal lamina (17). Here we demonstrate that each of the ordered layers contains a characteristic population of distinct glycoproteins. Deep-etch visualization of the molecules obtained by several different extraction protocols demonstrates that some wall components are fibrous and others are globular. Partial dissolution and/or reconstitution experiments, coupled with deep-etching, demonstrate several specific interactions between these components. Since several of the spatial arrays on the cell surface are very regular and two-dimenTHE JOURNAL OF CELL BIOLOGY . VOLUME 101 October 1985 155