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Cell Motility and the Cytoskeleton 41:297–307 (1998)
Flagellar Coordination in Chlamydomonas
Cells Held on Micropipettes
Ursula Rüffer* and Wilhelm Nultsch
Fachbereich Biologie der Philipps-Universität Marburg, Marburg, Germany
The two flagella of Chlamydomonas are known to beat synchronously: During
breaststroke beating they are generally coordinated in a bilateral way while in
shock responses during undulatory beating coordination is mostly parallel [Rüffer
and Nultsch, 1995: Botanica Acta 108:169–276]. Analysis of a great number of
shock responses revealed that in undulatory beats also periods of bilateral
coordination are found and that the coordination type may change several times
during a shock response, without concomitant changes of the beat envelope and the
beat period. In normal wt cells no coordination changes are found during
breaststroke beating, but only short temporary asynchronies: During 2 or 3 normal
beats of the cis flagellum, the trans flagellum performs 3 or 4 flat beats with a
reduced beat envelope and a smaller beat period, resulting in one additional trans
beat. Long periods with flat beats of the same shape and beat period are found in
both flagella of the non-phototactic mutant ptx1 and in defective wt 622E cells.
During these periods, the coordination is parallel, the two flagella beat alternately.
A correlation between normal asynchronous trans beats and the parallelcoordinated beats in the presumably cis defective cells and also the undulatory
beats is discussed. In the cis defective cells, a perpetual spontaneous change
between parallel beats with small beat periods (higher beat frequency) and bilateral
beats with greater beat periods (lower beat frequency) are observed and render
questionable the existence of two different intrinsic beat frequencies of the two
flagella cis and trans. Asynchronies occur spontaneously but may also be induced
by light changes, either step-up or step-down, but not by both stimuli in turn as
breaststroke flagellar photoresponses (BFPRs). Asynchronies are not involved in
phototaxis. They are independent of the BFPRs, which are supposed to be the basis
of phototaxis. Both types of coordination must be assumed to be regulated
internally, involving calcium-sensitive basal-body associated fibrous structures.
Cell Motil. Cytoskeleton 41:297–307, 1998. r 1998 Wiley-Liss, Inc.
Key words: high-speed microcinematography; beat pattern; beat period; breaststroke flagellar photoresponses; photoshock; undulatory beats; asynchronies
INTRODUCTION
Beating of the two flagella in Chlamydomonas is
generally coordinated. Two kinds of coordination are
found: (1) The bilateral flagellar coordination normal for
breaststroke beats (B-beats) during forward swimming.
With the synchronous recovery stroke (bends developing
from near the base to the tip), both flagella are brought to
the front of the cell. Then the flagella sweep backwards
simultaneously on both sides of the cell during the
effective stroke. (2) The parallel coordination of undulatory beats (U-beats) during backward swimming (shock
r 1998 Wiley-Liss, Inc.
response). In these beats there is no backward sweeping,
only alternating bends or waves traveling along the
Abbreviations used: BFPR ⫽ breaststroke flagellar photoresponse;
B-beats ⫽ breaststroke beats; U-beats ⫽ undulatory beats.
Contract grant sponsor: Deutsche Forschungsgemeinschaft.
*Correspondence to: Dr. Ursula Rüffer, Fachbereich Biologie der
Philipps-Universität Marburg, Botanik, Lahnberge, D-35032 Marburg,
Germany. E-mail: rueffer@mailer.uni-marburg.de
Received 29 April 1998; accepted 3 August 1998
298
Rüffer and Nultsch
TABLE I. Breaststroke Flagellar Photoresponses (BFPR) of
wt Chlamydomonas Cells by Step-Up (light-on) and Step-Down
(light-off) Light Stimulation: Front Amplitude Changes of Cis
and Trans Flagellar Beats*
Step-Up
Step-Down
Type
cis
trans
cis
trans
Phototaxis
(⫹)
(⫺)
⬍
⬎
⬎
⬍
⬎
⬍
⬍
⬎
Positive
Negative
*Flagellar beat pattern types BFPR (⫹) and (⫺). Inverse changes
increase (⬎) and decrease (⬍) of front beat amplitude in cis and trans
flagellum, and inverse changes by step-up and step-down light
stimulation, presumably causing phototactic steering, positive and
negative [see Rüffer and Nultsch, 1991, 1997].
flagella from base to tip. The inward bend of the one
flagellum (corresponding to the recovery stroke of Bbeats) is synchronous with the outward bend of the other
flagellum (corresponding to the effective stroke of Bbeats) and vice versa [Rüffer and Nultsch, 1995]. In both
cases, cis (near the stigma) and trans flagellum have equal
beat periods (or beat frequencies), they are synchronized.
Analysis of breaststroke beats in free-swimming
cells [Rüffer and Nultsch, 1985] as well as of those held
on micropipettes [Rüffer and Nultsch, 1987] revealed that
even without an obvious stimulation the usual synchrony
may be interrupted at irregular intervals by short asynchronies: During 2 or 3 beats of the cis flagellum, the trans
flagellum performs one additional beat. In some cells
asynchronies are very frequent, while in many others
there are none at all. In a few cells coordination was
completely lost and both flagella beat with different but
constant beat periods, the trans beat periods always being
shorter than the cis beat periods [Rüffer and Nultsch,
1987].
So far two kinds of flagellar photoresponses have
been analyzed: (1) The breaststroke flagellar photoresponses (BFPR), which concern changes in the flagellar
beat pattern of breaststroke beating, including changes of
beat amplitude and beat period [Rüffer and Nultsch,
1990, 1991]. The changes of beat amplitude are inverse in
the two flagella of wt cells and are induced by step-up as
well as by step-down light stimulation (Table I). They are
assumed to be the basis of phototaxis. These changes are
equal instead of inverse in cis and trans flagella of the
non-phototactic mutant ptx1 [Rüffer and Nultsch, 1997].
(2) The shock response (undulatory flagellar photoresponses), in which the mode of beating is changed from
breaststroke to undulatory [Rüffer and Nultsch, 1995]. In
this reaction both flagella change the beat pattern equally
in wt as well as in ptx1 cells.
Now a third complex of photoresponses shall be
presented, which concerns the coordination system. In the
studies of BFPRs hitherto published [Rüffer and Nultsch,
1990, 1991, 1997], only synchronous bilateral beating
was considered in order to separate the different responses. In addition to the BFPRs, also asynchronies (i.e.,
short disturbances of the bilateral coordination) were
found to be induced by light changes. A complete change
of the coordination type from bilateral to parallel takes
place in the photoshock response [Rüffer and Nultsch,
1995]. It is usually connected with the change of the beat
pattern. However, it will be shown in this paper that it is
an independent reaction: coordination changes between
bilateral and parallel may be observed in breaststroke as
well as in undulatory beating.
MATERIALS AND METHODS
For this study, most films hitherto recorded and
used for previous papers were analyzed with respect to
the coordination of the two flagella. Cells of Chlamydomonas reinhardtii wt strain 622E [Rüffer and Nultsch, 1987,
1990, 1991, 1995] and wt strain 137⫹ [Rüffer and
Nultsch, 1997] or of the non-phototactic mutant ptx1
[Rüffer and Nultsch, 1997] were held on micropipettes in
an open droplet of fresh culture medium while being
recorded mostly with red background light (OG 570,
SCHOTT, 1,900Wm⫺2). In some cases, the recording
light was white or blue (BG38, 1,900Wm⫺2). For the
analysis of light responses, either short lateral flashes
were introduced [Rüffer and Nultsch, 1995] or constant or
pulsed lateral light, white or blue (BG38, SCHOTT,
1,100Wm⫺2). The pulsed light was meant to simulate the
rotation of the free-swimming cells and was given with
pulses of about 1 Hz (1⁄2 s light on, 1⁄2 s light off) or of 2 Hz
(1⁄4 s light on, 1⁄4 s light off). Shocks were also induced by
introducing the full background light.
High-speed films were taken at 440–525 frames/s
using phase-contrast optics. Films were analyzed frame
by frame and flagella retraced by hand after projection
onto a drawing table. In the figures 1, 6, 9, 11, 13, outlines
of pipettes and cell bodies including the stigma are
schematic. The stigma is not visible in the phase-contrast
records but its position was determined before each
experiment, and lateral light or flashes were applied on
the stigma side.
RESULTS
Asynchronies in Breaststroke Beats
‘‘Spontaneous’’ asynchronies. The normal Bbeats of wt Chlamydomonas cells are synchronized such
that the two flagella perform recovery and effective stroke
about simultaneously (bilateral coordination). In numerous wt cells, this coordination is interrupted at irregular
intervals by short asynchronies [Rüffer and Nultsch,
1987] without an obvious external stimulation, though
the continuous background light may play a role. The
asynchronies are always of the same type: the trans
Flagellar Coordination in Chlamydomonas
299
Fig. 1. ‘‘Spontaneous’’ asynchrony in a wt 622E cell. The trans
flagellum (upper row) is performing one additional beat while the cis
flagellum (below) continues normal beating. Beats are shown beginning with the recovery stroke ⫽ weak dashed lines, then effective
stroke ⫽ solid lines, up to the first frame of the next recovery stroke ⫽
strong solid lines. Strong dashed lines ⫽ normal front amplitude.
Numbers denote frames. In the three asynchronous trans beats backward amplitude (see f18, f25) as well as front amplitude (see f21, f28)
are strongly reduced resulting in flat lateral beats. Bar ⫽ 10 µm.
flagellum performs one additional beat during 2 or 3 beats
of the cis flagellum, which keeps its normal mode of
beating (Fig. 1). The trans flagellum does not increase the
rate of movement during the asynchronies (sometimes it
is even decreased), but the 3 or 4 asynchronous beats are
flatter than usual; backward and front amplitude are
reduced. The reduction of the beat envelope (the total area
covered by the flagellum during a beat) is the cause for
the reduced periods of these beats. Often merely a
beginning desychronization is observed with the trans
flagellum going ahead (normally the cis flagellum is
slightly ahead), but after one to several beats the normal
synchronization is restored. The course of free-swimming
cells was found to be the same with and without single
asynchronies [Rüffer and Nultsch, 1985]. It is obvious
that the flat asynchronous trans beats are less effective
than normal beats, which appears to make up for the
additional trans beat.
Asynchronies Induced by Light Changes
Step-up and step-down responses. Many wt cells only
display synchronous beating throughout a record. However, a step-up or step-down light stimulation induced
one or a series of asynchronies in about 50% of these cells
before constant synchrony was resumed (Fig. 2A,B). In
cells with asynchronies before the light change, the
number of asynchronies was only slightly increased (Fig.
3Ab,Bb). The total of all cells investigated shows a
significant increase of asynchronies (Fig. 3Ac,Bc), mainly
due to the asynchronies induced in formerly synchronous
cells (Fig. 3Aa,Ba). All cells with clear step-up asynchronies (Fig. 3Aa) were found to have BFPRs of type(⫹) or
no response, while cells with clear step-down asynchronies (Fig. 3Ba) had strong BFPRs type (⫺).
Light induction of asynchronies and the parallelism
with the response type of BFPRs were confirmed by the
pulsed light experiments. In all cells reacting according to
the (⫹)-type of BFPRs, the majority of asynchronies
(Fig. 4A), in most cases all asynchronies (Fig. 5A), were
found in the light-phases, i.e., as step-up responses. In the
cells reacting according to the (⫺)-type of BFPRs,
asynchronies were predominently found in the light-off
phases (Fig. 4B, 5B), i.e., as step-down responses. Even
in cells without a tendency to ‘‘spontaneous’’ asynchronies, it must be expected that the last asynchronies of a
series are found in the next phase (compare Fig. 2). It
appears that only step-up or step-down asynchrony
responses are induced and not both responses in turn as
beat period and pattern changes. In both cases, asynchrony responses are found along with the same pattern
of BFPRs, as the step-up(⫹)BFPR equals the stepdown(⫺)BFPR (see Table I). The two reactions are
superimposed. Like the synchronous beats the smaller
trans beats in light-induced asynchronies are shifted to
the front of the cell in contrast to the lateral ‘‘spontaneous’’ asynchronous beats (Fig. 6A). If there are several
asynchronies in a series, this shift is gradually reduced in
300
Rüffer and Nultsch
Fig. 2. Asynchronies induced by step-up ¨ and step-down Ô light
stimulation in wt 622E cells. Cis flagellar beats are presented at the
scale given in A. In the asynchronies (As) the trans flagellum performs
one additional beat. sy ⫽ synchronous, b. ⫽ beats. Arrowheads
indicate reaction times. A: Do ⫽ red recording light only. Asynchronies induced by the onset of lateral white light (L). The flagellate shows
BFPRs type (⫹). B: Asynchronies induced by the set-off (D) of lateral
blue light (Lo, 2 min.). The flagellate shows BFPRs type (⫺).
Fig. 3. Number of asynchronies 1 s before a light change (left column)
and 1 s after a light change (right). A: Step-up (flash or lateral light on).
B: Step-down (lateral light off). a: Cells with synchronous beating
before the light change. b: Cells with asynchronies before the light
change. c: Total. (Aa): 19 cells, (Ab): 21 cells, (Ac): 63 cells, including
23 cells without any asynchronies. (Ba): 8 cells, (Bb): 3 cells, (Bc): 16
cells, including 5 cells without asynchronies. All cells in (Aa) had
BFPRs type (⫹) or none, all cells in (Ba) had BFPRs type (⫺). Wt
622E.
the following asynchronies, indicating that the two reactions are independent. This independence is confirmed by
the opposite shift of the asynchronous trans beats in the
exceptional asynchronies in the other phase (Fig. 6B). Moreover, BFPRs were observed without any asynchrony responses, or asynchrony responses without flagellar responses.
Reaction times of asynchrony responses varied
enormously (Fig. 7), between 40 and 250 ms for most
step-up responses and 70–250 ms for most step-down
responses. In step-down responses often reaction times
were even longer, up to 1 s (compare Fig. 2B). Therefore,
in the pulsed-light experiments some light-induced asynchronies might well be shifted into the next phase because
of a long reaction time.
Asynchronies after shock responses. After a shock
response the first few B-beats were usually synchronous,
but very often soon followed by one asynchrony or a
series of asynchronies before normal synchrony was
restored (Fig. 8). Asynchronies of this kind were found in
17/28 shocks; in 11 cells synchrony continued. In another
12 cells after the shock, asynchronies were produced till
the end of the record.
Coordination Changes of Undulatory Beats.
In the shock response, the flagella change their
beating mode from B- to U-beats and the position of the
beat envelope from lateral to frontal. They also change
Flagellar Coordination in Chlamydomonas
Fig. 4. Number of asynchronies in light-on (L) and light-off (D) phases of lateral pulsed light (1 or 2 Hz).
A: 16 cells with BFPRs type (⫹) (7 cells wt 622 E, 9 cells wt 137⫹), including 8 cells (2 ⫹ 6) without any
asynchronies. a ⫽ 1⁄2 s before the first step-up (2 cells). B: 46 cells with BFPRs type (⫺), including 9 cells
without asynchronies, wt 622E, a ⫽ 1⁄2 s before the first light change (7 cells, 8 cells with pulsed light before
the record was started are not included).
Fig. 5. Asynchronies in pulsed light. A: Asynchronies apparently induced by step-up light stimulation.
Pulsed light of 1 Hz. BFPRs type (⫹). Wt 137⫹. B: Aynchronies apparently induced by step-down light
stimulation. Pulsed light of 1 Hz. BFPRs type (⫺). Wt 622E.
301
302
Rüffer and Nultsch
Fig. 6. Superposition of asynchrony responses and BFPRs in a wt
622E cell with BFPRs type (⫺) in pulsed light (2 Hz). A: The smaller
beat envelopes of the asynchronous trans beats (as) are shifted to the
front during the D-phases as normal and like the synchronous beats (s).
B: In the exceptional asynchronies during L-phases, the asynchronous
trans beats are shifted to the back like the synchronous beats.
Fig. 7. Reaction times (ms) of step-up and step-down asynchrony
responses. Open circles ⫽ beginning of the first asynchrony after a
step-up light change in 43 cells (including the first light-on phase of
pulsed-light experiments); obelisks ⫽ beginning of the first asynchrony
after a step-down light change in 27 cells (including the first light-off
phase of pulsed-light experiments); closed circles ⫽ beginning of the
next asynchrony (not induced by light changes) after the moment 1⁄2 s
before a light-step up or down in all 70 cells with asynchronies after the
light change. Most cells (68%) had no asynchronies during this time
(⬎500 ms).
period and envelope size, which were continually increased during the shock responses [Rüffer and Nultsch,
1995].
Coordination Changes of Breaststroke Beats
the type of coordination. Usually the flagella then beat in
parallel (Fig. 9A): The ‘‘recovery’’ stroke of one flagellum is performed simultaneously with the ‘‘effective’’
stroke of the other flagellum and vice versa. In freeswimming cells, this coordination type is favoured by
hydrodynamics and by the vibration of the cells [Rüffer
and Nultsch, 1995]. However, a bilateral coordination of
U-beats is also found (Fig. 9B), even in free-swimming
cells but especially in cells held on micropipettes, where
vibration is missing. Outward waves (‘‘effective’’ strokes)
and inward waves (‘‘recovery’’ strokes) are performed
simultaneously in both flagella, as in typical B-beats.
In 43 shock responses of 622E cells held on
micropipettes, as much as 23% of the non-deactivated
U-beats were coordinated bilaterally (Table II). A multiple change of coordination type was often observed at
the beginning or/and the end of a response (Fig. 10). Only
9/43 cells did not show a temporary change to bilateral
coordination. Mere bilateral coordination was not found
in any case. Most changes in both directions were
performed by half an additional beat of the trans flagellum (Table II). In shock responses of wt137⫹ cells,
changes between the two coordination types were also
found. The coordination changes did not influence beat
In general, synchronized B-beats are coordinated in
the bilateral way with synchronous recovery and effective
strokes of both flagella. However, under certain circumstances periods of alternating B-beats of the two flagella
were observed with equal beat periods of cis and trans
flagellum, i.e., these beats were also fully synchronized.
While one flagellum performed the recovery stroke, the
other one performed the effective stroke (Fig. 11B). This
coordination corresponds to the prevailing coordination
type of U-beats; it is parallel. Changes to anormal parallel
coordination occurred very frequently in the phototaxis
deficient mutant strain ptx1, but they were also found in
some wt 622E cells.
Coordination changes in the mutant strain ptx1.
The Chlamydomonas mutant strain ptx1 has been investigated for its lack of phototaxis. Analysis of flagellar beat
patterns revealed that this deficiency is due to a loss of cis
flagellar peculiarities. Both flagella respond to changes of
light like wt trans flagella [Rüffer and Nultsch, 1997]. A
most striking phenomenon in the ptx1 films is the
perpetual change of coordination of the two flagella.
Frequent changes between bilateral and parallel coordination were found in most cells (Table III). During the few
beats of transition, either the cis or the trans flagellum
Flagellar Coordination in Chlamydomonas
303
Fig. 8. Asynchronies following a shock response. The photoshock had been elicited by the white recording
light. A 7th asynchrony was only started but synchrony was restored. U ⫽ undulatory beats, B ⫽
breaststroke beats. Wt 622E.
Fig. 9. Types of flagellar coordination in undulatory beats. The
photoshock response was induced by the onset of the blue recording
light. After 24 parallel beats, the flagella of a wt 622E cell changed to 9
bilateral beats by an additional trans half-beat. A: Parallel coordination: The inward wave ⫽ recovery stroke (f 1–4) of the trans flagellum
(left) is synchronous with the outward wave ⫽ effective stroke (f 1–4)
of the cis flagellum (right). B: Bilateral coordination: outward waves
(effective strokes) are synchronous in both flagella and also recovery
strokes (not shown).
performed one additional half-beat. A preference of either
flagellum was not found (Table III).
In the ptx1 cells, all parallel beats had significantly
shorter beat periods than the bilateral beats (Table III).
Moreover, in the cells with changing coordination, often
beat periods shorter than in wt cells were also found
during bilateral beating (up to 14.3 ms/b ⫽ 70 Hz), but
with still more reduced beat periods during parallel
coordination (up to 13.1 ms/b ⫽ 76.3 Hz). The reduced
beat periods of both flagella were the result of reduced
beat envelopes and not of accelerated beating. Both front
amplitude and backward amplitude were decreased (Fig.
11), just like in the asynchronous trans beats of normal wt
cells. Thus, coordination changes of B-beats (in contrast
to U-beats) were always connected with changes of beat
envelope and beat period. The short beat periods and
reduced beat envelopes during the parallel phases were
similar to those during undulatory beating in shock
responses. On the other hand, the parallel beats of the
trans as well as of the cis flagellum were similar to the
asynchronous trans beats in normal wt cells. These short
asynchronies, however, were completely missing in the
bilateral phases of ptx1 cells, and also in the few cells
with bilateral beating only. Even very short phases of
parallel beating were different from normal asynchronies
in that the cis flagellum also showed reduced beat
envelopes and beat periods.
Coordination changes of ptx1 cells during Bbeating were found under all light conditions. An influence of step-up or step-down light stimulation on the type
of coordination could not be excluded but was not clear
because of the many spontaneous changes (Fig. 12).
Independent of the coordination changes BFPRs were
found in response to step-up and step-down light stimulation: not only during bilateral coordination [Rüffer and
Nultsch, 1997], but also during parallel coordination (Fig.
13). The two kinds of pattern changes were quite
different. During coordination changes, the beat envelopes were increased or decreased in connection with
prominent changes of the beat period. During BFPRs, the
whole beat envelopes were shifted with mostly minor
changes of the beat period.
Coordination changes in wt 622E. The same
phenomenon of long periods of parallel coordinated
(alternating) B-beating was also observed in some wt
622E cells. In two independent series of records taken
during many years of filming numerous cells also displayed changes of coordination, and also one single cell
in an otherwise normal series. The cells of the two
anormal series were taken from phototaxis deficient
cultures. One series was taken from an exceptionally old
and dense culture. It did not show any phototaxis and no
transferring into fresh culture medium could restore the
ability of phototaxis (at least not during 2 weeks).
However, we did not succeed in producing such a culture
by aging again. All 5 cells recorded from this culture
showed a behaviour that corresponded exactly to that of
the ptx1 cells: (1) a change between more or less long
periods of bilateral and parallel coordination during
B-beating, (2) reduced beat periods (60–79%) and envelopes of both flagella during the parallel phases as
304
Rüffer and Nultsch
Fig. 10. Changing flagellar coordination of undulatory beats during a photoshock response (induced by the
white recording light) from the start of the response till the beginning of the second transition to B-beating
[see Rüffer and Nultsch, 1995]. ⫽: one parallel beat, ⬍: one bilateral beat. The coordination is changed by
additional trans half-beats. Beat periods ⫽ b.p. in ms/b increase gradually during the response,
independently of the type of coordination. Wt 622E.
TABLE II. Coordination and Coordination Changes during
Shock Responses (Undulatory Beating)*
Coordinaton
Parallel only
Bilateral only
Parallel ⫹ bilateral
Parallel
Bilateral
Cells
9/43
0
34/43
% Cells
77
23
changes
29
2
3
% Beats
21
0
79
1748
516
Changes of coordination
trans h-b
cis h-b
trans and cis h-b
trans h-b total
Beats
176
12
8:9
184/205
%changes
85
6
9
90
*During undulatory beating of wt cells (strain 622E), coordination was
predominantly parallel but not exclusively (only in 9 cells out of 43).
Changes between parallel and bilateral coordination and vice versa
were found in 79% of the cells and accomplished by additional
half-beats (h-b) of the trans flagellum, very rarely by additional
half-beats of the cis flagellum.
compared to the bilateral phases, (3) additional cis or
trans half-beats during the change, (4) no asynchronies
during bilateral beating as in normal wt cells, (5) changing priority of cis and trans flagellum during bilateral
beating, (6) equal BFPRs (in this case in response to a
flash) of cis and trans flagellum, and (7) ability to undergo
photoshocks. The second series was taken from a culture
which was grown in the normal way, but for unknown
reasons this culture was only weakly phototactic. Cells of
this culture had either normal B-beating and normal
inverse BFPRs or they were like ptx1 cells. No transitions
between normal and anormal were found, and the two
records that were made of each cell always showed the
same behaviour respectively. The single anormal cell in
the normal culture was just like the other anormal cells.
DISCUSSION
Temporary short asynchronies in the otherwise
synchronous breaststroke beating mode had already struck
the early investigators of flagellar beating of Chlamydomonas wt cells [Ringo, 1967]. Learning that asynchronies
may be elicited by light changes made a correlation with
phototaxis at first sight suggestive, but became more and
more improbable for many reasons. Phototaxis is achieved
with synchronous beating only. Asynchronies do not
importantly change the course of the cells [Rüffer and
Nultsch, 1985] probably because of the minor effectiveness of the flat and lateral asynchronous trans beats in
which the rate of movement is not increased. Even if
asynchronies were more effective in changing the swimming path of the cells, the long and variable reaction
times of asynchronies could not explain the precise and
quick responsiveness of the cells in phototaxis. Moreover,
asynchronies are only induced either by step-up or by
step-down light stimulation. In contrast, the combination
of step-up and step-down BFPRs together with the short
and invariable reaction times (moreover corresponding to
the rotation rate of the stigma to the flagellar plane) are
the best prerequisite for the exact and quick alignment of
the cells in phototaxis. Thus, it appears that the trans
asynchronies, though being flagellar light responses, have
no or little influence on the whole cell’s photobehaviour.
Close analysis of the flagellar coordination of
B-beats in the mutant strain ptx1, which has lost the
normal cis/trans differentiation [Horst and Witman, 1993;
Rüffer and Nultsch, 1997], gives valuable hints as to the
nature of these asynchronies. In ptx1 cells, and also in
several 622E cells, which for unknown reasons had also
lost the flagellar differentiation, asynchronies are replaced by longer periods of anormal beating of both
flagella that resembles the additional trans beats of wt
asynchronies. In both cases, the position of the beat
envelope is lateral and the size of the beat envelope and
hence the beat period is strongly reduced, but not the rate
of movement. The resemblance of these short trans and
cis beats of the ptx1 and the anormal 622E cells with the
asynchronous normal trans beats supports the suggestion
that cis flagellar specialization is lost in those cells
[Rüffer and Nultsch, 1997]. The two flagella are like wt
trans flagella. The longer periods of short beats in ptx1
may be explained by the missing regulation by the normal
not affected cis flagellum that leads to a quick return to
normal bilateral beating in wt cells.
During the periods of flat lateral beating, the
synchronization is not lost in the cis defective cells but
the coordination type is changed from bilateral to parallel.
Flagellar Coordination in Chlamydomonas
Fig. 11. Bilateral and parallel coordination of breaststroke beats in the
same ptx1 cell (red recording light). A: Bilateral coordination: cis and
trans flagellum perform the recovery stroke (left) and the effective
stroke (right) simultaneously. B: Parallel coordination: the trans
flagellum performs the recovery stroke while the cis flagellum per-
305
forms the effective stroke (left) and the recovery stroke of the cis
flagellum is synchronous with the effective stroke of the trans flagellum
(right). Front and backward amplitude of the parallel beats are reduced
in both flagella as compared to the bilateral beats, i.e., beat envelopes
are reduced and also the beat periods (from 19.2 to 13.5 ms/b).
TABLE III. Coordination and Coordination Changes during Breaststroke Beating
in the Mutant Strain ptx1*
Coordination
Cells
Bilateral only
Parallel only
Bilateral ⫹ parallel
Bilateral
Parallel
6/33
0
27/33
Changes of
coordination
cis h-b
trans h-b
cis and trans h-b
cis h-b total
trans h-b total
Beats
Beat period
% Cells
23.6 ms/b ⫽ 42 Hz
18
% Beats
% Beat P.
53
47
100
75
82
3,241
2,913
20.8 ms/b ⫽ 48 Hz
15.5 ms/b ⫽ 64 Hz
Cells
Changes
% Cells
12
9
6
71
110
114:141
185/436
251/436
44
33
22
% Changes
45:55
42
58
*In ptx1 cells, changes between bilateral and parallel coordination were found in most cells (82%) with almost
equally long periods of parallel beating. Changes were accomplished by additional half-beats (h-b) of either the cis
or the trans flagellum with no significant preference. Beat period (P.) values in ms/beat (and frequency in Hz) are
averaged from all respective cells. In every single cell, beat period was reduced during parallel coordination to
67–91% (average 75%).
This parallel coordination is anormal for B-beats, but
typical for U-beats. Considering also the smaller beat
envelope and beat period at the beginning of a shock
response, a partial correlation between B-beats coordinated in parallel and normal U-beats becomes apparent.
The difference between the two kinds of beating concerns
the beating mode (typical B-beats and typical U-beats)
and the position of the beat envelope with respect to the
cell (lateral in the B-beats and frontal in the U-beats).
Thus, asynchronies are correlated with parallel
B-beating and this is correlated with U-beating, suggesting a correlation between asynchronies and U-beating.
Such a correlation is supported by the occurrence of
‘‘spontaneous’’ asynchronies as well as of ‘‘spontaneous’’
shock responses (while BFPRs never occur spontaneously) and by the fact that shock responses are often
followed by a series of asynchronies. In flash experiments, either asynchronies or shock responses were
306
Rüffer and Nultsch
Fig. 12. Coordination changes of breaststroke beats in a ptx1 cell in pulsed lateral blue light of 1 Hz. L ⫽
light on, D ⫽ light off. The coordination type was changed by additional trans half-beats (in this case, but
not always) with concomitant changes of beat period (ms/b) and of beat envelope (as in Fig. 11). An
influence of the light changes on the changes of coordination is not obvious.
Fig. 13. BFPRs in phases of parallel coordination in a ptx1 cell. As
usual in ptx1 cells, flagellar pattern changes are equal in trans and cis
flagellum. A: During the light-on phases (L) of the pulsed blue light (2
Hz), beat envelopes of both flagella were positioned more laterally
(front amplitude decreased). B: During the light-off phases (D), beat
envelopes were shifted to the front (front amplitude increased). These
responses correspond to those during bilateral coordination [Rüffer and
Nultsch, 1997] and are type (⫺) for the trans flagellum.
found but no prominent BFPRs. It should also be
mentioned that asynchronies may be induced by mechanic stimulation when objects hit the flagella (data not
shown), which is known for shock responses [Kreimer
and Witman, 1994].
Asynchronies or coordination changes are reactions
in the coordination system, independent of other flagellar
responses like BFPRs or the change of the beating mode
and the position of the beat envelope in the shock
responses. If there are the two kinds of responses,
light-induced asynchronies and BFPRs, asynchronies are
always elicited along with the same type of BFPRs: either
(⫹)-step-up or (⫺)-step-down, both having the same cis
and trans flagellar pattern. BFPRs precede the light-
induced asynchronies (having much shorter reaction
times), but they are not the prerequisite for asynchronies.
Asynchronies also occur without BFPRs and in some
exceptional cases in the opposite light phase with an
opposite pattern of BFPRs. Both reactions are superimposed such that the position of the smaller trans beat
envelope is also shifted according to the type of BFPRs.
Possibly asynchronies and BFPRs type (⫹)-step-up and
type (⫺)-step-down are based on the same ‘‘physiological’’ (electrical?) state. In the cis defective cells, most
coordination changes of B-beats were completely independent of the light changes and of the BFPRs, and pattern
changes of both reactions were also superimposed. In the
shock responses, changes between parallel and bilateral
Flagellar Coordination in Chlamydomonas
coordination are also found, supporting the independence
of the coordination system.
Earlier investigations suggested two different intrinsic beat periods or beat frequencies of the two flagella cis
and trans. If the synchronization of the two flagella on
biflagellated wt cells is lost [Rüffer and Nultsch, 1987] or
one or the other flagellum is removed [Kamiya and
Hasegawa, 1987], the cis flagellum shows the normal
B-beat frequency while the trans flagellum beats with a
higher frequency ⫽ smaller beat period, which was also
found in the uni1 mutant with a single trans flagellum
[Brokaw and Luck, 1983]. Unexpectedly, in the cis
defective cells the two trans flagella had both the
characteristic beat frequencies, always coupled to either a
bilateral or a parallel coordination, respectively. A change
of coordination was also observed during undulatory
beating, but without a change of beat frequency. With
these findings it is difficult to understand a dominance of
either flagellum on the type of beating or on the type of
coordination.
An external synchronization of the two flagella in
Chlamydomonas has been considered, but it is not likely
for either type of coordination because of the abrupt
coordination changes during breaststroke or during undulatory beating and also at the beginning of shock responses [Rüffer and Nultsch, 1995]. A basically internal
synchronization must be assumed, though in freeswimming cells the synchronization in both types of
coordination may be stabilized by vibration or the
resistance and movement of the medium. Therefore,
whether there is a dominance of one flagellum or not,
basal-body associated fibrous structures must be involved
in the coordination of the two flagella. Suggestions have
been made as to the basal-body connecting fibers [Hyams
and Borisy, 1975], or to the nucleus-basal body connectors [Salisbury and Floyd, 1978]. These structures are
highly Ca-sensitive [Salisbury et al., 1986], containing
the Ca⫹⫹ modulated contractile phosphoprotein centrin
[Coling and Salisbury, 1987; Melkonian et al., 1988]. So
far centrin mutants as vfl-3 [Wright et al., 1983] or vfl-2
[Wright et al., 1989], showing various structural defects,
do not allow definite conclusions with regard to coordination. More work is needed in order to elucidate the role of
the basal-body associated fibers in coordination.
ACKNOWLEDGMENTS
We thank the I.W.F. Göttingen, especially the
camera-man Mr. K.H. Seack, for their cooperation and
Professor Paul Galland for kindly providing the working
facilities. This work was supported in part by the
Deutsche Forschungsgemeinschaft.
307
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