High-resolution electron microscopy of glycoproteins: the crystalline cell wall of Lobomonas.

Lobomonas piriformis is a member of an order of green algae (Volvocales) that have crystalline glycoprotein cell walls. As part of a program of investigation of these glycoproteins and their architecture we have studied the cell wall of Lobomonas by a variety of chemical, electron-microscopical and image-analysis techniques. Lobomonas and Vitreochlamys incisa show a very similar structure in their cell walls and represent I of the 4 classes into which all the structures of the wall of these algae that we have so far examined fall. The 2 classes that we have previously studied in detail, represented by Chlamydomonas reinhardii and chlorogonium elongatum, have a crystalline component of the wall that is a more or less smooth continuous surface overlying an amorphous inner wall layer. Although Lobomonas also has this 2-layer structure, the crystalline layer consists of distinct plates, each of which is built around a single, very coherent crystal lattice. The polar nature of the architecture of the cell wall is shown by sectioning and by examination of the cell-wall surface by metal-shadowing of carbon replicas, both of intact cells and of isolated cell-wall plates. There are great similarities in chemical composition between the glycoproteins of the cell wall of C. reinhardii and those of Lobomonas. Both has a large content of hydroxyproline in their amino acid composition and a sugar/hydroxyproline ratio of about 6.0, and both contain sugar sulphates. Lobomonas however has a large glucose content, whereas Chlamydamonas has almost none. Electron micrographs of walls stained with methylamine tungstate and shadowed specimens show that the Lobomonas crystal structure is entirely different from that of C. Reinhardii, and that there is a distinctly different structure in the centre of the plates from that at their edges, although the transition between the 2 areas occurs with no distortion of the crystal lattice. Computer image analysis has been used to calculate reconstructed images of the 2 areas, and by using minimal-dose techniques has yielded 2-dimensional maps of the negatively stained structure at a resolution of I.8 nm. The 2-sided plane group of both areas of the crystal is P2, and the centre area contains 2 distinct structural units, both centered on dyad axes, together with other more complex features. In the edge structure, one of the structural units appears unchanged, but the other unit has a considerably different appearance. The most likely interpretation of this is as a conformational or positional change in one of the subunits. However, because the underlying lattice is so accurately maintained across this transition, it seems probable that the basic structural arrangement that defines the lattice is common to the 2 areas. Some of the computational and mathematical techniques used in the image analysis have not been previously published and are described in detail and compared with published techniques in an Appendix.