Lifting the Curtain? The Microtubular Cytoskeleton of Oxyrrhis marina (Dinophyceae) and its Rearrangement during Phagocytosis

Protist. Vol. 149, 75-88, February 1998 © Gustav Fischer Verlag

Protist

ORIGINAL PAPER

Lifting the Curtain? The Microtubular Cytoskeleton of Oxyrrhis marina (Dinophyceae) and its Rearrangement during Phagocytosis Ingo Hohfelda and Michael Melkonian b,1 aBiozentrum Niederursel, Molekulare Zellbiologie (N200, 3.0G), Marie-Curie-Str. 9, 0-60439 Frankfurt, Germany bBotanisches Institut, Lehrstuhll, Universitat zu K61n, Gyrhofstr. 15, 0-50931 K61n, Germany Submitted November 17, 1997; Accepted December 30, 1997 Monitoring Editor: Robert A. Andersen

The cortical microtubular cytoskeleton of the colorless, phagotrophic dinoflagellate Oxyrrhis marina has been investigated by immunofluorescence and transmission electron microscopy. It consists of two systems, an anterior system comprising microtubular bands (of 2-4 microtubules each) which extend from a focal point at the cell apex to about three-quarters of the cell length where they either become transversely oriented (on the ventral right surface of the cell) or abut transversely oriented microtubules (on the dorsal and ventral left cell surface); and a posterior system in which microtubular bands extend from a focal point near the basal apparatus posteriorly around the antapex of the cell to become transversely oriented in the region where they meet the abutting anterior microtubular bands. The peripheral cytoskeleton of Oxyrrhis contains no continuous pole-to-pole microtubules and is thus basically similar to that of other dinoflagellates. Upon phagotrophic feeding the peripheral microtubular cytoskeleton undergoes reversible rearrangements. The non-permanent cytostome is located at the right ventral surface of the cell between the ventral ridge microtubules (vrm) and the groove of the longitudinal flagellum. During phagocytosis the anteriorly focused microtubular bands of the peripheral cytoskeleton near the right ventral surface of the cell are 'lifted' or 'pushed' towards the vrm to enable uptake of food organisms of diverse size and shape. Within minutes after phagocytosis the microtubular bands are relocated to their former position. We conclude that the organization of a peripheral microtubular cytoskeleton from two opposite focal points provided the dinoflagellates with the flexibility needed to evolve the multitude of phagocytotic mechanisms that characterize this group of protists today.

Introduction About half of all known dinoflagellate taxa do not possess plastids and thus, as heterotrophic organisms, feed on dissolved or particulate organic material (Gaines and Elbrachter 1987). But also in photo, Corresponding author; fax 49-221-470-5181; e-mail [email protected]

synthetic dinoflagellates including thecate forms, phagotrophy appears to be far more common than previously recognized (Biecheler 1952; Bockstahler and Coats 1993; Jacobson and Anderson 1996; Li et al. 1996). Different modes of heterotrophic nutrition occur in dinoflagellates: 1) osmotrophy, the uptake of dissolved organic substances, 2) phagotrophy sensu stricto, the uptake of particulate food - in-

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cluding the plasma membrane of prey organisms through a cytostome (cell 'mouth', the area of the plasma membrane involved in phagocytotic food uptake), 3) myzocytosis, the uptake of intracellular material of the prey through a cytoplasmic extension - the peduncle - leaving the plasma membrane and extracellular matrix of the prey behind, and 4) pallium feeding, the engulfment of prey by a cytoplasmic veil - the pallium - with digestion of the food taking place outside the dinoflagellate cell body (Elbrachter 1991; Schnepf and Elbrachter 1992). Members of the Blastodiniales, Gymnodiniales, Noctilucales, and Oxyrrhinales are described as phagotrophic organisms sensu stricto, Le. these organisms completely ingest prey through a cytostome (Elbrachter 1991). The cytostome is represented either by a well defined structure as in Noctiluca (Melkonian and Hohfeld 1988; Soyer 1970), or a specialized structure is lacking (a non-permanent cytostome according to Elbrachter 1991). In Oxyrrhis marina previous electron microscope investigations gave no indication of a structurally specialized cytostome (Dodge and Crawford 1974; bpik and Flynn 1989) and, therefore, the site of phagocytotic food uptake could not be located unequivocally in this taxon (Dodge and Crawford 1974). In a typical dinoflagellate, the two flagella arise from the side, one (the transverse flagellum) beating sideways around the cell, the other (the longitudinal flagellum) beating backwards. This arrangement is known as the dinokont condition (Taylor 1987). In dinokont dinoflagellates the flagella beat in surface grooves, the transverse flagellum lies within the girdle groove (or cingulum), the proximal part of the longitudinal flagellum lies in the sulcus. The cingulum runs circumferentially around the cell and essentially divides the cell into an anterior part, the epicone and a posterior part, the hypocone (Lebour 1925). The cell surface exposing the sulcus and flagellar insertion is termed ventral, the opposite side is termed dorsal. Further, a left and a right half of the cell can be distinguished (by convention view from the dorsal side of the cell to the outside). A schematic representation of these cell surface landmarks is shown in Fig. 29 for Oxyrrhis marina (Note that in Fig. 29 the specimens are oriented as if seen by an outside observer!). The cell surface of dinoflagellates is characterized by a cortex (amphiesma) consisting of a continuous outer membrane (the plasma membrane), subtended by a single layer of flattened vesicles (amphiesmal vesicles) that are usually appressed at their edges, and a longitudinal array of cortical microtubules located beneath the amphiesmal vesicles. The structure of the peripheral (cortical) micro-

tubular cytoskeleton of dinoflagellates has been relatively well studied (Dodge 1973), but only recently using immunofluorescence microscopy has the overall disposition of the peripheral microtubules in selected dinoflagellates been revealed (Brown et al. 1988; Perret et al. 1993; Roberts et al. 1988a, b; Roberts 1991; Roberts and Roberts 1991). Predominantly, the cortical microtubules in the dinoflagellate taxa studied are organized in three longitudinal arrays corresponding to the epicone, hypocone and girdle of the cell: an anterior array focused at the cell apex, a posterior array presumably focused near the cell antapex and an array consisting of very short microtubules located beneath the cingular depression [termed alb (anterior longitudinal microtubular bundle), plb (posterior longitudinal microtubular bundle), and clb (cingular longitudinal microtubular bundle) by Roberts and Roberts 1991]. These longitudinal arrays of microtubules are separated from each other in the cingular region by two transverse bands of microtubules [anterior transverse microtubular band (atp) and posterior transverse microtubular band (ptb) respectively; Roberts and Roberts 1991] demarcating the edges of the cingular groove. The origin of the transverse microtubular bands and their relationship to the sulcal microtubules and the posteriorly focused array of microtubules are not well understood (Roberts 1991). It is important to state that unlike the initial perception (Brown et al. 1988), the cortical microtubular system of dinoflagellates is not continuous from pole to pole, anteriorly and posteriorly focused arrays terminate at the cingulum (also refer to Roberts et al. 1992). A peripheral cytoskeleton of longitudinally oriented bands of microtubules has also previously been visualized in O. marina by immunofluorescence (Brown et al. 1988; Roberts et al. 1993). No large cell surface area is devoid of underlying microtubular bands. Since Oxyrrhis is capable of ingesting prey of almost its own size (Dodge and Crawford 1974; Droop 1966), the intriguing question arises as to how the cytoskeleton can be modified to allow phagocytosis of large prey in this organism. We describe the process of food ingestion in O. marina by using light and electron microscopy techniques, and we also locate the position of the cytostome. In addition, we document extensive rearrangements of the microtubular cytoskeleton during phagocytosis using immunofluorescence microscopy. The latter also yielded new information on the detailed structure of the peripheral microtubular cytoskeleton which makes a reinterpretation of existing data necessary and brings the Oxyrrhis cytoskeleton more in line with that of other dinoflagellates.

Oxyrrhis Cytoskeleton

Results

The Peripheral Microtubular Cytoskeleton of Oxyrrhis marina The peripheral microtubular cytoskeleton of O. marina reflects the dorsoventral symmetry of the cell

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(Figs 1-8; schematic presentation Fig. 29). The subamphiesmal microtubules of the dorsal cell half are arranged in regularly spaced longitudinal bands consisting of 2 to 4 microtubules each (Figs 9, 10). At the cell apex, the microtubular bands are focused in a point from which they radiate in a whirl-like, clockwise fashion (seen from the cell apex; Fig. 2;

Figures 1-4. Immunofluorescence images of the microtubular cytoskeleton in permeabilized cells of Oxyrrhis marina. All cells are oriented as if viewed from the outside. Scale bar: 10 IJm. 1. A ventral view of a cell. Two longitudinal arrays of microtubular bands (one focused at the cell apex, the other in the mid-ventral part) can be seen. The area of the tentacle is marked by arrowheads. An open arrow indicates the ventral ridge microtubules. 2. An apical view of a cell. The focal point of the anteriorly focused microtubules is seen (arrowhead). The ventral cell surface is located at the bottom of the micrograph where the two axonemes of the flagella have been immunolabeled. 3. A left dorsal view of a cell. The anteriorly and ventrally focused arrays of microtubules meet in a contact zone near the posterior third of the cell. The ventrally focused array of microtubules extends as a parallel array to the ventral surface (white arrow). 4. A right dorsal view of a cell. It can be clearly seen that the ventrally focused array of microtubules does not terminate at the cell antapex, but curves around to the ventral cell surface (white arrow). The contact zone between the two longitudinal arrays of microtubules is marked (white arrowhead). The axoneme of the transverse flagellum is immunolabeled.

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arrowhead}. In the posterior third of the cell (3-5 IJm from the antapex towards the dorsal half of the cell) these microtubules end bluntly at more or less transversely oriented microtubular bands (Figs 3, 4, 29) which originate at the ventral half of the cell (see

below). The arrangement of microtubules in the peripheral cytoskeleton is more complex on the cell's ventral half where the two heteromorphic flagella insert centrally (Figs 1, 5, 6, 8, 29). In the anterior region of the cell, microtubules are arranged in longi-

Figures 5-8 Immunofluorescence images of the microtubular cytoskeleton in permeabilized cells of Oxyrrhis marina. All cells oriented as if viewed from the outside. Scale bar: 10 ~m. 5. A ventral view of a cell. From the flagellar basal apparatus (arrowhead) extends a brightly fluorescent microtubular band (presumably the longitudinal microtubular flagellar root) along the left edge of the flagellar groove of the longitudinal flagellum (both flagella were lost during preparation). 6. Two cells in ventral view. The left cell is seen from the right ventral side, the anteriorly focused array of microtubules bends towards the cell's right and converges to a thicker band of microtubules near the the right edge of the flagellar groove. The right cell displays the focal area of the ventrally focused microtubules and the intercalating microtubules which extend anteriorly from the left edge of the ventrally focused microtubules (arrowhead). 7. Two cells, the left cell in dorsal view, the right cell with the left ventral surface exposed. The anteriorly focused array of microtubules can be seen to abut transversely oriented microtubules (arrowhead), representing the intercalating microtubules which extend from the ventrally focused array of microtubules. 8. Three cells, the left two cells in dorsal view, the right cell in ventral view. The right cell displays the focal area of the ventrally focused array of microubules. In addition, in the right cell both axonemes of the two flagella were retained in their in vivo orientation.

Oxyrrhis Cytoskeleton

tudinal bands as already described for the cell's dorsal half (Figs 1, 5, 6, 29). These bands are also focused at the cell apex in the stellate pattern described above (Fig. 2). Posteriorly these microtubular bands terminate in different ways depending on whether they are located in the cell's ventral left or ventral right region: in the ventral left region the microtubular bands terminate blunt-ended at trans-

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verse microtubular strands as described above for the dorsal microtubular bands (Figs 7, 29); in the ventral right region the microtubular bands converge to thicker bundles and run towards the cell's right at an angle of 45° to the cell axis; they successively terminate along this way (Figs 1, 5, 6, 29). In the cell's ventral mid-region all microtubular bands which are focused at the cell apex apparently sepa-

Figures 9-12. Electron microscope images of the peripheral microtubular cytoskeleton of Oxyrrhis marina. Scale bar: 1 IJm. 9. Cross section through a cell revealing the typical arrangement and spacing of the microtubular bands (arrowheads) of the two longitudinal arrays of microtubules with each band consisting of 2-3 microtubules. av: amphiesmal vesicle. 10. Tangential section through a cell with microtubular bands (arrowheads). Sometimes a microtubular band of the peripheral cytoskeleton may consist of up to 4 microtubules. 11-12. Two serial tangential sections through the cell surface close to the anterior end of the tentacle depicting only single microtubules (small arrowheads). Large arrowhead: transverse striated fibrous root (tsr).

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rate into the two groups as described above; none of the microtubular bands (dorsal or ventral) which are focused at the cell apex have been observed to extend continuously from pole to pole (Figs 1, 6, 7, 29). In addition to the system of anteriorly focused microtubular bands, the cell also displays two sets of microtubules which are exclusively located in the

posterior region of the cell. From a broad focal point near the flagellar insertion site and close to the base of the tentacle (also termed tentacular lobe by Kofoid and Swezy 1921 or ventral bulb by Roberts et al. 1993; Fig. 29), microtubules extend longitudinally along the ventral cell surface towards the cell antapex (Figs 1, 5, 6, 29). In an area adjacent to and

Figures 13-18. Immunofluorescence images of the microtubular cytoskeleton in permeabilized cells of Oxyrrhis marina during phagocytosis of Dunaliella bioculata. All cells oriented as if viewed from the outside. Scale bar: 10 IJm. 13. Cell in ventral view; early stage of phagocytosis. The cytoskeleton of a Dunaliella cell can be seen near the site of phagocytosis (white arrowhead). 14. The cytostome at the ventral right cell surface near the posterior end of the cell is revealed (white arrowhead). During preparation of the cell, the phagocytosed cell of Dunaliella has fallen out of the cytostome. At the left side of the relatively small cytostome a microtubular band of the anteriorly focused array of microtubules has remained in position, whereas the other microtubular bands of this array have been 'lifted' towards the cell's right. 15. The maximally opened cytostome (white arrowhead). The transversely oriented ventral ridge microtubules (small arrow) delineate the anterior edge of the cytostome. 16. A ventral view of a cell during phagocytosis with the cytostome (white arrowhead) revealed. Black arrowhead: a thin microtubular band (perhaps consisting of only 1 or 2 microtubules) from the anteriorly focused array of microtubules extends along the right edge of the flagellar groove. The transversely oriented ventral ridge microtubules are seen (black arrow). 17. A ventral view of a cell during phagocytosis similar to the cell picturred in Figure 16. The dislocation ('lifting') of the anteriorly focused array of microtubules towards the cell's right assuming a transverse orientation at the anterior edge of the cytostome is clearly seen. 18. A ventral view of a cell during phagocytosis. The cytostome (white arrowhead) is widely open. 'Lifting' of the anteriorly focused array of microtubules is restricted to the area posterior to the ventral ridge microtubules (black arrow).

Oxyrrhis Cytoskeleton

underlying the tentacle these microtubules are mostly single (Figs 11, 12) and only converge to bands (Figs 1, 6) posterior to the tentacle, whereas towards the right side of the tentacle area microtubules already form bands near the focal point (Figs 1, 6, 29). The ventrally focused microtubular bands extend beyond the cell antapex as a parallel array

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and on the dorsal cell surface in the posterior third of the cell bend towards the cell's left to assume a more or less transverse orientation (Figs 3,4,7,29). It is at these microtubules that in the dorsal cell half the anteriorly focused microtubular bands terminate (see above). Finally, the left cell surface in the posterior region of the cell is subtended by another set of microtubular bands which extend from along the left margin of the ventrally focused microtubular bands in an anterior direction to meet the anteriorly focused microtubular bands. In the contact zone these microtubular bands become transversely oriented towards the cell's right (Figs 6, 7, 29). Thus, the only area of the cell surface of O. marina which is not subtended by microtubules is the region of the flagellar groove of the longitudinal flagellum (2x10 IJm; Figs 1, 5, 6, 29). Two additional components of the microtubular cytoskeleton of O. marina can be clearly visualized by immunofluorescence: the ventral ridge microtubules (vrm) and the posteriorly directed microtubular root (pmr) of the basal apparatus (Hohfeld et al. 1994). The vrm extend from the basal apparatus for about 10 IJm towards the cell's anterior right (Figs 1, 5, 6, 29). The pmr bounds the longitudinal flagellar groove on its left side and, towards its distal end, cannot be clearly distinguished from the ventrally focused microtubular bands (Figs 1, 5, 6, 19).

The Microtubular Cytoskeleton during Phagocytosis

Figures 19-20. A cell of Oxyrrhis marina in a ventral right view after phagocytosis of a cell of Dunaliella bioculata. Scale bar: 10 ~m. 19. Immunofluorescence image of the microtubular cytoskeleton. The anteriorly focused array of microtubules has resumed its pre-phagocytosis arrangement (white arrow). White arrowhead: ventral ridge microtubules. 20. Corresponding phase contrast image to Figure 19 showing a phagocytosed cell of D. bioculata (arrowhead) in a location that reflects the site of the cytostome during phagocytosis. Arrow: longitudinal flagellum.

Phagocytosis in O. marina is an extremely rapid process taking 10-15 seconds and occurs while the cell is swimming at high speed around its prey in narrow circles. The organism is one of the fastest swimming flagellates achieving speeds of up to 400-700 IJm/s (Cosson et al. 1988). It proved impossible therefore, using standard video microscopy, to document this process in detail in live cells. What has been observed, however, can be summarized as follows: cells apparently come into contact with their prey by accident (Le. Dunaliella bioculata), either with the cell apex or the longitudinal flagellum. A cell then swims around the prey probing its surface several times with the longitudinal flagellum before it rapidly encircles the cell to be phagocytosed which, during this process, is held close to the posterior right cell surface of the predator. Ingestion of the prey follows within a few seconds near this site. We have obtained no evidence that the tentacle of O. marina is involved in the phagocytotic process. To analyze changes in the structure of the cell surface and the peripheral microtubular cytoskeleton of

Figures 21-26. Electron microscope images of a cell of Oxyrrhis marina during phagocytosis. Images from a series of cross sections through the cell in the region of the cytostome have been selected. The sequence starts (Fig. 21) from the cell's posterior and the viewer looks onto the cell from its posterior end. Images are oriented such that the

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Figures 27-28. Electron microscopy images of the phagocytotic process in Oxyrrhis marina. Scale bar: 1 ~m. 27. A cell of O. marina fixed during ingestion of a D. bioculata cell. The ventral surface of the dinoflagellate cell is located to the right of the micrograph. The ventral ridge microtubules (vrm) and the 'lifted' anteriorly focused cortical microtubules can be seen. 28. A cell of O. marina after phagocytosis of a D. bioculata ell. The ventral surface of the dinoflagellate is located at the bottom of the micrograph. The engulfed prey is enclosed within a food vacuole (arrow). The food vacuole is located close to the anterior end of the phagocytosis site as indicated by the ventral ridge microtubules (vrm).

O. marina upon phagocytosis, cells preying for 2 minutes on the flagellate green alga D. bioculata were fixed for immunofluorescence and electron microscopy. After this time various stages of prey capture or ingestion were found, allowing us to propose a putative sequence of events taking place during phagocytosis. The earliest stages of phagocytosis are characterized by the presence of D. bioculata in contact with the posterior right cell surface of O. marina (Fig. 13). During the next stage the peripheral microtubular cytoskeleton of O. marina in the contact area undergoes a conspicuous rearrangement: the anteriorly focused microtubular bands located in the ventral right region of the cell are displaced towards the cell apex exposing an area of the cell surface not subtended by microtubules representing the nonpermanent cytostome (Figs 14-18, 29). The microtubular bands curve to the cell's right only in a region posterior to the vrm, whereas anterior to the vrm all anteriorly focused microtubular bands remain longitudinally oriented during phagocytosis (Figs 14, 15, 17, 18, 29). The extent of microtubular bending seems to be variable resulting in cytostomes of different sizes (Figs 14-18). Interestingly, a single microtubular band located along the right rim of the

ventral surface is at the bottom of the micrographs. The ingested cell of D. bioculata (open arrow in Fig. 26) has fallen out of the cytostome during fixation/preparation for electron microscopy. Scale bar: 1 ~m. 21. The longitudinal flagellum (If) is seen in cross section within the flagellar groove in the left region of the micrograph. The arrowhead marks the beginning of the cytostome region which is characterized by the absence of amphiesmal vesicles underlying the plasma membrane. 22. Section 11 of the series. Basal body of longitudinal flagellum in cross section with flagellar roots (arrow: transverse striated fibrous root). 23. Section 17 of the series. The ventral ridge can be seen towards the bottom of the micrograph, the transverse flagellum is obliquely sectioned near the left corner of the micrograph (open arrow). 24. Section 19 of the series. The ventral ridge microtubules (vrm) are depicted. 25. Section 21 of the series. At the anterior end of the cytostome, the 'lifted' anteriorly focused cortical microtubules (em) can be seen. At the upper right of the micrograph a cell of D. bioculata is located which presumably had been ingested and fallen out of the cytostome. 26. Section 23 of the series. The 'lifted' cortical microtubules (em) are located near the anterior edge of the cytostome.

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longitudinal flagellar groove is not dislodged during cytostome formation and marks the left edge of the cytostome (Figs 14, 16-18, 29; black arrowhead). The formation of a non-permanent cytostome is apparentlya prerequisite for phagocytosis. After ingestion of the prey, the cytostome disappears and the microtubular bands resume their pre-phagocytotic arrangement (Figs 19, 20). This must occur within 2 minutes or less, as cells with a reformed peripheral microtubular cytoskeleton containing D. bioculata in food vacuoles were frequently observed in samples fed with D. bioculata for 2 minutes before fixation and analysis by immunofluorescence or electron microscopy (Figs 19, 20). Serial section electron microscopy has revealed further details of the cell surface in the region of the non-permanent cytostome and its positional relationship to the flagellar basal apparatus (Figs 21-26). In the early stages of the phagocytotic process, cells of D. bioculata sometimes dissociate from the cy-

dorsal

ventral

right

right vrm /

tostome upon fixation, dehydration or embedding for electron microscopy and the plasma membrane of the cytostome then often evaginates (as in Figs 21, 22; however, refer to plasma membrane invagination in Fig. 27). The plasma membrane in the region of the cytostome is not subtended by microtubular bands or amphiesmal vesicles as observed for almost all other surface areas of the cell (Figs 21, 22). The cytostome is located posterior to the vrm (Fig. 24) on the ventral right cell surface and is bound on one side by the flagellar groove of the longitudinal flagellum (Figs 21-23). Near the vrm a bundle of peripheral microtubules representing the dislodged anteriorly focused microtubular bands was always observed (Figs 25-27). The serial section analysis gave no indication for discharged trichocysts during early stages of phagocytosis suggesting that trichocysts are not involved in prey capture. Following phagocytosis the engulfed cell is enclosed within a food vacuole (Fig. 28).

ventral, feeding

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Figure 29. Diagrammatic presentation of the peripheral microtubular cytoskeleton of Oxyrrhis marina based on an anti-~-tubulin

immunofluorescence analysis of permeabilized cells (for details refer to the Results section). The peripheral cytoskeleton is shown as if viewed from the outside of the cell. The dorsal (dorsal) or ventral (ventral) cell surfaces are depicted, as is the ventral cell surface during phagocytosis (ventral, feeding). The left and right halves of each cell are also indicated (please note that by convention the designations left and right refer to a view from the dorsal side of the cell to the outside). The arrow indicates the displaced ('lifted') part of the anteriorly focused array of microtubules revealing the non-permanent cytostome. The ventral ridge microtubules (vrm) are also shown. The position of the longitudinal (If) and the transverse flagellum (tf) as well as the tentacle (asterisk) are shown as landmarks.

Oxyrrhis Cytoskeleton

Discussion The cortical microtubular cytoskeleton of Oxyrrhis marina has been previously studied by immunofluorescence microscopy on several occasions. However interpretations of the images obtained have been somewhat ambiguous, presumably due in part to the suboptimal conditions used for fixation (inclusion of 0.1 % glutaraldehyde) which led to a strong background fluorescence (Brown et al. 1988; Roberts 1991; Roberts and Roberts 1991; Roberts et al. 1992, 1993). Whereas, we confirmed the absence of both the longitudinal array of cingular microtubules (clb) and the transverse microtubular bands (atb, ptb), in accordance with the lack of a girdle in this organism, we encountered two distinct and separate sets of differently focused longitudinal arrays of microtubules and conclude in contrast to a previous study (Roberts et al. 1993) that there are no continuous pole to pole microtubules in Oxyrrhis. The anteriorly focused array of microtubules in O. marina closely resembles the system (alb) present in other dinoflagellates in that it is also split near the mid-ventral surface into a left and right part (for Gymnodinium spp. see Roberts et al. 1988b and Roberts and Roberts 1991; for Woloszynskia pascheri: Meinicke, H6hfeld and Melkonian, unpublished observations). However, compared to other dinoflagellates the anteriorly focused array of microtubules in O. marina appears to be in the mirror image orientation: in the ventral right region of the cell the microtubular bands are bent to the cell's right (Fig. 29), whereas in the same region in other dinoflagellates they are bent towards the cell's left (Fig. 9 in Roberts and Roberts 1991 ; Meinicke, H6hfeld and Melkonian, unpublished observations). Similarly, the abutting microtubular bands are located in the ventral left region of the cell in 0. marina (Fig. 29), whereas in other dinoflagellates they are located in the ventral right region of the cell. The differences in orientation of the anteriorly focused array of microtubules between Oxyrrhis and other dinoflagellates may be related to the presence or absence of a centrin fiber at the cell apices: whereas in all other dinoflagellates investigated a centrin fiber is located at the cell apex exactly in the focal area from which the microtubular bands radiate (Meinicke, H6hfeld and Melkonian, unpublished observations; for information on the presence of a centrin fiber at the cell apex in another dinoflagellate, see Roberts and Roberts 1991), it is apparently absent in O. marina (H6hfeld et al. 1994). The second, ventrally focused, array of microtubules in O. marina is predominantly located in the posterior third of the cell and presumably because it

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is less conspicuous than the anteriorly focused array has not been previously recognized. It is broadly focused near the flagellar insertion in the mid-ventral region of the cell and extends around the cell antapex to meet the anteriorly focused microtubules at the dorsal cell surface (Fig. 29). At the junction between the two arrays, the ventrally focused microtubules bend towards the cell's left and the anteriorly focused microtubules abut these almost transversely oriented microtubules (Fig. 29). Because of its mid-ventral focus, this array of microtubules presumably cannot make direct contact with the anteriorly focused microtubules in the ventral half of the cell: whereas the 'gap' in the peripheral microtubular cytoskeleton in the ventral left region of the cell is 'closed' by an intercalating array of microtubules (Fig. 29), there are no intercalating microtubules in the cell's ventral right region where, because they are considerably longer in this region of the cell, the anteriorly focused microtubules partially 'close' this 'gap' (Fig. 29). In the ventral right region of the cell the anteriorly and ventrally focused microtubules are separated from each other by the groove of the longitudinal flagellum. It is in this part of the cell that the non-permanent cytostome of O. marina is displayed (see below). We propose that the ventrally focused microtubules in O. marina are homologous to the posteriorly focused microtubules (plb) of other dinoflagellates and thus, that the posterior region of the Oxyrrhis cell covered by the ventrally focused microtubules and the set of intercalating microtubules (subtending the left cell surface in this region) is equivalent to the 'hypocone' of other dinoflagellates. Kofoid and Swezy (1921) already considered Oxyrrhis to be a 'primitive dinoflagellate' because of its 'imperfect girdle', 'incompletely differentiated sulcus' and 'apparent lack of differentiation of the transverse flagellum into the typical ribbon'. Molecular phylogenetic analyses on two highly divergent domains (01 and 08) of the nuclear-encoded LSU rONA gene in 13 taxa of dinoflagellates corroborated their view, as O. marina emerged as the earliest branching lineage in the dinoflagellates when the ciliate Tetrahymena thermophila was used as the outgroup (Lenaers et al. 1991). We envisage the following hypothetical scenario for the evolutionary transformation of an Oxyrrhis-type peripheral cytoskeleton to the more typical microtubular cytoskeleton of dinokont dinoflagellates: as the sulcus elongated, it pushed the ventral focal area towards the cell's posterior. This enabled all intercalating microtubules in the ventral left region of the cell to become focused in the same area as the ventrally focused microtubules, i.e. at the distal end of the sul-

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cus near the antapex of the cell. In addition, from the distal end of the sulcus a new set of microtubules may have developed, which extended (almost transversely) towards the cell's ventral right to close the 'gap' between the sulcus and the anteriorly focused array of microtubules and establish another contact zone between anteriorly and posteriorly focused microtubules. Differential growth of the microtubular arrays on either side of the sulcus would have led to the displacement of the respective contact zones along the anterior/posterior cell axis, whereas coordinated growth of the respective microtubular arrays would have resulted in contact zones being nearly at the same level at the ventral left and right cell surface. Since we regard the contact zone between the two longitudinal arrays of microtubules as the future site of evolution of the girdle, differential growth of microtubules (as described above) would have favoured the evolution of strongly helical girdles (as observed in Gyrodinium and Cochlodinium), whereas coordinated growth of microtubules would have favoured more circular girdles (as with Gymnodinium). The evolution of the microtubular systems of the girdle (i.e. the two transverse bands of microtubules plus the longitudinal array of cingular microtubules, see above) was perhaps related to the evolution of the sulcus, as these microtubular arrays are likely to have originated from the sulcal area (Roberts 1991; Meinicke, H6hfeld and Melkonian, unpublished observations). Although the above scenario for the evolution of the dinokont cortical cytoskeleton from an Oxyrrhistype ancestral cytoskeleton is entirely speculative, it suggests to us that there is no fundamental difference in the organization of the peripheral microtubular cytoskeleton between O. marina and other dinoflagellates, and that the differences observed can be readily explained by the lack of a typical sulcus and girdle in Oxyrrhis. This conclusion is corroborated by a comparison of the structure of the flagellar basal apparatus between O. marina and other dinoflagellates which is essentially identical, most of the basal apparatus components which are unique to the dinoflagellates (tsr, tmr, pmr, src, etc.; review: Roberts and Roberts 1991) are also found in Oxyrrhis. In addition, the 'ventral ridge fiber' (vrf) in O. marina (Roberts et al. 1993; H6hfeld et al. 1994) is presumably homologous to the 'flagellar collar' system of other dinoflagellates since both systems interconnect the two flagellar bases and contain the Ca2+-modulated contractile protein centrin (H6hfeld et al. 1994; Meinicke, H6hfeld and Melkonian, unpublished observations). Structural differences between the flagellar apparatus of O. marina and that of other dinoflagellates relate to the presence of unique organelles

(e.g. the 'tentacle' of Oxyrrhis; Roberts 1985) or the absence of a girdle (the transverse flagellum of O. marina lacks a centrin-containing paraxonemal fiber; H6hfeld et al. 1988; H6hfeld et al. 1994). The peripheral cytoskeleton of dinoflagellates (including Oxyrrhis) differs considerably from that of other protists such as the kinetoplastids and euglenoids (Triemer and Farmer 1991), retortamonads (Brugerolle 1991), heterokonts (Wetherbee et al. 1992), flagellate green algae (Lechtreck et al. 1989), ciliates (Cohen and Beisson 1993) or apicomplexans (Vivier and Desportes 1990) because it is neither focused on the basal bodies (kinetosomes) or flagellar roots nor does it extend continuously along the cell axis from pole to pole. The bipolar organization of the cortical microtubular cytoskeleton is, to our knowledge, a unique characteristic of the dinoflagellates. O. marina is a voracious predator feeding on a variety of prey including small flagellates, yeasts and diatoms; even freshly moulted amphipods can be attacked (Droop 1953; Droop 1966; Gaines and EIbrachter 1987). Ingestion of prey is very rapid (10-15 seconds; Opik and Flynn 1989) and because of the high swimming speed of O. marina the phagocytotic process could not be documented by video microscopy (see also Cosson et al. 1988). Using precise feeding periods and immunofluorescence microscopy we have localized the cytostome of O. marina to the ventral right cell surface in the posterior region of the cell. Previously, the cytostome of Oxyrrhis was thought to be located near the tentacle (Brown et al. 1988; Dodge and Crawford 1974). Brown et al. (1988) described a slit-like structure near the opening of the flagellar groove of the transverse flagellum which the authors termed 'the feeding apparatus with the two lips and the cytostomal fissure between'. Dodge and Crawford (1974) had previously suggested that the cytostome of O. marina might be located at the base of the tentacle and thus close to the flagellar basal apparatus. Since the ventral ridge microtubules (vrm) terminate in the inner of the two lips and because the vrm are connected to the contractile centrin-containing vrf (H6hfeld et al. 1994) it is possible that the slit-like opening, functions mainly to allow the vrm enough space to reorientate upon contraction of the vrf (H6hfeld et al. 1994 and below). One of the more significant observations of this study has been the documentation of the conspicuous rearrangements of the peripheral microtubular cytoskeleton upon phagocytosis. While we have no experimental data to suggest a mechanism for the displacement of the anteriorly focused microtubules in the ventral right region of the cell, we note that 'differential lifting' of the microtubules would allow the cell to adjust the size of the cytostome to the

Oxyrrhis Cytoskeleton

size of its prey. Since the anterior delimitation of the cytostome is marked by the vrm which distally (towards the cell's right) bend upwards (Figs 1, 5, 17, 19) the cell can phagocytose prey of almost its own size. Why the vrm prevent the longitudinal array of microtubules located anteriorly to them to become displaced or 'lifted' is also unknown, but could be related to the reorientation of the vrm (rotation of 90° from face to edge view) described previously (H6hfeld et al. 1994). It is conceivable that contraction of the centrin-containing ventral ridge fiber leads to reorientation of the vrm which could become appressed to the anteriorly focused microtubules thus preventing their dislocation. Conceptually the 'lifting' of the peripheral microtubules during phagocytosis may be thought of as a shortening of filaments extending from the distal part of the vrm to a microtubular band of the anteriorly focused array of microtubules located close to the flagellar groove of the longitudinal flagellum. Alternatively, the microtubules may simply be pushed aside passively as the prey is ingested. As in other 'naked' protists of sufficient size and/or elongated shape (e.g. the kinetoplastids) the athecate dinoflagellates utilize longitudinal arrays of cortical microtubules as the major mechanical support for the plasma membrane, and thus the rigidity of the whole cell (review: Bouck and Ngo 1996). We propose that the organization of the cortical cytoskeleton from two opposing foci enabled the dinoflagellates to evolve a multitude of phagocytotic mechanisms which exploit a large spectrum of prey types and sizes while maintaining its principal cytoskeletal function.

Methods The marine dinoflagellate Oxyrrhis marina Dujardin (UTEX LB 1974) was grown in modified Provasoli's artificial seawater medium (ASP-H; McFadden and Melkonian 1986) at 15°C in the dark using 1 I-Erlenmeyer flasks. The organism was regularly fed with a small quantity of the flagellate green alga Dunaliella bioculata Butcher (SAG 19-4) which was completely phagocytosed within 1 hour (Melkonian and H6hfeld 1988). For indirect immunofluorescence, cells of O. marina were fixed in freshly prepared 3% paraformaldehyde in MT-buffer containing 4% (wlv) NaCI (essentially as described by Katsaros and Galatis 1992) for 20 minutes. During this time, the cells were allowed to adhere to poly-L-Iysine-coated coverslips and, after gradually reducing the NaCI-concentration of the MT-buffer, were washed in PBS. The cells were permeabilized with 1% Nonidet P40

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(Sigma, Deisenhofen, Germany) in PBS for 10 minutes and finally washed in PBS. All subsequent steps were performed according to the protocol of H6hfeld et al. (1994) using a monoclonal anti-/3tubulin-lgG 2b (Boehringer, Mannheim, Germany), as the primary antibody diluted 1:200, and fluorescein-conjugated polyclonal sheep-anti-mouse-lgG (Boehringer, Mannheim), as the secondary antibody diluted 1:50. To analyse the microtubular cytoskeleton of O. marina during phagocytosis, cells were fixed according to the above protocol 2 minutes after the addition of the food organism D. bioculata. For standard thin-section electron microscopy of O. marina, cells were fixed and embedded as previously described (H6hfeld et al. 1994). Again, phagocytosis was analysed by fixing cells 2 minutes after mixing with D. bioculata.

Acknowledgements We thank Martina Meinicke (Cologne) for helpful discussions. IH was supported by a fellowship from the University of Cologne. This study was supported by the Deutsche Forschungsgemeinschaft.

References Biecheler B (1952) Recherches sur les Peridiniens. Bull Bio Fr Belg Suppl 36: 1-149 Bockstahler KR, Coats DW (1993) Spatial and temporal aspects of mixotrophy in Chesapeake Bay dinoflagellates. J Eukaryotic Microbiol 40: 49-60 Bouck GB, Ngo H (1996) Cortical structure and function in euglenoids with reference to trypanosomes, ciliates and dinoflagellates. Int Rev Cyto1169: 267-318 Brown DL, Cachon J, Cachon M, Boillot A (1988) The cytoskeletal microtubular system of some naked dinoflagellates. Cell Motil Cytoskeleton 9: 361-374 Brugerolle G (1991) Flagellar and cytoskeletal systems in amitochondrial flagellates: Archamoeba, Metamonada and Parabasala. Protoplasma 164: 70-90 Cohen J, Beisson J (1993) The cytoskeleton. In G6rtz H-D (ed) Paramecium. Springer, Berlin, pp 1-45 Cosson J, Cachon M, Cachon J, Cosson M-P (1988) Swimming behaviour of the unicellular biflagellate Oxyrrhis marina: in vivo and in vitro movement of the two flagella. Bioi Cell 63: 117-126 Dodge JD (1973) The Fine Structure of Algal Cells. Academic Press, London Dodge JD, Crawford RM (1974) Fine structure of the dinoflagellate Oxyrrhis marina III. Phagotrophy. Protistologica 10: 239-244

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Droop MR (1953) Phagotrophy in Oxyrrhis marina. Nature 172: 250 Droop MR (1966) The role of algae in the nutrition of Heteramoeba clara Droop, with notes on Oxyrrhis marina Dujardin and Phi/odina roseola Ehrenberg. In Barnes H (ed) Some Contemporary Studies in Marine Science. George Allen and Unwin Ltd, London, pp 269-282 Elbrachter M (1991) Food uptake mechanisms in phagotrophic dinoflagellates and classification: a discussion. In Patterson OJ, Larsen J (eds) The Biology of Free-living Heterotrophic Flagellates. Systematics Association Special Volume No 45. Clarendon Press, Oxford, pp 303-312 Gaines G, Elbrachter M (1987) Heterotrophic nutrition. In Taylor FJR (ed) The Biology of Dinoflagellates. Blackwell, Oxford, pp 224-268 Hohfeld I, Beech P, Melkonian M (1994) Immunolocalization of centrin in Oxyrrhis marina (Dinophyceae). J PhycoI30:474-489 Hohfeld I, Otten J, Melkonian M (1988) Contractile eukaryotic flagella: centrin is involved. Protoplasma 147: 16-24 Jacobson DM, Anderson DM (1996) Widespread phagocytosis of ciliates and other protists by marine mixotrophic and heterotrophic thecate dinoflagellates. J Phycol 32: 279-285

Opik H, Flynn KJ (1989) The digestive process of the dinoflagellate Oxyrrhis marina Dujardin, feeding on the chlorophyte, Dunaliella primo/ecta Butcher: a combined study of ultrastructure and free amino acids. New Phyto1113: 143-152 Perret E, Davoust J, Albert M, Besseau L, Soyer-Gobillard M-O (1993) Microtubule organisation during the cell cycle of the primitive eukaryote dinoflagellate Crypthecodinium cohnii. J Cell Sci 104: 639-651 Roberts KR (1985) The flagellar apparatus of Oxyrrhis marina (Pyrrophyta). J Phycol 21: 641-655 Roberts KR, Farmer MA, Schneider RM, Lemoine JE (1988a) The microtubular cytoskeleton of Amphidinium rynchocepha/um (Dinophyceae). J Phycol 24: 544-553 Roberts KR, Lemoine JE, Schneider RM, Farmer MA (1988b) The microtubular cytoskeleton of three dinoflagellates: an immunofluorescence study. Protoplasma 144: 68-71 Roberts KR (1991) The Flagellar Apparatus and Cytoskeleton of Dinoflagellates: Organization and Use in Systematics. In Patterson OJ, Larsen J (eds) The Biology of Free-liVing Heterotrophic Flagellates. The Systematics Association Special Volume No. 45. Clarendon Press, Oxford, pp 285-302 Roberts KR, Roberts JE (1991) The flagellar apparatus and cytoskeleton of the dinoflagellates. A comparative overview. Protoplasma 164: 105-122

Katsaros C, Galatis B (1992) Immunofluorescence and electron microscopic studies of microtubule organization during the cell cycle of Dictyota dichotoma (Phaeophyta, Dictyotales). Protoplasma 169: 75-84

Roberts KR, Roberts JE, Cormier SA (1992): The Dinoflagellate Cytoskeleton. In Menzel 0 (ed) The Cytoskeleton of the Algae. CRC Press, Boca Raton, pp 19-38

Kofoid CA, Swezy 0 (1921) The Free-living Unarmored Dinoflagellata. Univ Calif Press, Berkeley

Roberts KR, Rusche ML, Taylor FJR (1993) The cortical microtubular cytoskeleton of Oxyrrhis marina (Dinophyceae) observed with immunofluorescence and electron microscopy. J Phycol29: 642-649

Lebour MV (1925) The Dinoflagellates of Northern Seas. Mar Bioi Ass UK, Plymouth Lechtreck K-F, McFadden GI, Melkonian M (1989) The cytoskeleton of the naked green flagellate Spermatozopsis simi/is: isolation, whole mount electron microscopy, and preliminary biochemical and immunological characterization. Cell Moti! Cytoskeleton 14: 552-561 Lenaers G, Scholin C, Bhaud Y, Saint-Hillaire D, Herzog M (1991) A molecular phylogeny of dinoflagellate protists (Pyrrophyta) inferred from the sequence of 24S rRNA divergent domains 01 and 08. J Mol Evol 32: 53-63 Li AS, Stoecker DK, Coats DW, Adam EJ (1996) Ingestion of fluorescently labeled and phycoerythrincontaining prey by mixotrophic dinoflagellates. Aquat Microb Eco110: 139-148 McFadden GI, Melkonian M (1986) Use of Hepes buffer for microalgal culture media and fixation for electron microscopy. Phycologia 25: 551-557 Melkonian M, Hohfeld I (1988) Amphiesmal ultrastructure in Noctiluca miliaris Suriray (Dinophyceae). Helgol Meeresunters 42: 601-612

Schnepf E, Elbrachter M (1992) Nutritional strategies in dinoflagellates. A review with emphasis on cell biological aspects. Europ J Protistol 28: 3-24 Soyer MO (1970) Les ultrastructrure liees aux fonctions de relation chez Nocti/uca mi/iaris S. (Dinoflagellata). Z Zellforsch 104: 29-55 Taylor FJR (1987) The Biology of Dinoflagellates. Blackwell, Oxford Triemer RE, Farmer MA (1991) An ultrastructural comparison of the mitotic apparatus, feeding apparatus, flagellar apparatus and cytoskeleton in euglenoids and kinetoplastids. Protoplasma 164: 91-104 Vivier E, Desportes, I (1990) Phylum Apicomplexa. In Margulis L, Corliss JO, Melkonian M, Chapman OJ (eds) Handbook of Protoctista. Jones and Bartlett Publ, Boston, pp 549-573 Wetherbee R, Koutoulis A, Andersen RA (1992) The microarchitecture of the chrysophycean cytoskeleton. In Menzel 0 (ed) The Cytoskeleton of the Algae. CRC Press, Boca Raton, PP 1-17