J. Cell Sci. 31, 25-35 (i978) 25Printed in Great Britain © Company of Biologists Limited 1978

THE ULTRASTRUCTURE OF CELL DIVISION

IN EUGLENA GRACILIS

MARCELLE A. GILLOTT ANDRICHARD E. TRIEMERDepartment of Botany, Rutgers University, New Brunswick, New Jersey, U.S.A.08903

SUMMARY

The ultrastructure of mitosis in Euglena gracilis was investigated. At preprophase thenucleus migrates anteriorly and associates with the basal bodies. Flagella and basal bodiesreplicate at preprophase. Cells retain motility throughout division. The reservoir and theprophase nucleus elongate perpendicular to the incipient cleavage furrow. One basal body pairsurrounded by a ribosome-free zone is found at each of the nuclear poles. The spindle formswithin the intact nuclear envelope. Polar fenestrae are absent. At metaphase, the endosome iselongated from pole to pole, and chromosomes are loosely arranged in the equatorial region.Distinct, trilayered kinetochores are present. Spindle elongates as chromosomes migrate to thepoles forming a dumb-bell shaped nucleus by telophase. Daughter nuclei are formed by con-striction of the nuclear envelope. Cytokinesis is accomplished by furrowing. Cell division inEuglena is compared with that of certain other algae.

INTRODUCTION

Euglena is a unicellular alga which can be grown under a variety of culture condi-tions. It can be easily manipulated in the laboratory and several mutant strains areavailable. These attributes make it an ideal subject for study and over 2300 papershave been published on Euglena since 1970. Nonetheless, many questions about thisorganism remain unanswered.

The euglenoid flagellates have long been recognized as a distinct group of organisms.They were awarded class status by Fritsch (1935) and constitute either a separate classor division in most modern taxonomic schemes. The interphase nucleus, withcondensed chromatin, and the distinctive mitotic mechanism are often included inlists of peculiar euglenoid characteristics.

Leedale (1967, 1968) reviewed the various theories concerning euglenoid mitosisbased on light-microscopic observations dating back to 1895. In addition, somepreliminary observations on the ultrastructure of mitosis in Euglena gracilis arepresented in his latter report. He summarizes the evidence for and against the opera-tion of a typical spindle apparatus in Euglena. The present study will resolve some ofthe problems discussed by Leedale by presenting some previously unreported featuresof mitosis in E. gracilis.

26 M. A. Gillott and R. E. Triemer

MATERIALS AND METHODS

Euglena gracilis (Klebs) strain Z (Pringsheim) was grown photoheterotrophically on modifiedHutner's media (Vasconcelos et al. 1971). Cultures were placed on a shaker under continuouslight (1500 lux) at 20 °C.

Concentrated cells were fixed in 2 % glutaraldehyde plus 0-5 % neutralized tannic acid(pH 7-0) made up in Euglena growth media. Cells remained in fixative for 2 h in the dark at 4 CC.They were then washed 3 times in cold growth media before postfixation. Cells were incubatedin 2 % osmium tetroxide made up in growth media for 2 h in the dark at 4 °C. This was followedby a rinse in distilled water. Samples were then dehydrated rapidly in a graded ethanol series,followed by 2 changes of 100 % acetone, and embedded in Epon.

Silver sections were cut on a Sorvall MT 2-B ultramicrotome using a Dupont diamond knife.After poststaining with uranyl acetate and lead citrate, sections were examined on a SiemensElmiskop iA electron microscope at 80 kV.

Abbreviations on figuresbcclcve

fgkm

basal bodychromosomechloroplastcontractile vacuoleendosomeflagellumGolgikinetochoremitochondrion

msmtn

"gnpPpar

mastigonememicrotubulenucleusnuclear granulenuclear porepellicleparamylonreservoir

cf cleavage furrow

OBSERVATIONS

Euglena gracilis possesses a single emergent flagellum arising from one of the pair ofbasal bodies located at the base of the reservoir. The cells contain large amounts ofparamylon when grown under high light conditions; diskoid chloroplasts and mito-chondria are scattered throughout the cytoplasm (Fig. 1). In non-dividing cells, thepellicle appears in cross-section as a regular series of ridges and grooves (Fig. 1). Theinterphase nucleus is centrally located and chromatin remains condensed (Fig. 1)throughout the cell cycle. Few ribosomes are associated with the outer nuclearenvelope in both non-dividing (Fig. 1) and dividing cells (Figs. 2-10).

Prior to mitosis, the pellicle begins to replicate, as evidenced by the production ofsmall, newly formed strips alternating with the larger, fully formed pellicle strips

Fig. i. Interphase cell. Note the centrally located nucleus with its condensedchromatin. Numerous paramylon granules, mitochondria, and several diskoidchloroplasts are visible. A grazing section of reservoir can be seen in the upper right,x 3800.Fig. 2. Preprophase. The nucleus has migrated to the anterior of the cell and becomesassociated with the basal bodies. Note the ribosome-free zone surrounding the basalbodies, and the tubular nature of the Golgi secreting face (arrowheads). The pelliclehas begun to replicate as evidenced by the small protrusions (arrows) alternating withthe larger, fully formed pellicle strips, x 7500.Fig. 3. Basal body replication. Basal body replication has occurred, as indicated by thepresence of 4 flagellar cross-sections in the reservoir. Microtubules are present in thenucleus, x 7000.

Ultrastructure of cell division in Euglena

A

M. A. Gillott and R. E. Triemet

Fig. 4A, B. Prophase. Serial sections demonstrating the parallel elongation of thereservoir and nucleus. A basal body pair with associated ribosome-free zone is presentat each pole. The polar region of the nucleus is highly invaginated. Note the nuclearpores, cytoplasmic microtubules and mastigonemes in the reservoir, x 11200.

Ultrastructure of cell division in Euglena 29

(Fig. 2). These continue to develop during division (Figs. 2, 8, 10). A second cyto-plasmic indicator of mitosis is increased Golgi activity, indicated by the extensivetubular proliferation occurring at the secreting face (Fig. 2). Preprophase is charac-terized by the anterior migration of the nucleus; the nucleus curves around the basalbody pair and is separated from it by a ribosome-free zone (Fig. 2).

Mierotubules begin to form within the intact nuclear envelope at prophase (Fig. 3).The flagella and their basal bodies have replicated, as indicated by the presence of 4flagellar cross-sections in the reservoir (Fig. 3). During prophase, the reservoir andthe nucleus elongate parallel to each other and perpendicular to the incipient cleavageplane (Fig. 4A, B). The basal body pairs migrate to opposite ends of the expandingreservoir and associate with the poles of the elongated prophase nucleus (Fig. 4 A, B).Ribosome-free zones remain associated with the basal bodies at the poles (Figs. 4A,B, 6, 10). The invaginations of the polar regions of the nucleus formed at prophasepersist throughout the division cycle (Figs. 4A, B, 6, 7). Serial sections have failed todemonstrate the presence of polar fenestrae.

At metaphase, the chromosomes are loosely arranged in the equatorial region(Figs. 5, 6). Kinetochores, which can be observed at this stage, consist of 2 denselayers separated by a light layer (Fig. 5 and inset). The mierotubules attach to the outerdense layer of the trilayered kinetochore (Fig. 5 and inset). The inner dense layer isclosely associated with a less-condensed region of the chromosome (Fig. 5 and inset).Clusters of dense granules appear in the nucleus (Figs. 5, 7, 8). The endosome enlargesand elongates from pole to pole by late metaphase (Fig. 6). Although mierotubulesappear to be closely associated with the endosome (Figs. 6—9), no kinetochore-typestructure has been found.

The endosome assumes a dumb-bell shape as the chromosomes move toward thepoles during late anaphase (Fig. 7). Both continuous and chromosomal microtubulesare present. As the chromosomes approach the poles, the nucleus begins to constrict inthe central region, marking the beginning of telophase (Fig. 8). The close associationof the endosome with the central spindle fibres can be seen (Fig. 8). Elongation of thereservoir parallels that of the nucleus (Fig. 8). By late telophase the nuclear envelopehas constricted further, forming a dumb-bell shape (Fig. 9). The endosome remainsextended through the isthmus until the completion of telophase and the resultantformation of 2 daughter nuclei.

Cytokinesis proceeds from the anterior of the cell between the 2 daughter reservoirs.Light-microscopic observation of living E. gracilis cells reveals vigorous metabolyduring cytokinesis. Organelles can be seen to flow freely between the separatingdaughter cells. The nuclei remain closely associated with the basal bodies throughoutthis process (Fig. 10). Each daughter cell receives a portion of the newly synthesizedpellicle, indicated by the presence of large and small ridges (Fig. 10).

C EL 31

M. A. Gillott and R. E. Triemer

^ T 1

Ultrastructure of cell division in Euglena 31

DISCUSSION

In certain algae, e.g. Chlamydomonas (Triemer & Brown, 1974), and the zoosporesof Ulothrix (Floyd, Stewart & Mattox, 1972 a) and Klebsormidium (Floyd, Stewart &Mattox, 1972 ft), flagellar abscision occurs prior to mitosis. The basal bodies are thenfree to migrate to the nucleus and function as centrioles. Euglena, however, retains itsmotility throughout the division cycle. Therefore, the anterior migration of the nucleusis necessary for the basal bodies to assume a centriolar function. Leedale (1968) citesthis anterior nuclear migration as circumstantial evidence for basal bodies functioningas centrioles during mitosis, but he was unable to find ultrastructural evidence tosupport this hypothesis.

This study presents the first evidence for the association of basal body pairs withthe nuclear poles during division in Euglena gradlis. The curvature of the nucleus inE. gradlis required serial sectioning to demonstrate the presence of basal bodies atboth poles (Fig. 4A, B). Sommer & Blum (1965), studying Astasia longa, a closelyrelated euglenoid, found a basal body pair associated with only one pole of a dividingnucleus. Since the nucleus and reservoir exhibit parallel elongation in both E. gradlisand A. longa, and flagellar replication preceeds mitosis, it is probable that serialsections of Astasia would also reveal the presence of basal bodies at both poles.Pyramimonas, a prasinophycean alga, also retains its flagella during division, and theevents of prophase parallel those in the euglenoids (Pearson & Norris, 1975). Thereservoir and nucleus elongate and the replicated basal bodies associate with the poles.

Small densely staining granules, often clustered, appear in both interphase anddividing nuclei. These nuclear granules have also been observed by Leedale (personalcommunication). Their function is unknown.

The nuclear envelope remains intact throughout mitosis. This type of completelyclosed spindle has been found in 2 other algae, Vaucheria litorea (Ott & Brown, 1972)and Chlamydomonas moetvusii (Triemer & Brown, 1974). A closed spindle has alsobeen reported in several fungi and protozoa (Albugo, Berlin & Bowen, 1964; Blasto-cladiella, Lessie & Lovett, 1968; Saccharomyces, Robinow & Marak, 1966; Aspergillus,Robinow & Caten, 1969; Tetrahymena, Elliot, 1963; Blepharisma, Jenkins, 1967; andTrypanosoma, Vickerman & Preston, 1970). The trypanosomes are similar to theeuglenoids in certain mitotic features. The persistent endosome of T. rhodesiensebecomes ensheathed by the spindle which traverses the centre of the nucleus. Theendosome of E. gradlis is also closely associated with several spindle microtubules. Intrypanosomes, however, the chromosomes are attached to the nuclear envelope andthe basal bodies are not associated with the nucleus (Vickerman & Preston, 1970).

Fig. 5. Metaphase. Section through equatorial region near the periphery of thenucleus. Chromosomes are loosely arranged on the metaphase plate. Note thekinetochores and electron-dense granules, x 11400. Inset: Higher magnification of atrilayered kinetochore (outlined in Fig. 5). x 33000.

Fig. 6. Longitudinal section of nucleus. The endosome is elongated perpendicular tothe metaphase plate. Note the basal body with ribosome-free zone near the nuclearpole. X8550.

3-2

M. A. Gillott and R. E. Triemer

Vltrastructure of cell division in Euglena 3 3

Kinetochores, which had not been previously described at the ultrastructural levelin euglenoid flagellates, were observed at metaphase. Using light microscopy, Saito(1961) had observed ' V - and 'L'-shaped chromosomes during anaphase in E. viridis.He interpreted these chromosomal configurations as evidence for kinetochores. Sinceother authors failed to find evidence of kinetochores (see Leedale, 1968) it wasproposed that chromosomal movement occurred independently of the spindle. Thekinetochores observed in E. gracilis have a trilayered structure similar to that describedin Chlamydomonas (Triemer & Brown, 1974). The existence of typical kinetochores inE. gracilis provides evidence for the operation of a normal spindle apparatus as themechanism controlling chromosomal movements during mitosis.

Observations of the living cells with Nomarski optics indicates the presence of 2reservoirs during late anaphase-early telophase. The formation of 2 reservoirs duringanaphase has also been observed in E. gracilis by Leedale (1968), and in Astasia longa(Sommer & Blum, 1965) and Pyramimonas (Pearson & Norris, 1975). Initiation of thecleavage furrow between the flagella, and therefore between their attached basalbodies, guarantees the distribution of one basal body pair to each daughter cell.During cytokinesis, metabolic movements result in the flow of organelles between theseparating cells. Hence, the continued association of the nucleus with the basal bodiesmay provide a method of ensuring the passage of one nucleus to each daughter cell.

The presence of microtubules, as well as their position and orientation duringcleavage (furrowing vs. phragmoplast vs. phycoplast), has been accorded taxonomicand phylogenetic significance (Pickett-Heaps, 19726, 1975; Stewart, Mattox & Floyd,1973). Simple furrowing has been observed in Ulva (Lovlie & Braten, 1968), Tricho-sarcina and PseudendocIonium (Mattox & Stewart, 1974), and in Pyramimonas (Pearson& Norris, 1975). A few algae utilize phragmoplasts (Pickett-Heaps, 1967; Fowke &Pickett-Heaps, 1969; Marchant & Pickett-Heaps, 1973), while many of the other algaeemploy phycoplasts (Marchant, 1977; Pickett-Heaps, 1972a, 1975; Triemer & Brown,

1974)-Neither phragmoplasts nor phycoplasts are found in Euglena and microtubules are

not associated with the cleavage furrow. The twisting motions observed duringcytokinesis with the light microscope lends support to the hypothesis that the cleavagefurrow in Euglena follows the helical ' l ine' of one of the pellicle strips (Leedale, 1967;Hofmann & Bouck, 1976).

The results of our study indicate that Euglena possesses a typical mitotic spindle;basal body pairs are associated with the poles, and kinetochores attach the chromo-somes to spindle fibres. The endosomal elongation appears to be guided by the spindle

Fig. 7. Anaphase. Chromosomes have moved toward the poles. The endosome assumesa dumb-bell shape, x 9450.Fig. 8. Early telophase. Oblique section of the elongated endosome with associatedmicrotubules. An interzonal spindle is present. Note the elongated reservoir parallelto the nucleus, x 7750.Fig. 9. Telophase. Daughter nuclei beginning to reform. The elongated endosomestill spans the isthmus, x 8400.

M. A. Gillott and R. E. Triemer

tioFig* 10. Cytokinesis. Daughter nuclei remain associated with the basal bodies until the

completion of cleavage, x 6500.

fibres, which may attach directly to the endosome. The strongest remaining argumentagainst the operation of a normal spindle apparatus is the lack of inhibition bycolchicine. Leedale (1968) suggests that the failure of colchicine to inhibit chromo-somal movements is due to its inability to enter the cell. Studies by Silverman &Hikida (1976) on pellicle microtubules also suggest that colchicine may not penetratethe pellicle complex.

This work was supported in part by a Biomedical Research Support Grant and a ResearchCouncil Grant from Rutgers University. We also wish to acknowledge the Bureau of BiologicalResearch for providing the electron-microscopy facilities used in this research.

REFERENCESBERLIN, J. D. & BOWEN, C C. (1964). Centrioles in the fungus Albugo Candida. Am. J. Bot. 51,

650-652.ELLIOT, A. M. (1963). The fine structure of Tetrahymena pyriformis during mitosis. In The Cell

in Mitosis (ed. L. Levine), pp. 107-124. New York: Academic Press.

Ultrastructure of cell division in Euglena 35

FLOYD, G. L., STEWART, K. D. & MATTOX, K. R. (1972a). Comparative cytology of Ulothrixand Stigeoclonium. J. Phycol. 8, 68-81.

FLOYD, G. L., STEWART, K. D. & MATTOX, K. R. (19726). Cellular organization, mitosis andcytokinesis in the Ulotrichalean alga Klebsormidium. J. Phycol. 8, 176-183.

FOWKE, L. C. & PICKETT-HEAPS, J. D. (1969). Cell division in Spirogyra. II. Cytokinesis.J. Phycol. 5, 273-281.

FRITSCH, F. E. (1935). The Structure and Reproduction of the Algae. Cambridge: UniversityPress.

HOFMANN, C. & BOUCK, G. B. (1976). Immunological and structural evidence for patternedintussusceptive surface growth in a unicellular organism. J. Cell Biol. 69, 693-715.

JENKINS, R. A. (1967). The fine structure of division in ciliate protozoa. I. Micronuclear mitosisin Blepharisma. J. Cell Biol. 34, 463-481.

LEEDALE, G. F. (1967). Euglenoid Flagellates. New Jersey: Prentice-Hall.LEEDALE, G. F. (1968). The nucleus in Euglena. In The Biology of Euglena (ed. D. E. Buetow),

pp. 185-272. New York: Academic Press.LESSIE, P. E. & LOVETT, J. S. (1968). Ultrastructural changes during sporangium and zoospore

differentiation in Blastocladiella emersonii. Am. J. Bot. 55, 220-36.LOVLIE, P. E. & BRATEN, T. (1970). On mitosis in the multicellular alga Ulva mutabilis Foyn.

J. Cell Sci. 6, 109-129.MARCHANT, H. J. (1977). Cell division and colony formation in the green alga Coelastrum

(Chlorococcales). J. Phycol. 13, 102-110.MARCHANT, H. J. & PICKETT-HEAPS, J. D. (1973). Mitosis and cytokinesis in Coleochaete scutata.

J. Phycol. 9, 461-471.MATTOX, K. R. & STEWART, K. D. (1974). A comparative study of cell division in Trichosarcina

polymorpha and Pseudenoclonium basiliense (Chloropyceae). J. Phycol. 10, 447-455.OTT, D. W. & BROWN, R. M. (1972). Light and electron microscopic observations on mitosis in

Vaucheria litorea Hofman ex Agardh. Br. Phycol. J. 7, 361-374.PEARSON, B. R. & NORRIS, R. E. (1975). Fine structure of cell division in Pyramimonas parkeae

Norris and Pearson (Chlorophyta, Prasinophyceae). J. Phycol. 10, 113-124.PICKETT-HEAPS, J. D. (1967). Ultrastructure and differentiation in Chara sp. II . Mitosis. Aust.

J. biol. Sci. a i , 255-274.PICKETT-HEAPS, J. D. (19720). Cell division in Tetraedron. Ann. Bot. 36, 693-701.PICKETT-HEAPS, J. D. (19726). Variation in mitosis and cytokinesis in plant cells: its signifi-

cance in the phylogeny and evolution of ultrastructural systems. Cytobios 5, 59-77.PICKBTT-HEAPS, J. D. (1975). The Green Algae. Massachusetts. Sinauer Associates.ROBINOW, C. F. & CATEN, C. E. (1969). Mitosis in Aspergillus nidulans. J. Cell Sci. 5, 403-431.ROBINOW, C. F. & MARAK, J. (1966). A fiber apparatus in the nucleus of the yeast cell. J. Cell

Biol. 29, 129-151.SAITO, M. (1961). Studies in the mitosis of Euglena. I. On the chromosome cycle of Euglena

viridis Ehrbg. J. Protozool. 8, 300-307.SILVERMAN, H. & HIKIDA, R. S. (1976). Pellicle complex of Euglena gracilis: characterization by

disruptive treatments. Protoplasma 87, 237-252.SOMMER, J. R. & BLUM, J. J. (1965). Cell division in Astasia longa. Expl Cell Res. 39, 504-527.STEWART, K. D., MATTOX, K. R. & FLOYD, G. L. (1973). Mitosis cytokinesis, the distribution

of plasmodesmata and the cytological characteristics in the Ulotricales, Ulvales and Chaeto-phorales: Phylogenetic and taxonomic considerations.^. Phycol. 9, 128-140.

TRIEMER, R. E. & BROWN, R. M. (1974). Cell division in Chlamydomonas moetousii. J. Phycol.10, 419-432.

VASCONCELOS, A., POLLACK, M., MENDIOLA, L. R., HOFFMANN, H. P., BROWN, D. H. & PRICE,C. A. (1971). Isolation of intact chloroplasts from Euglena gracilis by zonal centrifugation.Plant Physiol., Lancaster 47, 217-221.

VICKERMAK, K. & PRESTON, T. M. (1970). Spindle microtubules of trypanosomes. J. Cell Sci. 6,365-383-

{Received 15 August 1977)