Plasma Membrane Coat and a Coated Vesicle-associated Reticulum of Membranes: Their Structure and Possible Interrelationship in Chara corallina THOMAS C . PESACRETA and WILLIAM J . LUCAS Department of Botany, University of California, Davis, California 95616 ABSTRACT Primary fixation with buffered glutaraldehyde plus 2 .0 mM CaC12 and 0.1% tannic acid results in the preservation of certain portions of the plasma membrane coat of Chara when seen with the electron microscope. Such a coat is not observable after fixation with glutaraldehyde alone . The coat appears to be present on all the above ground, vegetative cells of the male plant. Within complex invaginations of the plasma membrane, which are known as charasomes, the coat has two structural components, a central core that is either tubular or solid and a fibrous or granular peripheral region that surrounds the core. The coat material appears to be at least partially derived, via exocytosis, from the contents of single membrane-bound organelles known as glycosomes. Glycosomes seem to originate from within an assemblage of membranes and coated vesicles that can be described, in purely structural terms, as a partially coated reticulum. Such a reticulum is distinguishable from Golgi stacks because the reticulum (a) is not composed of stacked membranes, (b) is extensively involved with large, clearly detailed coated vesicles and coated invaginations, (c) is closely associated with glycosomes, and (d) is only slightly stained by the zinc-iodide-osmium tetraoxide reagent. Chara corallina is a member of the Characeae, a family of compressed by a turgor pressure of-0.7 mPa. While the plant large-celled algae that have been studiedby many investigators cell wall can resist turgor pressurealong the "smooth" portions with regard to processes such as ion transport (1), cyclosis (2), of the plasma membrane, it is doubtful that a conventional and cell wall growth (3). In an earlier series of articles from cell wall is present in the periplasm of the charasome (6). our laboratory (4-6), the ultrastructure of Chara was exam- Preliminary evidence that the periplasm of Chara does con- ined with special attention being given to studying the devel- tain some structural components has been presented (6). Such opment of the charasomes. Barton originated the term char- work did not consider the influence ofdivalent cations on the asome (7) to denote a multi-tiered complex of anastomosing structure of the charasome, nor was the structure of other tubules that are part of the plasma membrane of Chara. portions ofthe plasma membrane considered. In animal cells, Periplasm is an ancillary term that refers to the apoplastic proteoglycans in the extracellular matrix are associated with space that is present within the charasome. Instead of being the plasma membrane and absorb compressive loads (10). evenly distributed over the entire cell surface, charasomes are However, to our knowledge, little information is available located in a series of circumferential bands along the length concerning the existence of a plasma membrane associated ofthese cylindrical cells (4). Given the proper conditions (8), extracellular matrix (which we shall refer to as a "plasma such bands will acidify the surrounding solution; between membrane coat" to distinguish it from the plant cell wall) of bands, the solution that is adjacent to the cell will become plant cells. Robards (11), Levering and Thomson (12), and alkaline. The precise function of charasomes is not presently Olesen (13) have published micrographs of plant cell plasma understood. However, it has been shown that enzymes that membranes that have either dense particles or linings that are are capable of using ATP are present in the charasome mem- associated with the membrane to some degree. Robards (11) brane (9) and it has been suggested that proteins that are and Olesen (13) hypothesized that such particles were cellulose involved with chloride transport may be present as well (9). synthesizing enzymes. Levering and Thomson (12) showed One of the questions regarding the charasome concerns its an extensive lining with a circular substructure that was ability to maintain an anastomosing tubular form while being located within invaginations of the plasma membrane and THE JOURNAL OF CELL BIOLOGY " VOLUME 98 APRIL 1984 1537-1545 ® The Rockefeller University Press - 0021-9525/84/04/1537/09 $1 .00 1537 that was heavily stained when compared to the cell wall. Such were fixed in a solution that contained 3.0% formaldehyde (freshly made from a paucity of information may have been brought about by paraformaldehyde), 0.5% glutaraldehyde, 10.0 mM CaCIZ, and 50.0 mM so- dium cacodylate with the pH adjusted to 6.8 by adding a few drops of 1 .0 N the existence offactors similar to those that made visualization HCI. After a postaldehyde wash in distilled water they were placed in the ZIO of the lamina rara of animal cells difficult, until the proper solution described by Parker and Hawes (20), except that the temperature of fixation techniques were discovered (14). the solution was kept at 30 .0°C for 18.0 h. For osmium impregnation the cells In animal cells, proteoglycans of the extracellular matrix were placed in 2.0% Os0a at 30 .0°C for 18.0 h. Sections were viewed without are synthesized within the endoplasmic reticulum and the poststaining. Golgi complex, and are then further processed afterexocytosis RESULTS (10). Supportive evidence for the existence of a generally similar delivery system, in other plant cells, comes from Preservation of the Plasma Membrane Coat studies that show that exocytosis ofGolgi vesicles that contain While primary fixation with glutaraldehyde alone results in wall scales (15) and mucilage (16) can occur. It should be the absence of visible material within the periplasm (4-6), the noted that some ambiguity is present in contemporary scien- inclusion of tannic acid and calcium chloride in the fixative tific literature concerning the implication ofthe phrase "Golgi results in the preservation of the plasma membrane coat vesicles." Such ambiguity mainly stems from the studies of (PMC) both within the periplasm and on the external surface Novikoff et al. (17), who have interposed the concept of a of the plasma membrane outside the charasome (Fig. 1). GERL into previously constructed schemes of endomem- Because the PMC has been preserved, the periplasmic tubules brane flow. The GERL contains an assemblage of vesicles appear to be denser than the symplastic tubules; this is the and lamellae that are structurally similar to, but which some- reverse of the image that is seen after fixation with glutaral- times differ in their enzyme content from, the immediately dehyde alone. adjacent vesicles and lamellae of the Golgi complex. While Two structural components, a core and a matrix that sur- usage ofthe term GERL is becoming common in the scientific rounds the core, comprise the PMC (Fig. 1). In some in- literature, some workers still prefer to think of the GERL as stances, the core appears as a discrete rod or tube with a being part of the Golgi complex (18). diameter of -130.0-180.0 f1. Frequently, the core is associ- Here we describe the effect of fixative composition on the ated with radial extensions that bridge the space between the complex structure of the plasma membrane coat of Chara. In core and the plasma membrane. Matrix materials of variable addition, we present micrographs that support the contention structure always surround the core. Discrete particles are that at least some of the material of the plasma membrane evident in some areas ofthe matrix. In other cases, the matrix coat is derived from a partially coated reticulum of mem- is simply a dense, amorphous material that in some places branes rather than being directly derived from the Golgi obscures the structure of the core. stacks. Finally, we describe the distinctive structure and stain- Outside the charasome, the matrix appears as a dense ing characteristics of the partially coated reticulum and show fibrogranular material that is attached to the external surface its close association with both coated vesicles and glycosomes. of the plasma membrane; core material is only occasionally MATERIALS AND METHODS observed (Fig. 1). In areas where charasomes are not present and the plasma membrane is closely appressed to the cell Material and Growing Conditions: Plants of Chara corallina wall, it is difficult to perceive any structural distinction be- Klein ex Wild ., em. R.D.W. (= C. australis R. Br.), obtained from subcultures tween the PMC and the wall. But, when the cell is intention- that had been maintained in our laboratory for more than 5.0 years, were grown in the laboratory under fluorescent lamps at 21 .0-23.0 in 120-1 ally plasmolysed during fixation, the presence of the PMC containers in solutions containing 3.0 mM Na*, 0.2 mM K*, 0.1-0.2 mM and its attachment to the plasma membrane are evident (Fig. Cat *, 1.5 mM Cl-, and ~2.0 HC03 (pH 8.5-9.5). Only male plants were used . 2). Such a PMC is present on all the cell types of vegetative, In all cases, cells were exised from the parent plants during the afternoon, male Chara plants (with the possible exception of rhizoids, incubated in culture media for 1.0 h and then were transferred to our labora- tory's standard physiological testing solution that was composed of 1.0 mM which we have not studied). NaCl, 0.2 mM KCI, 0.2 mM CaSOa, and 1.0 mM NaHC0, (pH 8.0). Extensive We found that the preservation of PMC core material is testing by one of us (W. J. Lucas) has shown that Chara cells remain viable quite sensitive to the presence of calcium in the fixative.