J. Cell Sci. 9, 443-4S> ('97i) 443

Printed in Great Britain

THE INTRACELLULAR ORIGIN OF FLAGELLAR

HAIRS IN THE DINOFLAGELLATE

WOLOSZYNSKIA MICRA LEADBEATER

& DODGE

B. S. C. LEADBEATERDepartment of Botany, University of Birmingham, Edgbaston, Birmingham B15 zTT,England

SUMMARY

The arrangement of fine hairs on the longitudinal and transverse flagella of Woloszynskiavticra is illustrated and discussed. Dilations of the nuclear envelope and endoplasmic reticulumin W. micra, similar in appearance and location to those containing flagellar hairs in other algaland fungal zoids, are found to contain bundles of fibrils. These fibrils are approximately thesame width and length as the flagellar hairs. For the above reasons it is now considered thatthe flagellar hairs in the dinoflagellate W. micra are formed intracellularly within dilations ofthe nuclear envelope and endoplasmic reticulum and later deposited on the flagella.

INTRODUCTION

The intracellular origin of flagellar hairs has now been demonstrated in a widerange of motile cells. Manton, Rayns, Ettl & Parke (1965) first noted that the finecaducous hairs on the flagella of 2 species of Heteromastix (Prasinophyceae) wereproduced within vesicles of the Golgi apparatus. More recently it has been shown thatthe tubular flagellar hairs found on many algal and fungal zoids are produced withinvesicles of the endoplasmic reticulum (ER) and dilations of the nuclear envelope(Bouck, 1969; Leedale, Leadbeater & Massalski, 1970; Heath, Greenwood & Griffiths,1970).

The dinoflagellates differ from all other motile cells except the euglenoid flagellates(Leedale, 1967) in that they possess a single row of long fine hairs on one of theirflagella (Pitelka & Schooley, 1955; Leadbeater & Dodge, 1967a, b; Dodge, 1967;Dodge & Crawford, 1968). An intensive study of the fine structure of Woloszynskiamicra Leadbeater & Dodge (Leadbeater & Dodge, 1966; Leadbeater, 1967) revealedthe presence of large dilations of the nuclear envelope and ER that contained bundlesof fibrils of unknown significance. As will be shown below, the similarity in theposition and appearance of these dilations to those containing flagellar hairs in otherflagellates (see Leedale et al. 1970 etc.) together with the similarity in dimensions andappearance of the individual fibrils to the flagellar hairs suggests that in the dino-flagellate W. micra these also originate within the cell.

444 B. S.C. Leadbeater

MATERIAL AND METHODS

Two unialgal isolates of Woloszynskia micra Leadbeater & Dodge (Plymouth culturecollection nos. 207 and 366) were supplied by Dr M. Parke of the Marine Biological Association,Plymouth. Cultures of the isolates were maintained in Erdschreiber seawater at 20 °C under16 h light/8 h dark cycle.

For light microscopy a small quantity of culture was fixed in a solution of 2 % osmiumtetroxide in 01 M acetate veronal buffer at pH 70. Cells were viewed with anoptral contrastmicroscopy.

For electron microscopy of whole mounts, cells were concentrated by gentle centrifugation,fixed in a solution of 2% osmium tetroxide in acetate-veronal buffer at pH 7-0, washed andthen dried on Formvar-coated grids. Some specimens were shadowcast with gold/palladiumand others were negatively stained with sodium phosphotungstate at pH 7-0.

Material for embedding was centrifuged to form a pellet and then fixed for 15 h in ice-cold5 % glutaraldehyde in a balanced salt solution with acetate-veronal buffer at pH 78. This wasfollowed by washing in buffer, postosmication in 2 % osmium tetroxide in acetate-veronal atpH 7-8 (2 h), dehydration in a graded ethanol series, and embedding in Araldite. Sections werecut with a glass knife on a Porter Blum I ultramicrotome, stained with methanolic uranyl acetatefollowed by Reynold's lead citrate. Most observations were made on a Zeiss EM 9 microscopeat Birkbeck College, London. Supplementary observations were made on an AEI EM 6microscope in Birmingham.

OBSERVATIONS

Cells of Woloszynskia micra are approximately 9-15/6m long and 8-14 /tin wideand are divided almost equally into an upper epicone and lower hypocone by thealmost transverse girdle or sulcus (Fig. 2). Two flagella emerge from the cell in themid-ventral region. The longitudinal flagellum (about 10 /tm long) extends posteriorlywhilst the transverse flagellum (about 30 /tm long) encircles the cell within the girdle.

The longitudinal flagellum of W. micra bears a covering of short fine hairs (Fig. 3).On dried whole mounts the hairs appear to be borne bilaterally but whether this is anartifact caused by the method of preparation is not known. The hairs, approximately10 nm in width and 0-5 /tm in length, show no differentiation at the base or tip.

The sheath surrounding the transverse flagellum is expanded laterally and containsthe axoneme and 'striated strand' (see Leadbeater & Dodge, 1967a) separated bypacking material. Dried whole-cell mounts show that the axoneme undulates, simu-lating a sine curve when flattened, whilst the accessory striated strand follows a shorter,almost straight course (Fig. 4). The relationship between the axoneme and the striatedstrand can be seen best in fixed cells mounted in water and viewed with anoptralcontrast microscopy (Fig. 1). The axoneme winds around the shorter striated strandwhich runs along the centre of the helix. This arrangement results in the helicalmovement of the flagellum during swimming. The transverse flagellum bears aunilateral row of long (approximately 3-4 /tm), fine hairs which appear to be attachedto the sheath adjacent to the axoneme (Fig. 4). In shadowcast whole mounts (Fig. 4)the flexible hairs frequently clump together in groups of 2 or 3. When negativelystained the hairs are more rigid in appearance but are of regular width throughouttheir length (Fig. 5). In both shadowcast and negatively stained preparations theaverage width is 10 nm.

Origin of flagellar hairs 445

In sections examined by electron microscopy, most cells can be seen to containlarge dilations of either the nuclear envelope or the ER (Figs. 6-9, 11, 12) and thelatter may be closely associated with the Golgi apparatus (Fig. 9). These dilated profiles,which may be up to 5 fim in length, usually contain bundles of fibrils (Figs. 7-12)which are now interpretable as developing or mature flagellar hairs. This interpretationis based on the equivalent location and appearance of vesicles known to containdeveloping flagellar hairs in other algal and fungal zoids (see Bouck, 1969; Leedale etal. 1970; Heath et al. 1970 for list of species) and on the similarity, in appearance andwidth, between the individual fibrils within the vesicles and the flagellar hairs.

Table 1. Percentage of cells with fibril-containing dilations of the nuclear envelope afterbeing kept in different light regimes

Percentage of cells with fibril-containing dilations ofthe nuclear envelope after 3 days of:

Number of Continuous Alternating light TotalExperiment cells counted light and dark darkness

I

2

Average

2 8 0

2 3 0

2-8

3-°2 9

1 6

2-0

i -8

63945-9

S4'9

Although the individual fibrils cannot be clearly seen in permanganate-fixed cells(Fig. 6 and inset) the membrane surrounding the vesicles (v1 andw2, Fig. 6) and theirconnexion with the ER are obvious (Fig. 6, inset). On the other hand in cells post-osmicated after glutaraldehyde fixation the fibrillar nature of the contents is distinct(Figs. 7-12). In whatever plane a large dilation is sectioned, fibrils running in morethan one direction can usually be found. When observed in side view the fibrils areseen as delicate, more or less parallel lines sometimes marked by irregular transversestriations (Fig. 12) which may be artifacts. In transverse section the extreme thinnessof the hairs is more suggestive of a fine granular deposit (Figs. 8, 9, 12). As the fibrilsare so delicate no developmental stages have been observed. The approximate widthof mature fibrils is 10 nm (Fig. 10). This is substantially less than the width of thecomparable fibres in Olisthodiscus luteus Carter (Leadbeater, 1969) and the otherheterokont organisms enumerated below but agrees closely with the flagellar hairs ofthis organism. The length of individual fibrils varies according to the plane of sectionbut in median longitudinal section, fibrils measuring 5 fim have been recorded.

Under normal light conditions (16 h light/8 h dark) only a few cells possess dilationsof the nuclear envelope. In cultures that have been maintained in darkness for 3 daysprior to fixation the number of cells with perinuclear dilations increases from approxi-mately 2 to 55 % (see Table 1).

A section of a cell fixed after 3 days continuous darkness is illustrated in Fig. 7.The mitochondria are large, very little ER is present and there is an accumulation offibrillar material within the perinuclear space (arrow).

In cells grown under normal light conditions the dilations of the nuclear envelope

446 B. S. C. Leadbeater

appear to detach from the nucleus and come into close association with the Golgiapparatus (Fig. 9). These fibril-containing vesicles (/) also lie close to vesicles con-taining trichocyst precursors (t) (Fig. 11). The latter are probably of Golgi origin.However, on no occasion has any definite membrane contact been observed betweenfibril-containing ER vesicles and either the Golgi apparatus or vesicles containingtrichocyst precursors. Movement of the mature fibrils to the surface of the cell and theirsubsequent discharge on to the flagella has not been observed.

DISCUSSION

The general morphology of Woloszynskia micra has already been well documented(Leadbeater & Dodge, 1966, 1967a, b; Leadbeater, 1967). The very fine hairs ontransverse flagella have also been recorded in other dinoflagellate genera, for exampleGymnodinium sp. (Pitelka & Schooley, 1955), Aureodiniumpigmentosum Dodge (Dodge,1967) and Amphidinium carteri Hulbert (Dodge & Crawford, 1968). Recent observa-tions on dinonagellates collected from Norwegian waters show that the presence oflong, fine hairs is a standard feature of transverse flagella (B. Leadbeater, unpublishedobservations). The occurrence of fine hairs on the longitudinal flagellum does notappear to be so common. Pitelka & Schooley (1955) did not observe hairs on thelongitudinal flagellum of Gymnodinium sp. and they were not present on the longi-tudinal flagella of Aureodinium pigmentosum (Dodge, 1967) or Amphidinium carteri(Dodge & Crawford, 1968).

The average width of the hairs on both the transverse and longitudinal flagella is10 nm irrespective of the method of preparation. This is identical to the averagewidth of fibrils located within dilations of the nuclear envelope and ER. Furthermore,the fine fibrils found within dilations of the nuclear envelope in Amphidinium carteriare also approximately 10 nm in width (measurement taken from fig. 21 in Dodge &Crawford, 1968). The length of the hairs of the transverse flagellum is approximately5 fim and in exceptional circumstances when exact median longitudinal section of theintracellular fibrils is obtained, a length of 5 /tm has been recorded. At present, it isnot possible to distinguish between intrinsically short fibrils which might eventuallyform the short hairs of the longitudinal flagellum and oblique or tangential sections oflonger fibrils that will ultimately form the hairs of the transverse flagellum. Whetherthe hairs destined for a longitudinal flagellum are formed in separate dilations fromthose destined for a transverse flagellum is also unknown.

The presence in dinoflagellates of vesicles containing fibrils, sometimes arranged inbundles, has been recorded by several authors. Bouck& Sweeney (1966, fig. 8) observed'vacuoles with fibrous contents of unknown significance' in Prorocentrum micansEhrenberg. Dodge (1967, fig. 6) found a 'variably shaped fibrous body' which wasfrequently associated or adjacent to the nucleus in Aureodinium pigmentosum. InAmphidinium carteri the 'fibrous body' was found to develop from a dilation of thenuclear envelope (Dodge & Crawford, 1968). Leadbeater (1967) made a thoroughstudy of fibril-containing dilations in Woloszynskia micra. The fibrils were locatedwithin dilations of the nuclear envelope and the ER and their close association with

Origin offlagellar hairs 447

the Golgi apparatus and trichocyst precursors was well established. However, as thesignificance of these fibrils was unknown the record was incomplete. Following therecent observations on the intracellular origin offlagellar hairs (Bouck, 1969; Leedaleet al. 1970; Heath et al. 1970) it became obvious that there was a striking similarity inlocation and appearance between the dilations of the nuclear envelope and ER con-taining flagellar hairs in antherozoids of Ascophyllum nodosum (L.) Le Jol and Fucusspp. (Bouck, 1969); Olisthodiscus luteus Carter, Bumilleria sicula Borzi, Heterococctisspp., Tribonema spp. (Leedale et al. 1970); Saprolegnia ferax (Gruithuisen) Thuret,Dictyuchus sterile Coker, Synura caroliniana Whitehand, Cryptomonas sp. (Heath etal. 1970) and those containing fibrils in Woloszynskia micra.

The movement of the fibril-containing dilations in the cell under different lightconditions appears to be related to the fate of the ER. During dark treatment the ERdisappears from the cell cytoplasm and this probably prevents the fibril-containingdilations of the nuclear envelope from moving into the surrounding cytoplasm. Thisexplains why there is a considerable increase in the number of cells with fibril-containing dilations of the nuclear envelope. When the cells are returned to normallight conditions ER is re-formed and the dilations pass into the rest of the cytoplasm.

The close spatial association of the fibril-containing dilations in W. micra with theGolgi apparatus (Fig. 9; also figs. 6, 8 in Leadbeater & Dodge, 1966) and thetrichocyst precursors (Fig. 11) does not appear to imply structural continuity. Thefibril-containing dilations in Prorocentrum micans are similarly in close association withtrichocyst precursors (Bouck, 1969, fig. 7), and the ER vesicles containing flagellarhairs in Tribonema vulgare (Leedale et al. 1970, fig. 23) are in close association withcisternae and vesicles of the Golgi apparatus.

The final migration of the fibril-containing ER dilations to the cell surface and thesubsequent discharge of the fibrils on to the flagella has not been observed. This,unfortunately, is also the gap in the reports of Leedale et al. (1970) and Heath et al.(1970). Bouck (1969) only observed the outward migration of the vesicles containingpresumptive mastigonemes in Ascophyllum and Fucus but was unable to actuallyobserve the deposition of the hairs on the flagellum.

It now appears that the intracellular origin of flagellar hairs is standard for a widerange of algal and some fungal zoids. However the evidence in Woloszynskia micra isin no case complete. The absence of information on the mode of deposition has alreadybeen noted and chemical data are urgently required before equivalence can be claimedwith finality.

I would like to express my grateful thanks to Dr J. D. Dodge for supervising this work andreading the script; Dr M. Parke for kindly supplying the cultures of W. micra, and ProfessorI. Manton, F.R.S., for commenting on the plates and results. Finally I am grateful to S. R. C.for financial support.

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448 B. S. C. Leadbeater

REFERENCES

BOUCK, G. B. (1969). Extracellular microtubules. The origin, structure and attachment offlagellar hairs in Fucus and Ascophyllum antherozoids. J. Cell Biol. 40, 446-460.

BOUCK, G. B. & SWEENEY, B. M. (1966). The fine structure and ontogeny of trichocysts inmarine dinoflagellates. Protoplasma 61, 205-223.

DODGE, J. D. (1967). Fine structure of the dinoflagellate Aureodinium pigmentosum gen. etsp.nov. Br. phycol. Bull. 3, 327-336.

DODGE, J. D. & CRAWFORD, R. M. (1968). Fine structure of the dinoflagellate Amphidiniumcarteri Hulbert. Protistologica 4, 231-242.

HEATH, I. B., GREENWOOD, A. D. & GRIFFITHS, H. B. (1970). The origin of flimmer inSaprolegnia, Dictyuchus, Syruira and Cryptomonas.J. Cell Sci. 7, 445-461.

LEADBEATER, B. S. C. (1967). An Ultrastructural Study of the Morphology and Division of SomeMarine Dinoflagellates. Ph.D. Thesis, London University.

LEADBEATER, B. S. C. (1969). A fine structural study of Olisthodiscus luteus Carter. Br. phycol. J.4. 3-17-

LEADBEATER, B. & DODGE, J. D. (1966). The fine structure of Woloszynskia nticra sp.nov., anew marine dinoflagellate. Br. phycol. Bull. 3, 1-17.

LEADBEATER, B. & DODGE, J. D. (1967a). The fine structure of the dinoflagellate transverseflagellum. Nature, Lond. 213, 421-422.

LEADBEATER, B. & DODGE, J. D. (19676). An electron microscope study of dinoflagellate flagella.J. gen. Microbiol. 46, 305-314.

LEEDALE, G. F. (1967). Euglenoid Flagellates, pp. 1-242. Englewood Cliffs, N.J.: Prentice-Hall.LEEDALE, G. F., LEADBEATER, B. S. C. & MASSALSKI, A. (1970). The intracellular origin of

flagellar hairs in the Chrysophyceae and Xanthophyceae. J. Cell Sci. 6, 701-719.MANTON, I., RAYNS, D. G., ETTL, H. & PARKE, M. (1965). Further observations on green

flagellates with scaly flagella: the genus Heteromastix Korshikov. J. mar. biol. Ass. U.K.45, 241-255-

PITELKA, D. R. & SCHOOLEY, C. N. (1955). Comparative morphology of some protistan flagella.Univ. Calif. Publs Zool. 61, 79-128.

{Received 12 March 1971)

Fig. 1. Anoptral contrast photograph of a detached transverse flagellum showing thedrawn-out helix formed by the axoneme (a) winding around the striated strand (j).Micrograph 126, x 30000.Fig. 2. Shadowcast whole cell fixed with osmium tetroxide. The silhouette of theshrunken cell demonstrates the approximately equal size of the epicone and hypoconedivided by the transverse girdle. The transverse flagellum has looped over the epicone.Reversed print. Micrograph Gioo, x 4000.Fig. 3. Distal part of a longitudinal flagellum with a covering of fine short hairs. Partof a shadowcast whole mount. Micrograph D4, x 15000.Fig. 4. Part of a dried and flattened transverse flagellum. The axoneme (a) is undulatedwhilst the striated strand (s) follows a more or less straight course. The long fine hairsare attached to the flagellar sheath on the axoneme side. Micrograph G117, x 15000.Fig. 5. The long fine hairs of a transverse flagellum negatively stained with sodiumphosphotungstate. Micrograph D556, x 100000.Fig. 6. Permanganate-fixed specimen illustrating the general shape of the cell and thearrangement of internal organelles; 2 large dilations of the ER (D1 and ws) can beclearly observed. Micrograph F194, x 10000. Inset: higher power of the dilation vl

showing a connexion with the rest of the ER (arrow). Micrograph F194, x 25 000.

Origin of flagellar hairs 440

29-2

450 B. S. C. Leadbeater

Fig. 7. Section of a cell fixed after 3 days dark treatment. Note the dilation of thenuclear envelope (arrow) containing strands of fibrils. Micrograph D771, x 15000.Fig. 8. Dilation of the nuclear envelope containing 2 bundles of fibrils (nucleus, n).Micrograph D891, x 25000.

Fig. 9. Detached fibril-containing dilation close to a small stack of Golgi cisternae.Micrograph E876, x 25 000.

Fig. 10. Higher power of some of the fibrils from Fig. 9. Micrograph E876, x 100000.

Fig. 11. Three vesicles similar in appearance, 2 containing fibrils (/) and one con-taining a trichocyst precursor (t). Micrograph F421, x 25000.

Fig. 12. Large ER dilation containing fibrils in longitudinal and transverse (left)section. Micrograph E939, x 30000.

Origin of flagellar liairs