J. Cell Sri. 26, 1-8(1977)

Printed in Great Britain

EVIDENCE FROM SERIAL SECTIONS THAT

SOME CELLS CONTAIN LARGE NUMBERS

OF MITOCHONDRIA

PETER HEYWOODDivision of Biology and Medicine, Brown University,Providence, Rhode Island 02912, U.S.A.

SUMMARY

Three-dimensional reconstructions based on serial sections through cells of the chloro-monadophycean alga Gonyostomum semen Diesing indicate that there are many small unbranchedmitochondria in log-phase cells. These organelles are approximately ellipsoid in shape withtheir longest axis usually ranging from 07 to 2'O/an in length. Occasionally mitochondriareaching up to 3-6 fim in length have been observed: these have the same general morphologyas their smaller counterparts. Preliminary observations indicate that cells of a related alga,Vacuolaria virescens Cienkowsky, also possess many small unbranched mitochondria.

These observations are discussed in relation to recent reports that some cells contain a singlebranched mitochondrion or, at most, a small number of these organelles. It is suggested thatmitochondrial number may be related to cell volume and that large cells may contain manymitochondria.

INTRODUCTION

For some time it was widely assumed that, with few exceptions (Manton, 1959;Manton & Parke, i960), all eukaryotic cells contained large numbers of mitochondria.This view has recently been challenged (Hoffmann & Avers, 1973) as a result of evidenceobtained from serial sections which indicates that there is a single complex mito-chondrial reticulum in a fungus (Hoffmann & Avers, 1973), an alga (Atkinson, John &Gunning, 1974), and 2 species of protozoa (Paulin, 1975). Other protists examined inthis way had either one or a relatively small number of mitochondria per cell (Arnold,Schimmer, Schotz & Bathelt, 1972; Burton & Moore, 1974; Grimes, Mahler &Perlman, 1974; Grobe & Arnold, 1975; Keddie & Barajas, 1969; Schotz, Bathelt,Arnold & Schimmer, 1972) and this is also true for the one mammalian cell thus farinvestigated (Rancourt, McKee & Pollack, 1975). These interesting findings mightgive rise to an alternate assumption - that cells contain only small numbers of mito-chondria and that these are generally present as large branched structures. I herebriefly report that this is not the case for 2 large unicells, the phytoflagellates Gonyo-stomum semen Diesing and Vacuolaria virescens Cienkowsky.

P. Heywood

*

a' • f ^ ^ ^ ^ : ^ ^ ^

Fig. i. Profiles of 4 mitochondria (m) occur adjacent to the nucleus (n) and Golgi C?)in this section through a portion of Gonyostomum semen cytoplasm; 2 of the mito-chondria contain electron-translucent areas (arrows), x 15000.

Fig. 2. Typical cross-section through a mitochondrion of Gonyostomum semen.Regions of continuity between cristae and the inner mitochondrial membrane areindicated by arrowheads. Fibrillar material (arrows) which resembles the mito-chondrial DNA of other algae occurs in some of the electron-translucent parts of themitochondrion, x 60000.

Fig. 3. Mitochondria (m), part of a chloroplast (c), and numerous ribosomes are presentin this section of Vacuolaria virescens. In the absence of serial sections it is not possibleto determine whether the mitochondrial profiles in Figs. 1 and 3 represent sectionsthrough separate organelles or whether they are parts of a continuous structure,x 25000.

Some cells contain many mitochondria 3

MATERIALS AND METHODS

Cultures of Gonyostomum semen and Vacuolaria virescens (Cambridge Culture Collectionnumber LB 1195/1) were grown at 18 ± 1 °C on defined medium (Heywood, 1973) which wasaerated with a gas mixture of 4 % COj in air. Asynchronous populations in a log-phase ofgrowth were obtained by frequent transfer to new media of cells grown under continuousillumination of intensity 180 ft.-c. (194 x io3 lux) (supplied by Ecko daylight fluorescent tubes).

Cells were harvested by gentle centrifugation and were fixed for 1 h in a 2 % solution ofglutaraldehyde buffered with 0-07 M phosphate buffer to pH 6-5. After beingwashed thoroughlyin buffer they were postfixed in 1 % osmium tetroxide, dehydrated in a graded ethanol seriesand embedded in Epon. Ribbons of serial sections were transferred to single-hole copper gridscoated with Formvar and carbon. They were subsequently stained with aqueous uranylacetate and lead citrate and were examined using a Philips 20 iS electron microscope.

OBSERVATIONS

Gonyostomum semen and Vacuolaria virescens are large phytoflagellates which mayreach up to 80 /im in length. General details of their ultrastructure are available inearlier accounts (Heywood, 1968, 1972; Mignot, 1967). Mitochondria are especiallycommon in the layer of cytoplasm surrounding the nucleus (Fig. 1) but are also presentin more peripheral areas (Figs. 3, 16-31). They are approximately circular or ellipsoidin outline and are usually in the size range of 1-2 /im, though occasional ones have beennoted which reached 3-6 fim in length. Their tubular cristae are abundant and occupymuch of the volume of the mitochondrion; in favourable sections the continuitybetween cristae and the inner mitochondrial membrane is clearly apparent (Fig. 2).Cristae are absent from some regions of the mitochondria (Figs, i, 2, 9-12); at highmagnification some of these electron-translucent areas can be seen to contain finefibrils (Fig. 2) which resemble the mitochondrial DNA of other algae (Dodge, 1973;Evans, 1974).

Examination of many electron micrographs obtained from periodic fixations madeover a time span of several years never revealed any indications that mitochondriafrom cells of Gonyostomum semen and Vacuolaria virescens were large and extensivelybranched. Ultrathin sections of these cells often contain 20 or more mitochondrialprofiles per cell, but on the basis of single sections it is not possible to determinewhether these profiles are the result of the section passing through many small mito-chondria or whether they represent portions of a single mitochondrial reticulum.

Serial sections through portions of 23 cells of Gonyostomum semen were examinedbut no evidence was obtained for a mitochondrial reticulum. Serial sections throughportions of 2 of these cells were photographed to allow accurate calculation of mito-chondrial size. The sections had interference colours ranging from silver-gold togold and were therefore estimated to average 100 ran in thickness. Forty-two mito-chondria were measured: their longest dimension ranged from 0-90 to 1-96/im (witha single mitochondrion reaching 2-76 /im in length) while their shortest dimensionwas 0-52-1-33 /im. No evidence was seen for any extensive branching, though somemitochondria had irregular projections on their surface (e.g. Fig. 12). Figs. 4-15depict a typical series across two mitochondria.

P. Heywood

Some cells contain many mitochondria 5

The situation in Vacuolaria virescens appears to be substantially similar to that inGonyostomum semen. Although mitochondrial morphology has not been as systematic-ally investigated in this organism, where serial sections have been obtained they showmitochondria with similar dimensions to those of Gonyostomum semen (Figs. 16-31).

DISCUSSION

It has been known for some time that 2 flagellates contain only one mitochondrionper cell (Manton, 1959; Manton & Parke, i960). Recent studies employing serialsections have proved that these are not isolated instances: other protists contain eithera single mitochondrion (Atkinson et al. 1974; Hoffmann & Avers, 1973; Paulin, 1975)or a small number of mitochondria (Arnold et al. 1972; Schotz et al. 1972). In someorganisms the number is variable: some of the cells examined had a single mito-chondrion while others had several (Burton & Moore, 1974; Grobe & Arnold, 1975;Keddie & Barajas, 1969). Irrespective of the exact details and variations it is apparentthat those protists thus far systematically studied by serial sections had far fewermitochondria than had previously been assumed. The present report indicates thatthis is not the case in Gonyostomum semen and Vacuolaria virescens: it is estimated thatthese organisms usually contain in excess of a hundred mitochondria per cell. Theorganisms used in these experiments were taken from asynchronous log-phase culturesand consequently it is assumed that they included all stages of the cell cycle. Therewas no evidence for the type of alterations in mitochondrial shape during the cellcycle that have been reported in some other algae (Calvayrac, Bertaux, Lefort-Tran &Valencia, 1974; Osafune, Mihara, Hase & Ohkuro, 1972, 1975).

Since the issue of mitochondrial number is of far-reaching importance (Hoffmann &Avers, 1973), it is appropriate to consider whether the large number of mitochondriain Gonyostomum semen and Vacuolaria virescens is a peculiar feature of these organismsor whether it has wider implications. I suggest that the latter may be the case and thatamongst other factors, such as stage in the cell cycle (Calvayrac et al. 1974; Osafuneet al. 1972, 1975) and in development (Bromberg, 1974), the number of mitochondriamay berelated to the volume of the cell. All the cells that have been demonstrated tocontain small numbers of mitochondria are relatively small (Table 1) and it may beinappropriate to formulate general conclusions of mitochondrial number (Hoffmann& Avers, 1973) on this sample. Gonyostomum semen is much larger (Drouet & Cohen,1935; Hovasse, 1945): typically over 80% of the cells in laboratory cultures are40—55 /tm long by 30—40/tm wide (Heywood, 1968). This results in a cell volume ofapproximately 4000 fim3 which is more than an order of magnitude greater than any

Figs. 4-15. Sections through 2 mitochondria of Gonyostomum semen. Figs. 4 and 5 areserial, Figs. 5-15 are alternate sections in a serial series. Electron-translucent areas arepresent in Figs. 9-13. The electron-opaque structure labelled t in Fig. 4 is part of atrichocyst in transverse section. This structure is situated at approximately the sameposition in subsequent micrographs to aid in orientation. Electron-opaque materialbetween the mitochondria in Figs. 9-11 and adjacent to the trichocyst in Figs. 13-15is precursor material for trichocysts. The cytoplasm around the mitochondria containsrough endoplasmic reticulum and ribosomes. x 25 000.

P. Heywood

Figs. 16-31. Serial sections through a mitochondrion of Vacuolnria virescens. Thesection occurring between Figs. 27 and 28 has been omitted but otherwise the seriesis complete. This mitochondrion is situated in a narrow strip of cytoplasm surroundedby vacuoles. Endoplasmic reticulum and ribosomes are plentiful in the cytoplasm. Thenature of the electron-opaque material in Figs. 28-31 is not known, x 25000.

of the cells listed in Table 1 and is more than 3 orders of magnitude larger than someof them. Cells of Vacuolaria virescens have a similar volume (Heywood, 1968). Thesingle mitochondrion of Saccharomyces cerevisiae is 50-60 fim in length (Hoffmann &Avers, 1973) and that of Chlorellafusca attains a maximum length of 147 /tm (Atkinsonet al. 1974). If Gonyostomum semen were to possess a single mitochondrion it wouldhave to be highly branched and extremely long in order to serve the whole celladequately. The problems of accommodating such a complex structure and in

Some cells contain many mitochondria

Table i. Comparison of cell volume and mitochondrial number

Organism

Pityrosporum orbiculare(Fungus)

Micromonas pusilla (Alga)Micromonas squamata (Alga)

Saccharomyces cerevisiae diploidSaccharomyces cerevisiae diploid

Saccharomyces cerevisiae haploid(Fungus)

Chlamydomonas reinhardiiChlamydomonas reinhardiiChlamydomonas reinhardii

(Alga)

Chlorella ftisca (Alga)

Cell volume,

4-6-5-6

< IO

14-65

26 (mean)N.A.

13-8 (mean)

4459NA

188 (maximum)

Mito-chondrial

no.

i - 4

1

1

22 (mean)1

10 (mean)

9141-5

1

Reference

Keddie & Barajas, 1969

Manton, 1959Manton & Parke, i960

Grimes et al. 1974Hoffmann & Avers,

1973Grimes et al. 1974

Schfitz et al. 1972SchStz et al. 1972Grobe & Arnold, 1975

Atkinson et al. 1974

Mammalian lymphocyte 155-283 3-7

> 100

Rancourt et al. 197s

Gonyostomum semen (Alga) ~ 4000 > 100 Present reportNote: The number of mitochondria and most of the values of cell volumes were obtained

from the source listed adjacent to the name of the organism. The cell volumes of Micromonaspusilla and Micromonas squamata were calculated from their descriptions (Manton, 1959;Manton & Parke, i960). The cell volumes for Saccharomyces cerevisiae and Chlamydomonasreinhardii although not available (N.A.) in Hoffmann & Avers (1973) and Grobe & Arnold (1975)are assumed to be similar to those reported by other authors (Grimes et al. 1974; Schfttz et al.1972); the disparity in the reports of mitochondrial number in these organisms has been dis-cussed elsewhere (Grimes et al. 1974; Grobe & Arnold, 1975). Apparently there is no publishedinformation on cell volume for some of the organisms (Burton & Moore, 1974; Paulin, 1975)mentioned in the text.

controlling its growth during the cell cycle have probably selected in favour of manysmall mitochondria. Serial sections through other large cells should be examined totest this conclusion.

I am grateful to Mrs Julie Gross for her expert assistance in preparing the illustrations andto Mrs Jean Waage for typing the manuscript. This investigation was supported by a Bio-medical Sciences Support Grant from Brown University.

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(Received 23 December 1976)