Planta 150, 236 241 (1980)

P l a I l t a 9 by Springer-Verlag 1980

Variability of Mitochondrial Population in Chlamydomonas reinhardii Rudolf Blank, Edda H a u p t m a n n , and Carl-Gerold Arnold Institut ffir Botanik und Pharmazeutische Biologie der Universit~itErlangen-Niirnberg, SchloBgarten 4, D-8520, Erlangen, Federal Republic of Germany

Abstract. Several details have been published concerning the mitochondrial number and shapes at various stages of the synchronized vegetative and generative cell cycle in Chlamydomonas reinhardii. The present study, based on ultrathin serial sections and threedimensional reconstructions, completes these data. Quantitative analysis of serial micrographs makes it possible to give specific details of mitochondrial volumes in cells at early intermediate stages of the vegetative life cycle. Our investigations clearly show that mitochondria have a relatively wide range of sizes, within certain limits, and vary like the mitochondrial shapes; in fact, they vary in various cells at various stages as well as in several cells at the same stage and even in one and the same cell. Thus, we present a plastic insight into the dynamically changing micromorphology of the mitochondrial population in Chlamydomonas reinhardii.

statements about the volumes of single mitochondria, however, exist only from gametes which were submitted to gametogenesis from asynchronous batch-cultures at r a n d o m stages of the vegetative cell cycle (Sch6tz et al. 1972). Other quantitative volume analyses have been made only of the whole mitochondrial inventory, i.e., the chondriome: Volumes in the generative life cycle have been determined by means of serial sectioning studies (Grobe 1976); volumes in the vegetative stages have been determined on the basis of estimations from r a n d o m single micrographs (Fischer 1969). Therefore, to broaden our knowledge about the micromorphology of mitochondria, we carried out qualitative as well as quantitative investigations of certain stages during the vegetative and generative cell cycle of Chlamydomonas reinhardii by examining the entire cell in serial sections.

Key words: Cell cycle and mitochondria - Chlamydomonas - Mitochondria (micromorphology). Materials and Methods

Introduction The morphological variability of mitochondria as revealed by means of light microscopy has long been known but was re-discovered by using electron microscopy in biological investigations (see Arnold and Gaffal 1979 a). In the phytoflagellate Chlamydomonas reinhardii, mitochondrial micromorphology has been described in several stages of the vegetative (Osafune et al. 1975, 1976) and generative cell cycle (Arnold et al. 1972; G r o b e 1976; Grobe and Arnold 1975, 1977; Sch6tz 1972; Sch6tz et al. 1972), by use of serial sections and three-dimensional models. Quantitative

0032-0935/80/0150/0236/$01.20

Culture Conditions and Synchronizing Methods jbr Vegetative Growth. Good synchronization in Chlamydomonas reinhardii is described by Schl6sser (1966, 1976), but the strain 137c WT, which is kept in stock clones at our department, cannot utilize nitrate as a nitrogen source. Therefore, a new synchronizing method had to be developed for this study. Both mating types of the strain were used, but mt(+) achieved a greater rate of synchronization. In order to synchronize the vegetative cell cycle, the algae were grown in 300 cm ~ tubes with sterile liquid inorganic minimal medium as described by Loppes (1966); but the ammonium chloride concentration was changed (3.74 molm 3; 0.2 mg em 3), and trace elements were used according to Hutner et al. (1950). The algal solution was aerated from the bottom of the tubes with 3% carbon dioxide in air (flow velocity about 200 cm 3 rain-1). The phytoflagellates were synchronized in a Kniese-AlgenzuchtThermostat with a photoelectrically controlled dilution device (Kuhl and Lorenzen 1964; Lorenzen 1959; Pfau et al. 1971) under a light-dark regime with 14 h light (about 20,000 lx at the outer Plexiglas surface of the water bath) and 10 h darkness. At the beginning of each life cycle (onset of illumination), the cell solution

R. Blank et al. : Mitochondria in Chlamydomonas

237 was automatically density controlled and diluted to a preset density of about 1.6- 10 6 cells cm- 3, whereby fresh medium from a storage bottle was added via gas flooding from the bottom, thus forcing surplus algae from the top. We determined synchronism and cell number microscopically with a hemacytometer (Neubauer-Zfihlkammer) several times daily during a synchronization period. For mt(+), best results were found at a temperature of 30 ~ C (Fig. 1), whereas for m t ( - ) they were best at 34 ~ C. It should be noted that the course of development, as represented in the cycle in Fig. 1, applies to only about 70% of the cell population - this becomes concealed in the diagram in Fig. 1. This 70% degree of synchronization also applies only to the liberation of zoospores; the division of nuclei and chloroplasts are less synchronized (Mihara and Hase 1971); this probably applies to mitochondrial development too.

Induction of Sexuality. To study the mitochondrial inventory of Chlamydomonas reinhardii after gametogenesis under dark and cold

no.l ml

10~.]

~

'

"

3

h

I

Fig. 1. Developmental course of the vegetative life cycle of the Chlamydomonas reinhardii strain 137c WT m t ( + ) under synchronous conditions (top), and its diagram using an algal culture thermostat with an automatic daily dilution system (bottom)

7 CELLS: 5 AND11 MT IOCHONDRL~ BMROAS~cEHLEO G tATED,' LITTLE ~ . . . , . . ~

conditions, in which cells do not undergo gametogenic division and do not change morphologically, we used the method described by Isbiura (1976) which is based on his previous investigations (Ishiura and Iwasa 1973a, b, c). In the best phase of synchrony in severai trials, cells were harvested at two different stages in the vegetative life cycle ( t = 0 and t = 9), re-suspended in fresh nitrogen-free medium, and, after washing, re-adjusted to a cell number of about 2-106 cells cm -B. All preliminary steps were carried out under dim-green light, whereas the actual gametogenic process, lasting 20 to 60h, took place in total darkness at 10~ C. The percentage of pair formation reached a maximum of 20% after 24 h - this value, however, does not coincide with the values of Ishiura (1976).

fiAMETOGENESIS IN THE DARKICOLD ~{I[LLB:~.TO9 NITOCHONDRA i OVOIDORELOND , ATEO,SCARCELYBRANCHE0

o

VEGETATIVE CYCLE[h]

6~L ~\

//

v~

_~CELL__51 MITOCHONDRIA SBMEELoN~GATEDORBRANCHEO

L'TLCNgO?';

l~

/

(

MANYLITTLE,SOMEBRA,NEH~ l OSAFUNEETAL.I 19?6l

\

MOSTOVOIDORL.0 "

~

LANTM ITOCH(]NOIRIA,HIGHLYBRANCHED 2/3 CELL, 3 JS ~ANT MITOI -I0SAFUNEETAL ,1975 )

)

I kX

" PARTLY "NEHED[BSA'UNEETAL.,19• ]

A,~2~

I

~ ~

) /

XX

I CELL:1 NETWORKAND23 LITTLE OF~.HIGHLYBRANEHEDMITOEHO N D R i A / AND N I CREASEOFLITTLEONESWITH PRO\CEEDING T(MEAFTERFUSION ~ ( GROBEAN{] ARNOLD,197~)

DE ~ EASE

.~

~

~

/

GIANT ANDHIGHLYBRANCHED BESIDE LITTLEONES

, CELL: I ,ETWORK-/ 3 CELLS:INETWORKAND1TO&LITTLEFRAGMENTS ANB ARNOL~I~r~,5)

FRAGMENTS --

/

/

/

/

/ / ~

/ /

/

/

/

/

/

Fig, 2. Mitochondrial inventory in Chlamydomonas reinhardii at various stages of the vegetative and generative cell cycle, as studied by several authors (data from other authors cited with name and publication date)

238

Electron Microscopy. Cells were collected and washed with collidine buffer (50 tool m-3). After fixing in 2% glutaraldehyde in cold buffer for 90 min and three subsequent washings, they were postfixed in buffered 1% osmium tetroxide for 60 min and washed repeatedly with cold buffer. They were then dehydrated in ethanol series and propylen oxide, and were embedded in the medium described by Spurr (1969). Serial sections were made with a Diatome diamond knife in a Reichert OMU-2 ultramicrotome. Then they were put on hole grids coated with Pioloform films, double stained with uranyl acetate and lead citrate, and were observed and photographed with a Zeiss EM-10 electron microscope.

Measurementsand Model Building. After photographing a consecutive series of about 60 to 150 sections per cell, the profiles of plasmalemma and organelles were first traced from the micrographs onto transparent paper, according to the method of Sch6tz (1972). Then they were evaluated as described by Arnold and Gaffal (1979b), by means of superimposing the transparent papers and by numbering of each organelle profile. A few cells were examined quantitatively by measuring the areas of the profiles with a Kontron Digiplan-MOP and by multiplying them by the mean section thickness (between 65 and 85 nm), which was determined separately for each cell series (Arnold and Gaffal 1979b). This enabled us to find the values of the volumes of cells (within the plasmalemma) and organelles. We also reconstructed a three-dimensional model of the chondriome, the chloroplast, and the nucleus in a relatively young interphase cell (at t=0 of the vegetative life cycle) by scaling the mean section thickness of this cell series (75 nm) to a 3 mm thick polystyrene sheet. The profiles of the plasmalemma and the organelles were transposed via an enlarger from the transparent papers onto the polysterene; they were then cut out with a thermo-saw of Fischertechnik and glued together in the correct order to form a model with a scale of 40,000: t. The mitochondria were painted white, the nucleus red, and they were then arranged in and around the green chloroplast cup (Fig. 3) in the positions they had had at the time of fixation. To control the correct steric assembly of all profiles, we either superimposed all the transparent papers or the cut-outs of the polystyrene profiles of the cell and used the holes of the cut-out organelle discs as a stencil (Gaffal and Kreutzer 1977). Results In the course o f the s y n c h r o n i z e d vegetative life cycle o f Chlarnydomonas reinhardii, which is c h a r a c t e r i z e d by cell g r o w t h a n d division within 24 h (Fig. 1), m i t o c h o n d r i a increase s i m u l t a n e o u s l y with the cell by m e a n s o f m i t o c h o n d r i a l p r o l i f e r a t i o n , division, and, finally, d i s t r i b u t i o n o f the m o t h e r m i t o c h o n d r i a in the d a u g h t e r cells (Fig. 2). T w o cells w h i c h were studied at t = 0 o f the vegetative l i g h t - d a r k cycle c o n t a i n 5 a n d 11 m i t o c h o n d r i a . B o t h e l o n g a t e d (but n o t t o o m u c h b r a n c h e d ) a n d smaller f o r m s exist, which m a i n ly o c c u p y the p e r i p h e r y o f the cell a r o u n d the c h l o r o plast cup (Fig. 3). O u r investigations c o r r o b o r a t e the findings o f O s a f u n e et al. (1975) at t = 6, w h i c h m e a n s t h a t d u r i n g cell g r o w t h the n u m b e r o f m i t o c h o n d r i a increases to a b o u t 50 smaller m i t o c h o n d r i a l units which are either u n b r a n c h e d o r p o o r l y r a m i f i e d a n d o c c u p y m o r e central regions o f the cell. A t t = 9, they fuse to f o r m few b u t highly b r a n c h e d g i a n t m i t o c h o n -

R. Blank et al. : Mitochondria in Chlamydomonas dria s u r r o u n d e d by the c h l o r o p l a s t (Fig. 2), b u t they disintegrate into a larger n u m b e r o f p o o r l y b r a n c h e d m i t o c h o n d r i a at t = 14 to t = 16 in the course o f distrib u t i o n to the d a u g h t e r cells ( O s a f u n e et al. 1975, 1976). D u r i n g the d a r k p h a s e a p p a r e n t l y little or no change occurs. Therefore, it is p o s s i b l e t h a t the m i t o c h o n d r i a l p o p u l a t i o n in cells j u s t released f r o m the m a t e r n a l cell wall c o r r e s p o n d s to t h a t at t = 0 o f the vegetative cell cycle (Fig. 3). During gametogenesis under dark and cold conditions, the n u m b e r a n d f o r m s o f the m i t o c h o n d r i a in Chlamydomonas reinhardii r e m a i n m o r e or less constant (no difference b e t w e e n the m a t i n g types) in c o m p a r i s o n to the starting stage o f the vegetative cell cycle (Fig. 2). A f t e r g a m e t o g e n e s i s o f cells at t = 0 , which were s u b m i t t e d to i n d u c t i o n for 20 h, 4, 5, a n d 7 m i t o c h o n d r i a were c o u n t e d in six cells, a n d 6 a n d 9 m i t o c h o n d r i a were f o u n d in t w o cells after 60 h o f induction. T h r e e cells, which h a d been exp o s e d at t = 9 to d a r k - c o l d g a m e t o g e n e s i s for 20 h, c o n t a i n e d 8, 19, a n d 24 m i t o c h o n d r i a , w h i c h were d i s p e r s e d p r e d o m i n a n t l y in the centers o f the cells. As in the c o r r e s p o n d i n g vegetative cell stage, there are, in a d d i t i o n to s o m e smaller units, a few highly b r a n c h e d g i a n t m i t o c h o n d r i a , which can also be fused to a m i t o c h o n d r i a l n e t w o r k - in the cell with 24 m i t o c h o n d r i a , 1 n e t w o r k was f o u n d a n d , a d d i t i o n a l l y , 23 smaller m i t o c h o n d r i a l units (Blank a n d A r n o l d 1980). In crossing active gametes after light g a m e t o g e n e s i s o f cells at t = 9 , a netlike b r a n c h e d giant m i t o c h o n d r i o n was also detected, a n d in s o m e cells a d d i t i o n a l m i t o c h o n d r i a were d e t e c t e d as well ( G r o b e a n d A r n o l d 1975). A f t e r g a m e t o g a m y the n e t w o r k s o f p a r e n t s dissociate into small, either u n b r a n c h e d or p o o r l y b r a n c h e d m i t o c h o n d r i a ( G r o b e a n d A r n o l d 1977). T h e m a x i m u m n u m b e r o f single m i t o c h o n d r i a l units seems to be a r o u n d 50, which is the s a m e n u m b e r f o u n d at t = 6 o f the vegetative cell cycle (Fig. 2). T h r e e cells in the vegetative life cycle were investigated q u a n t i t a t i v e l y (Table 1); the c h l o r o p l a s t , h o w ever, was investigated in only one cell. The v o l u m e o f the c h l o r o p l a s t t a k e s u p n o t quite h a l f the cell (within the p l a s m a l e m m a ) , b u t the v o l u m e o f the c h o n d r i o m e is only a b o u t 3 % o f the cell or a b o u t half the nucleus. C o n s i d e r i n g the v a r i o u s s p o r u l a t i o n times, o u r values a r e in g o o d a g r e e m e n t w i t h the findings o f F i s c h e r (1969). W e c a n verify t h a t the cellular v o l u m e (within cell wall) increases a b o u t 3.5 times, whereas the c h o n d r i o m e increases a b o u t 3 times a n d the nucleus only a b o u t 2.5 times between t = 0 a n d t - - 6 . Interestingly enough, shapes a n d sizes o f the stage at t = 0 are in b r o a d a g r e e m e n t with those d e s c r i b e d by Sch6tz et al. (1972) for g a m e t e s after i n d u c t i o n f r o m a s y n c h r o n o u s b a t c h - c u l t u r e s at any stage o f the vegetative cell cycle.

R. Blank et al. : Mitochondria in Chlamydomonas

239

Fig. 3. Three-dimensional reconstruction of the mitochondria, the chloroplast, and the nucleus in a relatively young interphase cell of Chlamydornonas reinhardii, as analyzed from 60 consecutive sections of the whole cell. 11 mitochondria of various shapes (white) are arranged in and around the chloroplast cup according to their appropiate positions in the living organism. The nucleus (indicated by arrows) in the center of the celt is outlined by the chloroplast, (4 side views; apex; bottom)

240

R.

Table 1. Morphometric analysis of some volumes in three cells of Chlamydomonas reinhardii at two different stages of the vegetative life cycle, as revealed from 60 to 97 consecutive sections of entire cells. (The first cell listed is the same one as in F i g . 3) Volumes in gm 3

at t=O

at t=O

at

Cell Nucleus Chloroplast Chondriome Mitochondria

64.36

61,94

217.32

3.56

4,09

a

25.65

1

t=6

9s

a

1.67

1,96

5.36

0.01

0.02

0.02 0.03

2

0.01

0,05

3

0.01

0.43

0.05

4

0.02

0.55

0.05

5

0.03

0.91

0.05

6

0.07

-

0.06

7

0.13

-

0.06

8

0.21

-

0.06

9

0.25

-

0.07 0.07

10

0.35

-

11

0.58

--

0.07

12

--

--

0.07

13

-

-

0.08

14

-

-

0.08

15

-

--

0.08

16

-

-

0.08

17

-

-

0.09

18

-

-

0.09

19

-

-

0.10

20

-

-

0.10

21

--

-

0.11

22

-

-

O. 1 2

23

--

--

0.12

24

--

--

0.12

25

--

-

0.13

26

-

-

0.13

27

-

-

0.13

28

-

-

0.13

29

-

-

0.13

30

-

--

0.14

31

-

-

0.14 0.14

32

--

--

33

--

-

0.14

34

--

-

O. 1 6

35

-

-

0.16

36

-

-

0.17

37

-

-

0.19

38

-

-

0.19

39

-

-

0.21

40

--

--

0.25

41

--

-

0.28

42

-

-

0.29

43

-

-

0.42

Chloroplast volume not determined

The volumes of single- mitochondria vary from 0.01 to 0.91 lam 3 (for t = 0 ) and from 0.02 to 0.42 ~tm 3

(for t=6). These data show clearly that in the two cells at t = 0 mitochondria are present which are larger than 0.5 g m 3, whereas in the cell at t = 6 they are smaller (see Table 1). This reveals that an increase in mitochondrial number leads to an increase in mitochondria which are smaller than 0.1 ~tm 3.

Blank et al. : Mitochondria in Chlamydomonas

Discussion During the vegetative and generative life cycles of Chlamydomonas reinhardii, mitochondria increase simultaneously with the cell. However, the process of mitochondrial proliferation and division does not correspond to the classic scheme of maturing, enlarging, and dividing of a relatively compact form, i.e., of an organism, an organ or, perhaps even an organelle, as might be the case for the plastid in some singlecelled protists (beautifully described in all its intricate complexness for the colourless flagellate Polytoma papillatum by Gaffal 1978; Gaffal and Schneider 1978). The mitochondrial behavior in the course of the cell cycle is indeed of greatest complexity, presumably because the chondriome does not take on a relatively stiff, motionless, and compact form, but is constantly morphodynamically changing. Thus, the mitochondrial inventory is perpetually breaking up and coalescing. Single units, which can be composed of only one lobule, form larger and manifoldly lobulate mitochondria by means of aggregation; these aggregates can continue to fuse so that giant mitochondria or even mitochondrial networks result, which may again disintegrate into smaller mitochondria. Even if changes in the chondriome follow a certain periodic trend in the course of the life cycle, so that the mitochondrial population of a definite cell cycle stage has certain characteristics, the alterations of mitochondrial shapes are not confined to their circuit within the cellular life cycle, but occur permanently in each cell at any stage by means of a dynamically changing morphology. Just like the variety of mitochondrial shapes, the mitochondria also have a wide range of sizes from one stage to another; in fact, they vary in various cells at various stages as well as in several cells at the same stage and even in one and the same cell. We believe, in agreement with Gaffal (personal communication), that the mitochondrial micromorphology is very delicate and that the mitochondria are those organelles which are the most sensitive indicators to outside influences.

References Arnold, C.G., Gaffal, K . P . ( 1 9 7 9 a ) Extranuclefire Vererbung. Die Morphologie extranucle~irer Erbtr~iger im Verlauf des Lebenszyklus. F o r t s c h r . B o t . 4 1 , 2 1 2 - 2 2 0 Arnold, C.G., Gaffal, K . P . ( 1 9 7 9 b ) Die rfiumliche Struktur von Mitochondrien und Plastiden. Biol. in unserer Zeit 9 , 4 5 - 5 1 Arnold, C.G., Schimmer, O . , S c h 6 t z , F . , B a t h e l t , H . ( 1 9 7 2 ) Die Mitochondrien von Chlamydomonas reinhardii. Arch. M i k r o biol. 81, 50-67 R., Arnold, C . G . ( 1 9 8 0 ) Variety of mitochondrial shapes, sizes, and volumes in Chlamydomonas reinhardii. Protoplasma

Blank,

104, 187-191

R. Blank et al. : Mitochondria in Chlamydomonas Fischer, B. (1969) Morphometrische Bestimmung der Zellkonstituenten von Chlamydomonas reinhardii Dangeard w/ihrend eines Entwicklungszyklus. Diss. Thesis, G6ttingen Gaffal, K.P. (1978) Configural changes in the plastidome of Polytornapapillatum after completion of cytokinesis and during fusion of the gametes. Protoplasma 94, 175 191 Gaffal, K.P., Kreutzer, D. (1977)The mitochondria of Polytoma papillatum at two different stages of the vegetative cell cycle. Protoplasma 91, t67 177 Gaffal, K.P., Schneider, G.J. (I978) The changes in ultrastructure during fertilization of the colourless flagellate Polytoma papillaturn with special reference to the configural changes of their mitochondria. Cytobiol. 18, 161-173 Grobe, B. (1976) Untersuchungen zum Mitochondrienverhalten in der Zygote von Chlarnydornonas reinhardii. Diss. Thesis, Erlangen Grobe, B., Arnold, C.G. (1975) Evidence of a large, ramified mitochondrium in Chlamydomonas reinhardii. Protoplasma 86, 291294 Grobe, B., Arnold, C.G. (1977) The behaviour of mitochondria in the zygote of Chlarnydornonas reinhardii. Protoplasma 93, 357-361 Hutner, S.H., Provasoli, L., Schatz, A., Haskins, C.P. (1950) Some approaches to the study of the role of metals in the metabolism of microorganisms. Proc. Am. Phil. Soc. 94, 152-170 Ishiura, M. (1976) Gametogenesis of ChIamydornonas in the dark. Plant Cell Physiol. 17, 1141-1150 Ishiura, M., Iwasa, K. (1973a) Gametogenesis in Chlamydomonas. I. Effect of light on the induction of sexuality. Plant Cell Physiol. 14, 911-921 Ishiura, M., Iwasa, K. (1973b) Gametogenesis in Chlamydomonas. II. Effect of cycloheximide on the induction of sexuality. Plant Cell Physiol. 14, 923 933 Ishiura, M., Iwasa, K. (1973c) Gametogenesis in Chlamydomonas. III. Daily fluctuation of sex-competence. Plant Cell Physiol. 14, 935-939 Kuhl, A., Lorenzen, H. (1964) Handling and culturing of Chlorella. In: Methods in cell physiology, vol. I, pp. 159-187, Prescott, D.M., ed. Academic Press, New York Loppes, R. (1966) Damage induced by methyl methanesulfonate (MMS) in Chlarnydomonas reinhardi. Z. Vererbungsl. 98, 193202

241 Lorenzen, H. (1959) Die photosynthetische Sauerstoffproduktion wachsender Chlorella bei langfristig intermittierender Belichtung. Flora 147, 382-404 Mihara, S., Hase, E. (1971) Studies on the vegetative life cycle of Chlarnydomonas reinhardi Dangeard in synchronous culture. I. Some characteristics of the cell cycle. Plant Cell Physiol. 12, 225-236 Osafune, T., Mihara, S., Hase, E., Ohkuro, I. (1975) Electron microscope studies of the vegetative cellular life cycle of Chlarnydornonas reinhardi Dangeard in synchronous culture. III. Three-dimensional structures of mitochondria in the cells at intermediate stages of the growth phase of the cell cycle. J. Electron Microsc. 24, 247-252 Osafune, T., Mihara, S., Hase, E., Ohkuro, I. (1976) Electron microscope studies of the vegetative cellular life cycle of Chlarnydomonas reinhardi Dangeard in synchronous culture. IV. Mitochondria in dividing cells. J. Electron Microsc. 25, 261-270 Pfau, J., Werthmfiller, K., Senger, H. (1971) Permanent automatic synchronization of microalgae achieved by photoelectrically controlled dilution. Arch. Mikrobiol. 75, 338-345 Schl6sser, U. (1966) Enzymatisch gesteuerte Freisetzung yon Zoospore bei Chlarnydornonas reinhardii Dangeard in Synchronkultur. Arch. Mikrobiol. 54, 129-159 Schl6sser, U.G. (1976) Entwicklungsstadien- und sippenspezifische Zellwand-Autolysine bei der.Freisetzung von Fortpflanzungszellen in der Gattung Chlarnydomonas. Ber. Dtsch. Bot. Ges. 89, 1-56 Sch6tz, F. (1972) Dreidimensionale, magstabsgetreue Rekonstruktion einer gr/inen Flagellatenzelle nach Elektronenmikroskopie yon Serienschnitten. Planta 102, 152-159 Sch6tz, F., Bathelt, H., Arnold, C.G., Schimmer, O. (1972) Die Architektur and Organisation der Chlamydomonas-Zelle. Ergebnisse der Elektronenmikroskopie yon Serienschnitten und der daraus resultierenden dreidimensionalen Rekonstruktion. Protoplasma 75, 229-254 Spurr, A.R. (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43

Received 21 May; accepted 18 July 1980