three-dimensional reconstruction of the [PDF]

J. Cell Set. H 213-219 (1986)

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THREE-DIMENSIONAL RECONSTRUCTION OF THE CHONDRIOME OF THE UNICELLULAR RED ALGA RHODELLA RETICULATA SHARON BROADWATER AND JOE SCOTT Department of Biology, College of William and Mary, Williamsburg, Virginia 23185, USA

SUMMARY Three cells of the unicellular red alga Rhodella reticulata were serially sectioned and photographed in a transmission electron microscope in order to analyse the organization of the mitochondrial system, or chondriome, which, on the basis of cursory examination, appeared to consist of an interconnected network of one to a few organelles. The chondriome of all three cells was traced and superimposed on acetate paper and a three-dimensional model using balsa wood was constructed of one cell. The chondriome was found to consist primarily of one large, anastomosing mitochondrion located principally at the cell periphery. In addition, it appears that some cells can contain a few small mitochondria that are not connected to the main body of the chondriome. This is the first study to reveal the three-dimensional nature of the chondriome in a red alga.

INTRODUCTION

Studies leading to our present concept of the chondriome have been reviewed by Ware & LoPresti (1975) and Pellegrini (1980). In general, investigations have revealed the great plasticity of mitochondria and have shown that the number and morphology of the organelle appear to be quite variable from one cell type to another. In addition, it has been shown that the number of mitochondria per cell is often much less than expected on the basis of examination of electron micrographs. Although a number of phylogenetically diverse organisms have been studied, Beech & Wetherbee (1984) have indicated a paucity of work on the chondriomes in algae that do not contain chlorophyll b. To our knowledge there has been no detailed study of the chondriome of any red algal cell. The present work reports some of the results of studies on the shape and number of mitochondria in a unicellular, olive-coloured, coccoid red alga described as a new species of the genus Rhodella (Deason et al. 1983).

MATERIALS AND METHODS Cultures of Rhodella reticulata were obtained from the University of Alabama (courtesy of Temd Deason and Gary Butler), were grown in a 1:1 (v/v) mixture of von Stosch enriched sea water (von Stosch, 1964) and RILA Marine Mix (RILA Products, Teaneck, NJ) and maintained at 20°C on a 16:8, light:dark, photoregime using cool-white fluorescence bulbs (6—12/iEinstein m~ z s~'). Cells were prepared for electron microscopy according to procedures used previously in Key words: chondriome, mitochondria, red algae, Rhodophyta, Rhodella, three-dimensional reconstruction.

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this laboratory (Schornstein & Scott, 1982) and were serially sectioned, stained with lead citrate and photographed with a Zeiss EM 9S-2 electron microscope. Profiles of mitochondria of three cells at a magnification of 60000 were traced directly from a photographic enlarger onto acetate paper and one of the three cells was traced onto 1/4 inch balsa wood as well. The images traced onto acetate paper were superimposed to ensure steric correlation. Errors were due to differences in section thickness (average = 100 nm), tangentially cut profiles and an inability to obtain constant triangulation loci. Various organelles (nucleus, pyrenoid, chloroplast lobes, etc.) were used in aligning sections, but all of the above change somewhat with each section and are, therefore, inferior to outside triangulation points, which were not available. The balsa wood was cut with a band saw and the profiles were aligned using the acetate sheets prior to construction of the model.

RESULTS

Fig. 1 is a section through the central region of a typical cell of R. reticulata. The cells are predominantly uninucleate with the nucleus occupying an eccentric position. The bulk of the cell is taken up by the plastidome, which consists of a single chloroplast whose peripheral lobes are connected by narrow isthmuses to a large, central pyrenoid (see Deason et al. 1983, for a detailed ultrastructural description). As seen with the electron microscope, mitochondrial profiles are generally found at the outer regions of the cell, although occasional branches or small segments may sometimes be found in the cell interior. In many of our fixations the mitochondrial profiles appeared extremely osmiophilic (Figs 1,2). This particular feature made the task of recogni2ing and following the mitochondrial network relatively easy. R. reticulata cells vary in si2e from 8 to 30 /im in diameter, with most ranging from 8 to 15 /im. Three smaller cells were serially sectioned, resulting in an average of 110 sections per cell. In all cases, the greater portion of the chondriome was composed of a single, filamentous, highly branched mitochondrion located near the cell periphery. In the reconstructed chondriome there were two small, oval mitochondria (Figs 3, 4), one measuring 0-3/im X 1-0/im and the other measuring 0-5/im X 1-4/im. One of the traced cells had no 'extra' mitochondria; the other had only one small mitochondrial segment that was not attached to the rest of the chondriome. In all cases, the small mitochondrial 'pieces' were found near the central region of the cell. In the reconstructed chondriome the pieces were located near the nucleus (Figs 1, 3, 4). In one case the orientation of the piece was such that had it continued in length, it would have joined with a segment of the main mitochondrion that extended towards it. Fig. 1. Medial section through Rhodella reticulata cell used in chondriome reconstruction (shown in Figs 3, 4). The mitochondrial profiles could be mistaken for a number of single organelles that are either dividing or fusing. The arrowhead denotes a profile of one of the two small mitochondria that are separate from the primary mitochondrion. The approximate position of this section in the model is marked by the single arrowheads in Fig. 4. p, pyrenoid; nu, nucleus. X14 500. Fig. 2. Different section through same cell as shown in Fig. 1. The reticulate nature of the chondriome (arrowheads) is more obvious in this view. The approximate position of this section in the model is marked by the double arrowheads in Fig. 4. X14500.

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Figs 1 and 2 are sections from the cell used as a model for the reconstruction and are correlated with their position on the reconstruction (Figs 3, 4). The mitochondrion can vary greatly in width, some regions being as small as 50 nm wide. In some sections, the predominantly closed-loop, reticulate nature of the chondriome is fairly obvious (Figs 1, 2); in others (not shown), the profiles appear to bear little relation to each other. DISCUSSION

This study and reconstruction represent the first of their kind in a red alga, although primarily single, large, branched mitochondria have been shown in many other cell types: the yeasts Saccharomyces (Hoffman & Avers, 1973; Stevens, 1977) andPityrosporum (Keddie & Brarjas, 1969, 1972), the algae Chlamydomonas (Grobe & Arnold, 1975), Chlorella (Atkinson et al. 1974; Dempsey et al. 1980), Euglena (Pellegrini, 1980; Pellegrini & Pellegrini, 1976), Pleurochrysis (Beech & Wetherbee, 1984) and Polytoma (Gaffal & Kreutzer, 1977), the protozoa Blastocrithidia and Trypanosoma (Paulin, 1975), theflagellatePolytomella (Burton & Moore, 1974) and the phycomycete Olpidiuim (Lange & Olson, 1976). The stage of the cell cycle may have an effect on mitochondrial shape and number. Several studies indicate that single, branched mitochondria occur during the growth phase of the cell but several to many ovoid and, or, less-branched mitochondria are typical of dividing cells and young zygotes (Bromberg, 1974; Calvayrac et al. 1972; Gaffal & Kreutzer, 1977; Grimes et al. 1974; Grobe & Arnold, 1977; Stevens, 1977, 1981; Tanaka et al. 1985). Not all cells, however, seem to display gross morphological changes corresponding to the cell cycle (Koukl et al. 1977; Pellegrini, 1980), and the presence or absence of such changes does not appear to have phylogenetic significance. Along the same lines, cristae morphology, which is considered by many to be of great taxonomic importance, appears to bear no relationship to the number and shape of mitochondria in a cell (Beech & Wetherbee, 1984). The chondriome of R. reticulata is primarily a single anastomosing mitochondrion. The indications are that its shape is highly mutable and that segments can break off and re-fuse. The latter is inferred from finding a variable number of small mitochondria in the three serially sectioned and analysed cells, the very narrow widths seen in several regions of the mitochondrion and from the orientation of one of the small extra mitochondria that suggested its breakage from the much larger, predominant one. Observations of living Euglena gracilis (Leedale & Buetow, 1970) and serial reconstruction of this species (Pellegrini, 1980) support this view. It may be only chance that all three of the chondriomes of R. reticulata cells sectioned in this Fig. 3. Photograph of reconstructed chondriome, dorsal view. In this view, each tier reveals the mitochondrial profiles seen in the respective serial section. The two small, ovoid mitochondria (arrowheads) can be seen. They help to define the region where the nucleus is located (asterisk). Fig. 4. Photograph of reconstructed chondriome, lateral view. The view of the model here is at right angles to the plane of sectioning. The single arrowheads indicates the approximate position of the section shown in Fig. 1, and the double arrowheads indicate the approximate position of the section shown in Fig. 2.

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study were primarily composed of a single mitochondrion. One might expect to find one to three large, branched mitochondria with only a few small ovoid ones. This possibility is inferred from the apparent structural plasticity of the chondriome and from results of previous studies on other unicells. All of the R. reticulata cells sectioned appeared to be in interphase. No mitotic cells were found, so a comparison with cells in other stages of the cell cycle could not be made. There have probably been more three-dimensional models made of mitochondria than of all the other organelles combined, and the reconstructions have covered a wide variety of organisms. The wealth of information obtained provides an accurate account of a cell's chondriome and seems to indicate that the morphology and number of mitochondria may be a useful indicator of stages of the cell cycle, but is unfortunately of relatively little use in indicating phylogenetic and taxonomic relationships. We thank Jewel Thomas and Bill Saunders for their assistance with this study. We also thank Temd Deason and Gary Butler for providing us with cultures of Rhodella reticulata and information on proper culture conditions. Financial support was provided by NSF grant BSR 83-07714 to J. Scott.

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(Received 21 January 1986 -Accepted 9 April 1986)