Structural, biochemical, and inhibition studies of cell wall formation in the unicellular marine coccolithophorid alga Hymenomonas carterae

Abstract

<p>The wall in the coccolith-bearing stage of the life cycle of the marine coccolithophorid alga, Hymenomonas carterae, consists of several ultrastructurally distinct units: (1) columnar material, (2) "glue", (3) "crystalline vesicles", (4) 2-3 layers of scales, (5) coccoliths, and (6) coccolithonets. A technique based on the use of a nonionic detergent (Triton X-100) was developed to isolate the cell wall from coccolith-bearing cells. This cell wall was characterized with respect to its spatial arrangement, degradation, and the in vitro assembly of some of its components. The approach was to carefully describe the wall ultrastructure, partially solubilize and isolate the component parts, and biochemically characterize them. Treatment with 10 M LiCl and 10 mM EDTA dissociated the cell wall into soluble and insoluble fractions (SF and ISF). Cytochemical studies using ruthenium red and catonized ferritin as well as qualitative biochemical analysis (including EDTA extraction, colorimetric sugar determinations, pectinase digestion, and uronic acid assay) strongly suggested the presence of pectin-like acidic polysaccharides in the columnar material, "glue", the periphery of scales, and the distal surface of the coccoliths. Such tests also indicated that the wall is held together by hydrogen bonds, hydrophobic links, and primarily ionic interactions. Acidic polysaccharides of the cell wall were isolated. These molecules possess sedimentation coefficients of 1.55, 1.82, and 4.25. Polyacrylamide gel electrophoresis patterns of the EDTA soluble fraction of coccoliths along with self-assembly characteristics and ultrastructural evidence indicated that the polyanionic carbohydrate molecule with a sedimentation coefficient of 1.82 is a part of the coccolith. This molecule demonstrated a crystalline lattice structure with center to center spacing of 60-65 (ANGSTROM) and appeared to be a proper candidate for subunits of the small Golgi product, the coccolithosomes, which participate in the formation of the calcareous coccolith rim;Cell wall regeneration and kinetics of wall formation were monitored by light and electron microscopy following wall removal. Protoplasts were released after enzymatic treatment with macerozyme and cellulase. Electron microscopic observations revealed that the entire cell wall had reappeared within about 24 hours after removal of the cells from the enzyme mixture. Cell wall formation was inhibited by coumarin (1,2-benzopyrone) and 2,6-dichlorobenzonitrile (DBN). In H. carterae, both compounds reversibly inhibit wall formation as well as cytokinesis. Coumarin affected protoplasts more drastically than DBN, although, in the presence of DBN, cytokinesis completely stopped while nuclear division was unaffected. In contrast, the antimitotic effect of coumarin stopped nuclear division.</p>