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: firstname.lastname@example.org 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]. 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