499
Journal of Cell Science 108, 499-507 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
Behavior of mitochondria in synchronized cells of Chlamydomonas
reinhardtii (Chlorophyta)
Tomoko Ehara1,*, Tetsuaki Osafune2 and Eiji Hase3
1Department of Microbiology, Tokyo Medical College, 6-1-1 Shinjuku, Tokyo 160, Japan
2Department of Life Sciences, Nippon College of Physical Education, Kamosida, Aobaku,
3Laboratory of Chemistry, Faculty of Medicine, Teikyo University, Hachioji, Tokyo 192-03,
Yokohama 227, Japan
Japan
*Author for correspondence
SUMMARY
Cells of Chlamydomonas reinhardtii Dangeard were synchronized under a 12 hour:12 hour light:dark regimen.
Behavior of mitochondria in these cells was studied by
fluorescence microscopy using a mitochondrial membranebinding fluorescent dye, dimethylaminostyrylmethylpyridiniumiodine (DASMPI), as well as by electron
microscopy.
Following time courses of change in frequency of occurrence of five typical morphologies of mitochondria in synchronized cells, strikingly dynamic behavior of mitochondria was demonstrated. The five types are (A) a giant global
mitochondrion with large matrix and peripherally
localized cristae, a part of which is in close contact with the
nucleus, (B) a mitochondrion composed of thick-corded
bodies connected to each other, a part of which is in contact
with the nucleus, (C) thin-corded forms with a few
branches, (D) small lump forms scattered in the cytoplasm,
and (E) stringy forms with intricate branchings extended
throughout the cytoplasm. During the early half of the light
period, changes of C→B→C→D occur, while the inversely
sequential changes of D→C→B→C proceed during the
later half of the light period. The appearance of the B-type
mitochondrion is accompanied by a transient decrease of
O2-uptake activity of cells. The early appearing B-type
mitochondrion is temporarily turned into a giant A-type
mitochondrion, concomitant with discharge of membranes
into the cytoplasm and their retake by the A-type form in
the process of reversion to B-type. In the reversion process,
partitioning membranes are also formed in the large
matrix of A-type mitochondrion. Toward the end of the
light period, stringy E-type mitochondria are formed from
C-type ones, and apportioned between two dividing protoplasts during an early phase of the dark period. The E-type
forms are cut into D-type forms immediately prior to the
following cell division into four daughters.
Possible significance of the formation of B- and A-type
mitochondrion is discussed.
INTRODUCTION
dimensional models. Numerical, morphological and topographical heterogeneity of the chondriosome in Polytoma
papillatum have also been shown by Gaffal and Schneider
(1980) by electron microscopy using the serial-sectioning
technique.
In this work, behavior of mitochondria in synchronized cells
of Chlamydomonas reinhardtii was re-examined by fluorescence microscopy using a mitochondrial membrane-binding
fluorescent dye, DASMPI, as a specific vital stain as well as
by electron microscopy with improved methods. Following
changes in frequency of occurrence of typical morphologies of
mitochondria in synchronized cells, a characteristic pattern of
stage-dependent changes in mitochondrial morphology was
demonstrated.
Cell of Chlamydomonas reinhardtii synchronized under alternations of 12 hour light period and 12 hour dark period in a
mineral medium have been shown to increase in size during
the light period and divide during the dark period (Mihara and
Hase, 1971, 1978; see Fig. 1, upper, and Fig. 3A). During the
12 hour dark period, the nuclear division, chloroplast division
and cell division occur successively, and zoospores are
liberated toward the end of the dark period (Mihara and Hase,
1978; Ehara et al., 1990a). Behavior of chloroplast and its
nucleoids in synchronized Chlamydomonas cells has been
studied in our recent work (Ehara et al., 1990a).
Changes of mitochondrial morphology in synchronized cells
of Chlamydomonas reinhardtii have been studied by electron
microscopy in our previous work (Osafune et al., 1972a,b,
1975b, 1976). Arnold and his colleagues (Blank and Arnold,
1980; Blank et al., 1980), have shown various shapes, sizes and
volumes of mitochondria of Chlamydomonas reinhardtii by
electron microscopy with the use of serial sections and three-
Key words: Chlamydomonas reinhardtii, mitochondrion (giant)
synchronized culture
MATERIALS AND METHODS
Chlamydomonas reinhardtii (Dangeard) was obtained from the Algal
Culture Collection (IAM C-9) of the Institute of Applied Microbiol-
500
T. Ehara, T. Osafune and E. Hase
ogy, the University of Tokyo, and has been maintained in Department
of Microbiology, Tokyo Medical College.
Synchronized culture
Algal cells were cultured under alternations of 12 hour light period
and 12 hour dark period (Ehara et al., 1990a). The light intensity was
8000 lux at the surface of the flat culture container. Details of procedures were the same as those described by Mihara and Hase (1971).
DASMPI-staining of mitochondria
The staining was performed in accordance with the report by BereiterHahn (1976). A portion of culture was taken out and DASMPI
(purchased from Accurate Chemical and Scientific Corp, New York)
was added to give a final concentration of 70-100 µg/ml. The mixture
was aerated with 1.5-2.0% CO2-containing air, and illuminated or
placed in the dark as in the original culture. After 5-8 minutes, it was
centrifuged at 200-400 g for about 2 minutes. The pellet was washed
with culture medium, and DASMPI-stained mitochondria were
observed with an Olympus fluorescence microscope (Ehara et al.,
1984; Osafune et al., 1987; Ito-Kuwa et al., 1988).
Electron microscopy
Cells were fixed with 1% (v/v) glutaraldehyde and post-fixed with
0.5% (w/v) OsO4 (in water at 4°C). In some experiments (see Fig. 8),
1% glutaraldehyde was replaced by 1% (w/v) paraformaldehyde plus
0.2% glutaraldehyde. Other procedures were as described previously
with Euglena cells (Ehara et al., 1984).
Computer-aided stereographic representation of
mitochondrial morphologies
A cell was serially cut into about 90 sections, unless otherwise
mentioned. These cell sections were photographed at an appropriate
magnification, and organelles in each section were drawn on tracing
paper, as described previously (Osafune et al., 1989). Using Nikon’s
Cosmozone 2SA programme, these data were entered on a digitizing
table. The three-dimensional data in the computer were viewed on a
color display (NEC) and the image was rotated in space until a proper
angle of view was obtained. From the data in the computer, total
volume and surface area of mitochondria, chloroplast and nucleus
were estimated (Table 1).
Fig. 1. Growth and daughter cell liberation on synchronized cells of
Chlamydomonas reinhardtii Dangeard (upper) and change in O2uptake activity of cells (lower). Vertical bars represent the range of
variation and circles the mean values in four experiments.
RESULTS
The mitochondrial morphologies observed in synchronized
cells were tentatively classified into five types, A to E. These
are (A) a giant global mitochondrion with large matrix and
peripherally localized cristae, a part of which is in close contact
with the nucleus (Figs 4B, 5 and 10C), (B) a mitochondrion
composed of thick-corded bodies connected to each other, a
part of which is in contact with the nucleus (Figs 4A,C and 8),
(C) thin-corded forms with a few branches (Fig. 10A), (D)
small lump forms scattered in the cytoplasm (Fig. 4F), and (E)
stringy forms with intricate branchings extended throughout
the cytoplasm (Figs 4D,E, 9 and 10E,F). Fig. 2 shows changes
in frequency of occurrence of these five types of mitochondrial morphology during the 12 hour light period. A characteristic pattern of change in mitochondrial morphology
emerges from the data in Fig. 2. During the early half of the
light period, changes of C→B→C→D occur, while the
inversely sequential changes of D→C→B→C proceed during
the later half of the light period. Interestingly, the curves
showing the earlier changes and those representing the later
changes are approximately symmetrical with respect to the
middle of the light period.
The appearance of the B-type mitochondrion is accompanied by a transient decrease of O2-uptake activity of
cells (Fig. 1, lower). The early appearing B-type mitochondrion is temporarily turned into a giant global form of A-type.
The C-type mitochondria formed toward the end of the light
period are changed into E-type ones shortly before and after
the beginning of dark period, as seen from Figs 2 and 3B-a.
As seen from Fig. 3 showing changes of mitochondrial morphology in dividing cells during the 12 hour dark period, the
Fig. 2. Time course of change in morphology of mitochondria in
synchronized cells during the 12 hour light period. The morphologies
of mitochondria are classified into five types (A, B, C, D and E), as
schematically represented in the figure. The frequency of appearance
of each type was estimated by observing about 200 cells taken from
a culture at one hour intervals, unless otherwise described. Vertical
bars represent the range of variation and lines the mean values in
four experiments.
Behavior of mitochondria in Chlamydomonas
501
Table 1. Changes in total volume and surface area of mitochondria and other organelles per cell during an early phase of
the Chlamydomonas cell cycle
Time of cell sampling
in hours after the onset
of light period
0.5 hours
1.5 hours (A*)
1.5 hours (B*)
4 hours
13 hours
Total volume in µm3
Mitochondria
Chloroplast
Nucleus
Cytoplasm
2.2
36.1
4.6
33.8
2.1
22.2
4.4
26.4
3.7
28.1
5.5
35.3
2.7
49.8
5.5
55.6
10.2
191.9
18.7
159.2
Total
76.7
55.1
72.6
113.6
380.0
40.6 (126)
144.4
18.6
224.5 (697)
483.4
41.6
Total surface area in µm2
Mitochondria
Chloroplast
Nucleus
32.2 (100)
107.3
14.9
20.7 (64)
74.8
14.2
16.1 (50)
100.5
18.6
*A and B correspond to the three-dimensional models B and C, respectively, in Fig. 10.
The data were obtained from the three-dimensional models of cell organelles in Fig. 10. See Materials and Methods and text for explanation.
E-type mitochondria are apportioned between two dividing
protoplasts (see Fig. 4E). Immediately before or after partitioning of four daughter cells in a mother cell, the E-type
mitochondria are rapidly cut into D-type ones (Fig. 3B-b and
c). The D-type mitochondria fuse into C-type ones in four
divided daughter cells (Figs 3B-c, 4F and G). Subsequently the
C-type mitochondria begin to be turned into B- and A-type
shortly before the onset of the following light period. The
appearance of an A-type giant mitochondrion from a B-type
one was also observed with maximum frequency at an early
time of the dark period extended beyond 12 hours (data not
shown). This indicates that the formation of an A-type mitochondrion is an essential event occurring early in the algal cell
cycle, and not the result of the dark to light transition.
Concurrently with the formation of the giant form, a membranous structure (MS) appears in the cytoplasm, as shown in
Fig. 5. In the reversion of A-type to B- and C-types, concentric membranes appear in the large matrix of A-type mitochondrion as an extension of similar membranes existing in the
cytoplasm, as seen from Fig. 6. Further, partitioning
membranes appear in the large mitochondrial matrix as an
extension of the existing mitochondrial outer membrane, as
seen from Fig. 7.
Figs 10A,B,C and D illustrate, in three-dimensional models,
the change from C-type mitochondria to B-type followed by
A-type and the reversion of A-type to C-type. Comparing a
single B-type mitochondrion composed of thick body (Fig.
10B) with two thin-corded C-type mitochondria (Fig. 10A), it
is inferred that corded bodies of C-type mitochondria fuse to
form a B-type mitochondrion, which subsequently is turned
into a giant global form of A-type form.
Table 1 shows changes in total mitochondrial volume and
surface area per cell, together with the data for other
organelles, observed during an early phase and at the 13th hour
after the onset of the light period. During the early 4 hour
period, total volume of mitochondria per cell remains nearly
constant, while total surface area decreases by about 50% temporarily when the A-type giant mitochondrion appears with
maximum frequency. This is consistent with the morphological change that corded and branched forms (C- and B-type) of
mitochondria are turned into the global A-type form (Fig.
10A,B,C and D) without alteration of total volume. The mito-
Fig. 3. Time course
of cell division (A)
and change in
morphology of
mitochondria (B)
during the 12 hour
dark period. V, Nondivided cell; V
| ,
divided into two
daughters; and ~,
V
divided into four.
Symbols for different
types of mitochondria
are the same as in
Fig. 2. The data in A
show the mean values
in four experiments.
502
T. Ehara, T. Osafune and E. Hase
chondria observed at the 13th hour after the beginning of the
light period in Table 1 are mostly E-type existing in cells
immediately before or during the division into two daughter
protoplasts, as seen from Figs 3, 4D and E, and Fig. 10E and
F. These E-type mitochondria are about 4 times larger in total
volume and 7 times larger in total surface area, when compared
Fig. 4. Fluorescence microscopic profiles of DASMPI-stained mitochondria. (A) B-type mitochondria with thick corded bodies in cells taken
from a culture 1.5 hours after the onset of the light period. (B) A giant A-type mitochondrion in a cell taken from a culture 1.5 hours after the
onset of the light period. (C) A B-type mitochondrion in a cell taken from a culture at the 10th hour of the light period. Note that thick bodies of
the mitochondrion surround the nucleus (N). (D) E-type mitochondria with complicated branchings extended throughout the cytoplasm in cells
taken from a culture one hour after the beginning of the dark period. (E) E-type mitochondria are being apportioned between two dividing
protoplasts. The cell sample was taken from the same culture as in D. (F) D-type mitochondria in four divided daughter cells taken from a
culture 8 hours after the beginning of the dark period. (G) C-type mitochondria in four divided daughter cells immediately before separation
taken from a culture at the end of the dark period.
Behavior of mitochondria in Chlamydomonas
with the mitochondria in cells observed shortly after the onset
of the light period.
As seen from a three-dimensional model in Fig. 10E, E-type
503
mitochondria are stringy forms with intricate branchings
extended throughout the cytoplasm. When Fig. 10E and F
showing E-type mitochondria with and without elimination of
Fig. 5. An electron microscopic profile of a giant A-type mitochondrion with large matrix and peripherally localized cristae in a cell taken from
a culture 1.5 hours after the onset of the light period. Symbols used in this and subsequent figures: CP, chloroplast; M, mitochondrion; MS,
membranous structure; N, nucleus. Note that a part of the giant mitochondrion is in close contact with the nucleus (arrow), and that a
membranous structure (MS) is present near the mitochondrion.
Fig. 6. Two examples of serial sections through an A-type giant mitochondrion containing concentric membranes continued to similar
membranes existing in the cytoplasm. The cell sample was taken from a culture 2 hours after the onset of the light period. For explanation of
symbols see legend to Fig. 5.
504
T. Ehara, T. Osafune and E. Hase
the image of chloroplast body are compared with each other,
it is seen that most parts of mitochondrial bodies are present
within a space delimited by the cup-shaped chloroplast, but
some stringy mitochondrial arms penetrate the chloroplast,
their distal ends reaching the cell wall. Examinations of serial
sections of cells containing E-type mitochondria revealed that
Fig. 7. Partitioning membranes (arrows) formed in the large matrix of an A-type mitochondrion in the process of conversion to B-type one. The
cell samples were taken from the same culture as in Fig. 6. For explanation of symbols see legend to Fig. 5.
Fig. 8. Two examples of serial sections through a B-type mitochondrion in a cell taken from a culture 9 hours after the onset of the light period.
Note that separate mitochondrial bodies on one section are continuous on the other section. Cells were fixed with 1% paraformaldehyde plus
0.2% glutaraldehyde. For explanation of symbols see legend to Fig. 5.
Behavior of mitochondria in Chlamydomonas
505
However, the mode of mitochondrial division remains to be
seen, since it is not clear whether the E-type is a single, continuous network of mitochondrial membranes or it consists of
plural units complicatedly branched membranes.
Fig. 9. One of the serial sections through E-type mitochondria in a
cell taken from a culture 13 hours after the onset of the light period
(one hour into the dark period). Note that a part of the mitochondrial
body exists beneath the cell wall (arrows), while most parts are
present within a space delimited by a cup-shaped chloroplast. For
explanation of symbols see legend to Fig. 5.
there are openings in the wall of the cup-shaped chloroplast
body, and stringy mitochondrial arms pass through these
openings of the chloroplast (Fig. 9, arrows).
DISCUSSION
Synchrony of mitochondrial behavior in
synchronized cells
Fig. 11 summarizes schematically the observations described
above. However, when the data in Fig. 2 are scrutinized, it is
seen that all mitochondria in synchronized cells do not follow
the same sequence of morphological changes as described in
Results.
The cells containing D-type mitochondria exist with a
frequency of about 20% during an early few hours of the light
period and a few hours before the end of the light period. These
D-type mitochondria appear not to be changed into B-type
during one cell cycle, at least: it is possible that the change of
these D-type mitochondria into B-type occurs during the
following cell cycle. This implies that the morphological
changes of mitochondria are not completely synchronous in
synchronized cells. As seen from Fig. 3B-a and b, however, all
the D-type mitochondria are turned into E-type mitochondria,
via C-type, shortly before and after the end of the light period.
The E-type mitochondria are apportioned between two
dividing protoplasts. Accordingly, the mitochondrial division
is synchronous and closely coupled with the cell division.
Dynamic changes in mitochondrial membranes
Marked changes in mitochondrial morphology observed in
synchronized cells mean dynamic changes of their membranes.
As shown in Table 1, the total surface area of mitochondria in
a cell dramatically decreases, while the total volume remaining
constant, concurrent with the formation of a giant global form
(A-type) from corded mitochondria (C- and B-type), and soon
returns to the initial level concomitant with the reversion of the
global to the corded forms. As seen from Fig. 5, membranous
structures are formed in the neighborhood of the giant A-type
mitochondrion, while concentric membranes appear in the
large matrix of the giant mitochondrion in the process of
reversion to the B-type. These concentric membranes are continuous with similar membranes existing in the cytoplasm, as
seen from Fig. 6. These observations suggest that when thincorded mitochondria (C-type) fuse into a thick-corded form (Btype) followed by the conversion into the giant global form (Atype), excess membranes are discharged into the cytoplasm,
and that these membranes are retaken into the large matrix of
the A-type mitochondrion when it is turned into the B-type
form. As shown in Fig. 7, partitioning membranes are formed,
as extension of the existing outer membrane, in the large matrix
of the A-type mitochondrion in the process of reversion to the
B-type form. It may be presumed that the concentric
membranes are those of mitochondrial cristae, while the partitioning membrane constitutes the outer membrane of newly
produced B-type mitochondrion.
Possible significance of the formation of B- and Atype mitochondrion
The question arises as to the significance of the occurrence of
B-type mitochondrion with two maximal frequency in synchronized cells. The result that thick bodies of a B-type mitochondrion are in close contact with the nucleus, frequently surrounding it (Fig. 4C), suggests that some interactions occur
between the two organelles. We have shown previously (Ehara
et al., 1990b) that a giant aggregate of chloroplasts is temporarily formed by connection of individual bodies at an early
and late stages of the cell cycle of Euglena gracilis Z, and that
a part of the chloroplast aggregate is in close association with
the nucleus. Recent immunoelectron microscope studies in our
laboratory (Osafune et al., 1993) revealed that there occurs an
association between DNA molecules from the nuclear chromosomes and those from the chloroplast nucleoids through bridges
formed between the two organelles, suggesting the occurrence
of genomic interactions between the two organelles. Experiments to see whether such DNA molecular interactions occur
between the B-type mitochondrion and the nucleus in synchronized cells of Chlamydomonas are in progress.
The early appearing B-type mitochondrion is temporarily
turned into the A-type one with a large matrix and peripherally
localized cristae. The formation of a similar giant global mitochondrion from corded forms and its reversion to the latter by
producing partitioning membranes in the large matrix has been
observed during the cell cycle of Euglena gracilis Z in synchronized culture in our previous work (Osafune, 1973; Osafune
506
T. Ehara, T. Osafune and E. Hase
Fig. 10. Three-dimensional models of mitochondria of different morphologies. The magnifications are not the same. Yellow, mitochondria;
green, chloroplast; red, nucleus; blue, cell wall. (A) C-type mitochondria in a cell taken from a culture 30 minutes after the onset of the light
period. (B) A B-type mitochondrion in a cell taken from a culture 1.5 hours after the onset of the light period. (C) A giant A-type
mitochondrion in a cell taken from a culture 1.5 hours after the onset of the light period. (D) C-type mitochondria in the process of division into
small lump forms (D-type) in a cell taken from a culture 4 hours after the onset of the light period. (E and F) E-type mitochondria with (E) and
without (F) elimination of the image of chloroplast body in a cell taken from a culture 1 hour after the beginning of the dark period. To
construct this model, 135 serial cell sections were used. Fig. 9 shows one of these serial sections. Note that some mitochondrial arms penetrate
the chloroplast, their distal ends reaching the cell wall.
Behavior of mitochondria in Chlamydomonas
Fig. 11. A schematic representation of morphological changes of
mitochondria in synchronized cells of Chlamydomonas reinhardtii.
See Mihara and Hase (1978) and Ehara et al. (1990) for the nuclear
and chloroplast divisions.
et al., 1975a,c). Recent preliminary immunoelectron microscopic localization of mitochondrial DNA in Euglena gracilis Z
showed that DNA molecules gather in the large matrix of the
giant mitochondrion, while they are separately localized in the
reticulate form, suggesting that direct interactions among
otherwise separately existing mitochondrial DNA molecules
occur in the large matrix (Ehara et al., 1993). Examinations of
similar phenomenon in Chlamydomonas are in progress.
We thank Messrs Y. Tanaka and Y. Ikeda for their technical assistance. This work was supported by grants from the Kazato Research
Foundation (1988, to T.E.) and the Ministry of Education, Science
and Culture in Japan (Nos 02304007 and 05640736 to T.O.).
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(Received 20 December 1993 - Accepted, in revised form,
27 June 1994)