Analysis of the mechanism of dinoflagellate flagella contraction-relaxation cycle
Monique Cachon a, Claude Greuet b, Jacky Cosson a, Philippe Huitorel"
a Groupe de Motilitd Cellulaire, URA 671 du CNRS, Universit~ P e t M Curie, Observatoire Ocdanologique, 06230 Villefranche-sur-Mer; b Centre de Biologie Marine, Laboratoire de Cytophysiologie des Protistes, Universitd de
Nice-Sophia Antipolis, 06108 Nice Cedex, France (Received 11 May 1992; accepted 29 September 1992)
Summary - Dinoflagellates possess two flagella. One of them, the longitudinal flagellum, retracts from time to time in some species, such as Ceratium and Peridinium. Additional structures which run along the axoneme seem to be responsible for this particular beha- viour. The retraction which is rapid (less than 60 ms) may be subdivided into several steps: i) the undulating movement stops; ii) the flagellum appears then as a jagged line during 20 ms; iii) finally a rapid retraction (20 ms) takes place, the flagellum being folded 20 times inside the cylindrical flagellar pocket. The measurements on video-records suggest that the R-fibre shortens to 30°7o of its original length. The contraction and relaxation mechanism of nanofilaments is proposed to be through coiling and uncoiling depen- dent on Ca 2+ concentration.
protist / flagellar contraction / nanofilaments
Introduct ion
Dinof lage l la tes possess two flagella, one long i tud ina l and one t ransverse , whose p roper t i e s are no t yet ful ly under - s tood [ 1 -10 ] . These f lagel la appea r to bea t accord ing to s inusoida l waves [11] bu t in fact the t ransverse has a heli- cal bea t while the long i tud ina l has a p l ana r wave. H o w - ever, in Ceratium and Peridinium, the long i tud ina l f lagel lum, which is one o f the thickest f lagel la ever obser - ved, shows an or ig ina l p rope r ty : it is ab le to re t rac t and relax like a spr ing [1, 4, 10, 12].
Next to the axoneme, and running parallel to it, are asso- c ia ted f i l amentous s t ructures [5, 10] made o f cont rac t i le bu t non-ac t in f i laments , the nanof i l amen t s [8, 9] also observed in the cy top la sm o f Dinof lage l la tes [13-16]
w h i c h might be respons ib le for this special behav iour : the r e t r a c t i o n o f the long i tud ina l f lagel lum.
Materials and methods
Biological materials
The marine organisms were collected from surface layers with a fine meshed net in the Villefranche-sur-Mer bay during the win- ter and spring months and from Sugashima Bay (Japan) from September to November 1991. Individual cells were isolated with small pipettes (about 50/~m in diameter) and maintained in Petri dishes containing sea water till video-records or electron- microscopical fixations were made.
Several species of Ceratium have been studied: our observa- tions were mainly made on Cfurca Ehrenberg, C tripos Mtiller, C gravidum Gourret and C limulus Gourret. These species are almost flat and their two flagella originate from the middle of the ventral area out of a widely opened cylindrical pocket (fla- gellar pocket).
Motility observations
These were made using Leitz and Reichert microscopes equipped with differential interference contrast (DIC) Nomarski optics. Video recordings were made with a Panasomic CCD camera (F. 15) combined with a Hamamatsu real time image processor (DVS-3000) and a Sony Umatic or a Panasomic Super VHS video tape recorder. Some sequences were obtained with a strobosco- pic light source operated at 50 Hz. Photographs of still frames on monitor were obtained on Technical Pan 2415 Kodak film exposed for I /8 s. A NAC high speed video camera was also used at 200 frames/s.
Electron microscopy
Fixation was performed mostly according to Maruyama [5]. The cells were fixed with a 0.1-M phosphate buffered fixative (pH 7.4-7.8) containing 507o glutaraldehyde, 0.8-1 M glucose at room temperature for 1 h. Then they were washed in a buffer contain- ing 0.3 M phosphate and 0.8 M glucose. After treatment by 207o OsO 4 in phosphate-glucose buffer for approximately l h a decreasing graded series of phosphate-glucose solutions were used, before a progressive dehydratation. Finally, they were embedded in Spurr's low viscosity medium [17]. The sections were stained wiht 907o uranyl acetate in methanol followed by lead citrate and examined with a Hitachi H603 electron microscope.
Flagella are generally preserved in situ with difficulty, because the fixative itself acts as a contracting agent and the calcium as a triggering retraction agent. To preserve flagella the organisms were trapped among fibres of nucleohistones [18], this method allowing also an easier handling of the Protists.
Ca2+-free artificial sea water was used to prevent contraction of flagella (477 mM NaCl, 97 mM KC1, 20.9 mM MgCl 2, 27.6 mM MgSO4, 5 mM ethyleneglycol his (fl-aminoethylether) N,N-tetraacetic acid (EGTA) and 30 mM Tris-HCl (pH 7.6). The living organisms were briefly washed in this medium prior to fixation.
34 Monique Cachon et al
Fig 1. The longitudinal flagellum (fl) is visualized extended (a), straight across the flagellar pocket (fp) the organism, transferred into Ca2+-free sea-water, is observed by DIC optics contrast microscopy (x 1200) or contracted (b) and folded inside the flagellar pocket (living organism; video recording of DIC images, x 1500) scale bar = 10 tzm.
Computer image treatments of electron micrographs
The electron micrographs have been digitized with a scanner and processed on a computer to extract 'skeletons' of the structures by further contrast enhancement.
We used the GT 4000 Epson Colour Image Scanner connec- ted to an IBM PC operated under EpscanIlI. The digitized ima- ges were then treated by varying several parameters, using the Paintbrush software.
Results
Video-microscopical observations
The longitudinal flagellum originates from an unusually long (1.5/zm) basal body located at the bottom of a deep cylindrical flagellar pocket (about 6/zm in diameter and 16/~m in length). It can be easily observed (fig la) with DIC optics. The two cartoons of figure 2 show the beha- viour of the flagellum in the relaxed state (fig 2A) and the contracted state (fig 2B). On one side of the basal body (see the diagram; fig 2), the cytostome is located with its aperture close to the base of the flagellum. A large pusule opens on the other side and contracts periodically to eject waste products and liquid.
Analysis of video-recordings shows that sinusoidal waves are normally propagated along this flagellum (fig 3a), and that they appear as perfect sine waves com- posed of arcs and straight segments in between curved portions.
As the cells are heavily armoured (thecate), they do not move rapidly. However, it is easier to study the behaviour of their flagella when the cell bodies are stuck on the glass surface because the flagellum remains in focus. The wave amplitude is limited in the flageliar pocket but its frequency is the same as outside the pocket, the aperture of the pocket playing the role of a node of beating.
Spontaneously, and perhaps under the action of exter-
nal agents [4] such as mechanical shocks, the flagellum retracts suddenly inside the flagellar pocket (figs lb, 2B). It disappears inside the pocket in less than three video frames of 1/50 s that is, in less than 60 ms, its distal extre- mity often remaining outside and uncoiled.
Even though the retraction appears to be rapid, we could visualize different stages of retraction by studying many video sequences. It can be subdivided into four steps:
1) the sinusoidal waves stop (fig 3b); 2) a shiver propa- gates from the base of the flagellum towards its distal tip within 20 ms though Maruyama said the retraction was in the reverse direction. Bending points appear every 9 ~zm (fig 3b). A jagged line is thus observed located in a plane, each segment of 9 ~m becoming two segments of 4.5 tzm in length (figs 3c, 4a); 3) the next step of the retraction takes place in 20 ms (figs 3d, 4b, c). The flagellum shows 20 folds, the length of each segment remaining constant throughout the contraction process. The distance between a bend of the flagellum and the next one shortens down to about 0.6 ~m. The folds are adjacent to each other and in the same place (fig 4b); 4) soon after the complete retrac- tion the fold topology changes: the 3rd fold slides under the 1st fold, the 4th fold under the 2nd fold... (fig 4c). Finally the flagellum is inside the pocket, its folds being tightly squeezed together. The planes in which they are located define a right-handed helix as was already sugges- ted by Maruyama [4].
The duration of the contracted state is variable from one second to several minutes. When the organism is still in sea water, the flagellum contracts very rarely. Conversely, after a long time of observation, when it is exhausted, the flagel- lum may then contract very frequently without interruption.
In contrast to the contraction, the relaxation is much slower (about 1/5 s): the flagellum unfolds slowly from its base propagating sinusoidal waves progressively towards its tip, still folded and crumpled, until the very end of the relaxation process (fig 5).
The flagella which have been broken by manipulations regrow in 2 - 3 h.
Analysis of the mechanism of dinoflagellate flagella contraction-relaxation cycle 35
Fig 2. These two schematic drawings propose an interpretation of the behaviour of the longitudinal flagellum of Ceratium furca. A. The flagellum emerging from its basal body (bb) located between the cytostome (cyt) and the pusule (pu), extends from the flagel- lar pocket (Fp) while it is relaxed. B. While it is contracted, the flagellum is located entirely inside the flagellar pocket which appears then to be almost closed by a thin membranous operculum. Ax, axoneme; Rf, R-fibre; Pfr, paraflagellar rod; fr, flagellar rootlets coiled around the pusule; Sp, spines of the theca. The arrowheads indicate the pathways of the food particles.
Structures
As already observed [5, 10] the axoneme is flanked by three additional structures, the paraflageIlar rod (Pfr), the stria- ted fibre (Sf) and the R-fibre (Rf).
Structure o f the paraflagellar rod The Pfr of Ceratium is closely similar to that of Oxyrrhis [8, 9] except for some additional small elements responsi- ble for periodical links which connect it on one side to the axoneme and on the other side to the R-fibre (fig 6b, c, d, e). The links between the axoneme and the Pfr are clo- ser to each other than those between the axoneme and the Rf. The Pfr runs parallel to the proximal three-quarters of the axoneme. It is almost a cylinder, the wall of which is made of a filamentous network. Transverse sedtions of the Pfr show that it is composed of two different hemi- cylinders, joined through a dense ribbon on its Rf side.
Both hemi-cylinders contain eight layers of filaments. One element, the outer Pfr, is crescent-shaped and appears more developed (45-50 nm in width) than the other one, the inner Pfr (25 nm in width) which is straight and thin- ner (fig 6b). The filaments of the two elements are per-
pendicular to each other so that these structures appear as a lattice-work with rhomboidal meshes when superim- posed (fig 6d). Both hemi-cylinders are attached to one of the nine axonemal doublets: this doublet could not be identified unambiguously in Ceratium contrary to what web,previously wrote [10], whereas in Oxyrrhis it is dou- bler 4 (for a detailed description in O marina see Cachon et al [8, 9].
The dense ribbon of the Pfr is also made of nanofila- ments tightly arranged and showing a regular periodical structure (fig 8a). This periodicity is due to units of 50 -52 nm in width. Each unit represents three bands, two dark and one clear, each of them being 16 nm and one thin clear band being 2.5 nm. Each 16 nm dark band shows a very thin clear band in its middle (less than 1 nm).
Periodical links are observed between the Pfr and the Rf (fig 6b, c, d, e). So when the Rf contracts,, the distance between two links is progressively shortened, this inducing the following of the Pfr and of the axoneme (fig 7).
Structure o f the striated fibre (Sf) Another structure is associated to the axoneme approxi- mately along 3/4 of its length: the striated fibre (fig 8b)
36 Monique Cachon et al
0 , 0 0 " -:~:: "~ :~
Fig 4.a. Detail of a perfect jagged line visible just before the retraction. (x 1600). b, c. Two micrographs of a longitudinal flagellum highly retracted showing 19-20 bends (× 1800) scale bar = 10 ffm.
Fig 3. The retraction of the longitudinal flagellum has been stu- died on many video sequences in order to obtain a reconstruc- tion of the successive steps of the contraction. (The whole sequence lasting 80 ms.) The observations have been made at room temperature (about 23°C). a. Normal beating (sinusoidal waves). The arrow indicates the direction of the waves, b. Begin- ning of the shiver, the first bending 6urves appear every 9/~m. e. The flagellum appears as a jagged line with bending every 4.5 /~m. d. The retraction is progressing. The arrow indicates the direction of the wave propagation ( x 1800), scale bar = 10 tzm.
already observed by M a r u y a m a [5]. In cross-section it is square or tr iangular ( 30 -35 nm in width). It has always been observed in the inner side o f the concavity o f the axo- neme when folded, and appears to be made o f short seg- ments (about 500 nm) attached regularly by their ends (fig 8b). The regularity o f the length o f the segments made us suppose that the Sf is really linked to the axoneme but not only kinked because o f the bend of the axoneme. Because the Sf is always at tached to the doublet which follows
Analysis of the mechanism of dinoflagellate flagella contraction-relaxation cycle 37
41 " •
/
/
Fig 5. The re-extension of the flagellum is much slower (the whole process lasts about 1/5 s; T, 23°C). It progressively unfolds (× 1800), scale bar = 10 tzm.
immediately the doublet linked to the rod which may change, it obligatorily runs parallel to it.
Longitudinal sections show that this fibre is a thin bundle of small filaments which show a periodical struc- ture. We have measured the periodicity in two different stages showing a 48-nm or 42-nm unit, with alternation of dark and clear bands. In the 48-nm configuration the dark band is 12 nm and the clear band 36 nm thick while in the 42-nm configuration the dark band is 14 nm and the clear band 28 nm. This corresponds to a shortening of the Sf segments by 12.5°/0, by thickening of the dark bands of 16°70 and shortening of clear band by 22070. These variations suggest the contraction of Sf segments by thick- ening of dark bands at the expense of clear bands.
Structure o f the R-fibre (R f) An additional large filamentous bundle runs along, but not parallel, to the axoneme which is always located on the same side as described by Maruyama [6, 7] (fig 9a, b) and Cachon et al [10] (figs 6, 8c). It was called R-fibre by Maruyama [5]. The Rf is attached to the inner face of the membrane near the base of the flagellum. In this region, the flagellum is wider and its membrane is seen almost perpendicular to the axoneme (fig 8c). Very thick at its origin (0.5 tzm in diameter), the Rf appears to be 300 nm in diameter at its distal extremity, located about three-quarters of the flagellar length f rom its base. It is made of a large number of filaments ( 2 - 4 nm in diame- ter) running parallel to its axis.
Nanofilaments of the Rf arise f rom dense bodies (6 nm wide and 12 nm apart) at the inner side of the membrane (fig 8c, small arrows). The filaments are paired. They are not stretched, nor close to each other and seem to be loo- sely twisted. At about 130 nm from their origin, they cross each other. At the point of filament crossings, which are all located at the same level, a transverse dense 10-nm layer appears perpendicular to the axis of the bundle. It seems to correspond to different local arrangement or coiling of the filaments in register. At this level on both sides of the Rf, fibrillar (?) extensions can be observed (fig 8c). In some oblique sections many coilings of the nanofilaments can be seen (fig 6a).
The precise organization of the filaments in the Rf varies with the fixation conditions, the Rf being probably fixed in different contraction states, due to the fixative acting as a triggering agent in the presence of the Ca 2+ ions (11 mM) contained in sea water. The flagellum appears highly contracted in its flagellar pocket in presence of Ca 2÷ ions but relaxed when the fixation is done in Ca 2+ free artificial sea water containing 5 mM EGTA. These extreme conditions allowed us to observe that the Rf shows a very thin transverse striation when relaxed (but is the relaxation completely realized?) (cfd iagram fig 7). In the presence of an intermediate Ca 2÷ concentration, the Rf may also appear partially contracted, partially relaxed. In this case when the bundle appears periodically structured on sections, we cannot interpret which state is kinetically preceding the other, the contraction being too fast to allow the fixation of the structure at a precise stage.
Two situations may be observed within a structural unit repeated along the length of the R fiber: only two thin transverse striations or one thin and one thicker transverse striation. In this last situation we have measured the perio- dicities shown by different micrographs taken on three independent specimens presumably fixed in a slightly dif- ferent contraction state (table I).
This shows that there is a shortening of total striated
38 Monique Cachon et al
Fig 6.a. Oblique section of the Rf in the periphery of which one can easily observe coiled filaments (small arrow heads). The core of the bundle appears darker than its periphery (x 80000), scale bar = 0.25/zm. b, c, d, e. Relationships between the axoneme (Ax), the Sf, the Pfr and the Rf. The nanofilaments of the Rf are seen in continuity with those of the dense ribbon of the Pfr (small arrow heads) (x 80 000), scale bar = 0.25/zm.
Analysis of the mechanism of dinoflagellate flagella contraction-relaxation cycle 39
. . ~'~,.,t:=
• .
• " I i ,
r",~ C
A
Y B T
A T
~Z
A
T X
Fig 7. The contraction of the longitudinal flagellum has been schematized by different diagrams from the relaxed state (A) to the contracted state (D) A > B > C> D. The R-fibre has been drawn straight to make the understanding easier, x, y, z represent virtual points located along the axoneme on the same side of the R-fibre.
periodicities ( - 25 nm) which corresponds in fact to a shor- tening of clear bands ( - 20 and - 10 nm) while the dark bands thicken (+ 3 and + 2 nm). So a shortening of total striated periodicities indicates a contraction of Rf.
There is no periodical structure observed when the Rf is higly contracted (fig 6a). In that case, sections show that the filaments are helically organized and that they form tubes of 10-12 nm in diameter. A dense axial core is often observed which would mean that the filaments of the axis would be arranged tighter in the central part of the Rf.
The Rf appears periodically connected to the Pfr. The filaments of the Rf and the filaments of the dense ribbon of the rod are in continuity as observed in oblique sections (fig 6b -e ) .
Discussion The longitudinal flagellum of Ceratium seems tq play an important role in the capture of prey [10]. Thanks to the various contraction-relaxation cycles of its associated struc- tures, small food particles are captured between the folds of the flagellar membrane and carried into the flagellar pocket in the direction of the cytostome (cf the schematic drawings of fig 2). Such a behaviour is possible because the associated structures which run along the axoneme are contractile like myonemes.
The thinnest associated structure, the Sf, could be res- ponsible for the regularity and periodicity of the axoneme folds. Each fold of the axoneme (4-4.5 ~m) shows a num- ber of striated segments (0.5 ~m) in the concavity of the curvature. In a contracted flagellum which is totally inside the flagellar pocket (fig 8c), the radius of curvature of the axoneme (0.7 ~m) is in agreement to the measurement made on video pictures (fig 4b) (0.6/zm). The Sf could also strengthen the axoneme, avoiding an excessive fol- ding and thus its breaking by defining its precise folding. To be able to remain always inside the concavity of the axoneme, its links cannot be maintained on the same doublet•
As the Pfr is involved in the avoiding reaction as demonstrated in other dinoflagellates [9, 34], we suggest that the rapid and complete retraction of the flagellum is, at least, partly due to the large Rf. Its periodical links with the Pfr and thus the axoneme, induce the regular folds of the flagellum.
In the Rf, the thin layers would correspond to one cross- coiling of nanofilaments and the large layers to several coil- ings. Between two thin layers, one can finally suggest that four filaments might constitute the apparent single fila- ment, issued from the dense bodies close to the membrane, which could be made of two couples of two twisted fila- ments intimately coiled. Measurements of electron- micrographs showing the Rf in differents states of con-
40 Monique Cachon et al
Fig 8.a. Longitudinal section of the dense ribbon of the Pfr (which is linked to the axoneme) (x 80 000), scale bar = 0.25 ~zm. b. The Sf segments (0.5 t~m in length) are regularly linked to the axoneme (small arrow heads) and always located inside its concavity (x 60000), scale bar = 0.25 ~zm. c. Rf at its origin, attached to smalldense bodies (small arrow heads) located on the inner side of the flagellar membrane, the nanofilaments present a periodicity which is able to vary according to the state of contraction. ( x 100 000) scale bar = 0.25 t~m (Fm: flagellar membrane).
traction (fig 9) suggest that the contraction of the Rf is due to a conformational change in the nanofilaments which would be coiled six or eight times tighter in the dark bands.
Measurements on video-records have given rise to the schematic drawings (fig 7) which show that the distance between two bands of the axoneme (A) located on the same side of the Rf of a completely relaxed flagellum is roughly 30% greater than the distance (D) between the same two bands (A/D) of a contracted flagellum, this being in agree- ment with Maruyama [5]. The nanofilaments of the Rf are thus able to shorten to an extent which induces the com- plete twisting of the whole bundle, and the super coiling of the flagellum in less than 60 ms.
Other organelles such as the myonemes of Acantharia [19-23] appear similar both in their structure and beha- viour. They are able to shorten to 3 0 - 4 0 % of their length within 10-15 ms. These myonemes play, in these orga- nisms, a role in buoyancy regulation by changing the cell volume.
In the Heliozoa Sticholonche zanclea [24], an approxi- mately similar degree of contraction (a shortening to roughly 30% of the length) has been observed in the nano-
filaments which are located at the base of the axopods which are responsible for the swimming of this marine organism.
Roberts [33] reviewed many examples of nanofilaments found in Protists, Ciliates [25-27, 38], Dinoflagellates [13-16, 34], Acantharia [20, 22], in Heliozoa Sticholon- che zanclea [24] green algae [28, 29, 35, 37], as well as in Metazoan cells [30].
After video image treatment of digitized electron micro- graphs (fig 9a), it was possible to visualize the 'skeletons' of bundles of nanofilaments (fig 9b): we could draw the individual filaments (fig 9c): their contraction and relaxa- tion mechanism is proposed to proceed by coiling and uncoiling [20, 24, 31] under dependence of the Ca 2+ con- centration. It has not been possible so far to visualize the successive steps of the cycle of contraction and relaxation. Therefore, a hypothetical-cycle is suggested: 1) the fully relaxed state in which it is likely, even though never obser- ved, that no dark bands are present; 2) the beginning of the contraction would correspond to the appearance of thin transverse bands as a result of the coiling of paired filaments; 3) these bands would progressively thicken while contraction is progressing; and 4) the final contraction
Analysis of the mechanism of dinoflagellate flagella contraction-relaxation cycle 41
b
C
Fig 9. The micrograph (a) (× 150 000) has been digitized (b) so that it was possible to extract the individual filaments from this image and draw them (c). This interpretation shows the enhancement of the crossing of the nanofilaments and indicates that the filaments are always paired.
Thick dark band 15 17 18 + 20 + 3 Thick clear band 100 90 80 - 20 - 20 Thin dark band 10 10 12 + 20 + 2 Thin clear band 30 22 20 - 33 - 10 Total 155 140 130 - 13 - 25
would then correspond to the meeting o f all d~rk bands composed o f tightly coiled filaments. The relaxation does certainly not proceed inversely o f the contract ion.
Actin filaments (microfilaments) and non-act in fila- ments (nanofilaments) produce similar final results (a con- traction) and occur in periodical structures: the organelles which possess them are able to contract and to relax. But there are many features which distinguish their contrac-
t ion mechanisms. The acto-myosin system works accor- ding to a sliding mechanism which involves A T P hydroly- sis. The nanofi lament one proceeds by a Ca 2+ dependent 'coil coiling model ' , no A T P hydrolysis being directly involved in this mechanism. As for the nanofi laments , a sudden rise o f Ca 2+ concentra t ion in the cytosol o f the flagellum would initiate the contrac t ion o f the bundle o f nanofi laments by coiling o f the individual filaments upon
42 Monique Cachon et al
binding of Ca 2+ to Ca2+-binding proteins, as described for spasmin, centrin etc. This rise in Ca 2+ concentration is transient, these ions being pumped back out, which per- mits the relaxation of the organelle. Maruyama's experi- ments [6] on the permeabilized flagellum of Ceratium are the most demonstrative in favor of a direct effect of the rise in Ca 2 + concentration to trigger the contraction. He concluded that the undulation is generated by the axoneme using ATP hydrolysis and that the retraction can be indu- ced by Ca 2+ without any requirement for ATP. The mechanism of contraction could be explained, as shown by polarizing microscopy by Febvre et al [23] in the case of the myonemes of Acantharia, through the pitch varia- tions of the filaments coiling.
Physiological experiments still have to be done to bet- ter understand how such organdies made of nanof'daments are regulated in coordination with the flagellar beat. Bio- chemical studies have also to be made before knowing how many different binding proteins control the function of these nanofilaments [32].
Acknowledgments
This work has been supported by CNRS (LIRA 671). We also have to thank Andr~e Collomb for her technical help.
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