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New ultrastructural data on the morphogenesis of the test in the testacean Arcella vulgaris
Europ.J. Protisto\. 26, 132-1 41 (1990) Octob er 19, 1990
European Journal of
PROTISTOLOGY
New Ultrastructural Data on the Morphogenesis of the Test in the Testacean Arcella vulgaris Jean-Pierre Mignot and Igor B. Raikov* Zoologie et Protistologie, Universite Blaise Pascal Clermont II, Aubiere Cedex, France
SUMMARY Transmission electron microscopic studies of dividing Arcella vulgaris Ehrbg. have shown that: - The growth of the thecagenous bud originates at a particular cytoplasmic area where the nuclei and the thecagenous granul es concentrate : - The extrusion of the thecagenous granules is preceded by secretion of a mucous substance on the surface of the bud, likely to take part, thereafter, in the control of the test form: - The morpho logy of the test results from the combined acrion of two types of pseudopodia: (a) the thecagenous bud that moves the thecal alveoli newly assembled at its surface in centrifugal direction ; (b) the extralocular pseudop odia which form a tran sitory dome which limits this expansion; - Some thecagenous granules are utilized to form a clasp system to maintain the position of the daughter test against the mother test during the time of the cytoplasmic movements between the two.
Abbreviations DT MF MU N PD TA TB TG TW
= daughter test = microfilaments = mucous substance = nuclei = pseudopodial dome
test alveoli thecagenou s bud = thecagenous granules = test wall = =
Introduction Due to the extraordinary variety of form and structure of their tests, the testaceans have for a long time interested biologists for either tax onomic, phylogenetic or ecological reasons, their organization being sometime s closely adapted to certain environm ental conditions. Ho wever, the mechanisms of morphogenesis of the test are still little * Permanent address: Institute of Cytology, 4 Tikh oretsky Avenue 194064 Leningrad, USSR 0932-4739/90/0026-0132$3.50/0
known, and that calls for investigations of how does the cell control the construction and shaping of the replica shell, which generally involves a special form of secretion: extrusion and arrangement of thecagenous granules. The use of either transmission or scanning electron microscopical techniqu es has contributed to the knowledge of this mechanism in genera which form a purely organic test as Arcella [11-13, 17, 18, 22, 23] and Centropyxis [14, 19, 20], or in genera which form a test made of idiosomes as Netzelia [21], Euglypha [16,24) and Tracheleuglypha [26], or else in genera with a test involving exogenous elements as Lesquereusia [3], Difflu-
gia, Heleospora, Cucurbitella, Netzelia, Phryganella [15, 25,27,28] . The Arcella model which has certain advantages, test easy to cut, and culture s easily grown, has been the most studied. The investigations of Netzel et al. have provided the essential dat a on morphogenesis of the test due to cinematographic recording s, which permitted then to establish a precise chronology of the pro cess, SEM observations of the forming test and TEM investigation of sections. However, some points remained obscure, for instance, the exact role of the transitory pseudop odi al dome. © 1990 by Gustav Fischer Verlag, Stuttgart
Test Morphogenesis in Arcella . 133 H aving sta rted an ult rastructural study of the nuclear division in Arcella, we had the cha nce to find images which allowed us to complete the description made by Netzel and co-workers and to reconsider some interpretation s. This is thus the aim of the present pu blication.
Material and Methods The species used was Arcella vulgaris Ehrbg., very common in freshwater of the Auvergne region and cultivated in the laboratory in small Petri dishesin filtered water from the sampling place and baker's yeast for food. Cultures were maintained by transfers into fresh medium every three weeks, and the cultures were fed once a week with a suspension of fresh yeast. To obtain cells in exponential growth, the food was added every two or three days. The duration of cell division, which comprisesthe building up of a new test, is very short: 26 min after Netzeland Heunert [23], and the period of formation of the test is only about ten minutes. Thus, theprobability to find favorablestagesin non-synchronized cultures isvery low. To increasethis probability, wehave fixed the cells at the time when light microscopic observations showed a maximum of dividing individuals. Individuals were sectioned continuously with a diamond knife, except for occasional intercalation of semi-thin sections. A light microscopical examination of the semi-thin sections permitted us to select the grids with division stages. As fixative, we used 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), followed by 1% osmium tetroxide in the same buffer. Both fixation and postfixation timeswere 30 min at room temperature. Embedding was done first into agar gel, then in Epon. The sections were conventionally stained with aqueous uranyl acetate and lead citrate, and studied with a Siemens Elmiskop IA or a JEOL 1200 electron microscopes.
Results
It is known that the test of Arcella has a cap-l ike form and that its wall s show a honeycomb-like texture. It is form ed by juxtaposition of polyhedrical alveoli (Fig. 1) between which sma ller wells int ercalat e, being remn ants of cyto plasmic finger-like protrusion s [17, 18]. At first con sidered to be composed by glycosylated compounds, the wa lls of the alveoli lat er proved to con sist of a keratin-like pr ot ein [11, 12, 17, 32]. Th e formation of a new test is the first mani festati on of cell division. The test is built fro m prefabri cat ed elements, the th ecagenous granules (Fig. 2), which are all of the same type and Golgi derivatives, as sho wn by N etzel [17, 18]. Prior to division , the thecagenou s granules concentrate in the median cytoplasmic territory, i.e. near th e pseudosto me (Fig. 3) . Th e tw o nuclei, which were situated at the oppos ite sides of the cell bod y during inte rphase, also join this territory which is easy to distingu ish fro m the rest of the cyto plasm du e to its hom ogeneous appear ance. Th e formation of the new test sta rts with the gro wth of the th ecagenou s bud, followed by th e thecagenou s gra nules being co ncent rated near the summit of the bud (Figs. 4 an d 5). According to Netzel [13, 17, 18], the y become ar ra nged in a single layer and thereafter extruded syn-
chronously; at the sa me time they swell to form alveoli. In each corner, cyto plasmic villosities prevent a complete fusion of adj acent alveoli, and these lat er produce the square or tri angular wells visible in Fig. 1. We shall not redescribe these stages perfectly interpreted by Netzel. We would only indicate that the extrus ion of th e th ecagenous alveoli is pre ceded by secretio n of a hom ogeneo us substance which is rather electron-tr an spar ent. This substa nce, of unknown chemica l composition, caps the thec agenous bud and also spreads aro und the pseud ostome of th e mother cell test on its outer side (Figs. 4 and 5). At a shomew ha t more adva nced stage (Fig. 6), the alveoli have alrea dy fused, p roducing a con tin uo us stratum typical of the test wall . At thi s time ho wever, the new test does not have either the definitive for m or size. Pseudopods push the newly secreted test wall in centrifugal dir ection. One can see bundles of microfil am ent s in the pseudopodial cyto plasm, with occas iona lly some thicker filam ents (Fig. 7). Th ese pattern s are likely to be acto myosin complexes which ar e for med wh en pseud opodial gro wth is very active. Another ph ase probabl y fund am ental for test formation is the appeara nce of a pseudopodial (cytoplas mic) dom e, first observed by N etzel [13 ] in Arcella vulgaris. It ha s been demon strated, by cinema tography, that thi s is a tr ansient formation whi ch lasts for only a few minutes [23]. Beautiful SEM micrographs, thereafter obtained with Arcella dentata [22], have con firmed the formati on of this peculiar circular pseudopodium which does not seem to be completely continuous around the thecagen ous bud. We had the oppo rtunity to observe these stages on both semithin and thin sections . On e almost sagittal section, pr esented as Fig. 8, clearl y shows that the dome pseudopodium ha s a relati vely homogeneous stru cture and th at it sta rts from the pseudopod ial trunk just after the latter emerges from the pseudostom e. The pseud op odium then curves to envelop the thecagenou s bud outside the newl y formed daughter cell test. Its inner surface is rather smoo th, but its outer surface is far less regular (Figs. 8 and 9 ). Fiber bundles (actomyos in complexes?) can be observed in the dom e pseud op odium in some sections (no t shown). Inside the pseud opodial dom e, the sur face of the thecagenous bud is rather irregular (Fig. 8). It forms fold s and outgrow ths. H owever, the new test wall always remai ns closely adjacent to th e sur face of th e cyto plasm and follow s all its sin uos ities. Certain prep arations (e.g. in Fig. 9) show a kind of thin pellicle stretched over the outer surface of the pseudopodial dome. The daughter test once form ed, the two tests remain applied to each other with th eir pseudostomes. During this time and before actual cell division , there ar e shuttling movements of the cyto plasm between cells. Th ese movement s were st udied in detail by Netzel and Heunert [23 ] who also suggested th at th e tran sfer of the dau ghter nucle i into the daughter cell occurs during the se movements . Th e study of sagittal sections of cells which have reach ed this stage shows th at the mother cell test is fastened to the da ughter cell test by a curi ou s clasping system. Thi s system has the shap e of tw o clasps applying to the mar gins of the
134 . Mignot and Raikov
•
. '
..
TW
Fig. 1. Tangential section of the test showing the alveoli (TA) and the intercalary wells (arrows), x 8800. - Fig. 2. Thecagenous granules (TG) in situ. Note the non-uniformity of their internal structure, X 16500. - Fig. 3. Sagittal section of the moth er cell sho rtly before the growth of the thecagenous bud, showing thecagenous granules (TG) around the two nuclei (N), X 2300.
Test Morphogenesis in Arcella . 135
,
MU
MU
Figs. 4 and 5. Growth of the thecagenous bud (TB), marked by a concentration of thecagenous granules (TG), emission of a mucu slike secretion product (MU), Fig. 4: x 2300; Fig. 5: x 10000.
136 . Mignot and Raikov
•
DT
Fig. 6. Beginning of formation of the daughter test (DT); expansion of the test wall under the pressure of the pseudopod ia, x 1800.Fig. 7. Detail of the intraloc ular pseudopodia showing bundles of cytoskeletal microfilaments (MF), x 15500.
Test Mo rph ogenesis in Arcella . 137
Fig. 8. Sagitta l section thr ou gh th e da ughter cell at the time of development of the pseud opodial dome (PO) which seems to be inte rrupted at th e summit (arr ow ). N ote the difference between the smooth inner face an d the ru gged outer face of th e dome. Insid e the dom e, th e thecagcnous bud (TB) is covered by th e new daughter test wall (DT) whi ch begins to flatt en again st the int ern al sur face of the dom e; arrows show pr esum ed dir ections of cyto plasmic flow, X 3200.
138 . Mignot and Raikov
2JJm Fig. 9. Detail of one of the phases of test morphogenesis, showing that the external pseudopodial dome (PD) and the internal thecagenous bud (TB) constitute two complementary interfaces of a mould. Note the thin layer (arrows) against which the pseudopodium applies, x 9600.
two pseudostomes from the inside (Fig. 10), but in reality it must have the shape of a short sleeve inserted into the two pseudostomes; this is also suggested by views of transverse sections through the connecting region (not shown). The connecting sleeve is formed late in division by an accumulation of incompletely swollen thecagenous alveoli (Fig. 10) which probably originate from a final reserve of thecagenous granules. About the end of the division of the cytoplasmic body, this complex is disrupted (Fig. 11), which permits the separation of the two daughter cells. Discussion Following our observat ions, the first point to stress is the migration of the nuclei into the cytoplasmic zone destined to form the thecagenou s bud . It is generally admitted that this migration precedes nuclear division which end s by the transfer of half of the daughter nuclei into the future daughter cell. But one has to remember that the nucleu s is often associat ed to a morphogenetic territory during various processes of cytomorphogenesis. It is the case, e.g., in Euglena, where the nucleus approaches the reservoir at
the time when the replication of the cuticular bands begins [10]. It is also the case in unicellular green algae like Micrasterias, where it has been shown that a correct reconstruction of a hcrni-cell after cytokinesis, in other words, the restoration of the cellular symmetry, implies a migration of the nucleus which is controlled by microtubuies [5-8]. It rema ins to be found out wh y the participation of the nucleus is necessary. The second point concern s the transitory formation of a pseudopodial dome. Our micrographs generally confirm the observations of N etzel [13, 18] and Netzel and Grunewald [22]. The y supp ose that this dome of cytoplasmic origin may have var ious functions : - formation of a support against which the thecal alveoli would flatten; - isolation of the thecagenous bud preventing it from attaching to the substrate; - creation of a space which would prevent dispers al of thecagenous granule s and whose contents would induce the transformation of granule s into thecal alveoli. Netzel's later observ ations with SEM on A. dentata [22], as well as our TEM data (e.g., Fig. 8) demonstrate that the space delimin ated by the cup-shaped pseudopodi-
Test Morphogenesis in Arcella . 139
. 10
11
TW- ~~
Fig. 10. Cell in late division showing the clasping system (arrows) between the two tests, x 2300. - Fig. 11. Separation of the two daughter cells, showing an early cytoplasmic constriction (arrow) and the rupture of the clasping device between the tests (asterisk), x 2300.
140 . Mignot and Raikov um is not completely closed, at least most of the time of the existence of the cytoplasmic dome which is already very short. On the other hand, our micrographs show that the test alveoli are formed and fused before the development of the dome (Fig. 6). Therefore we should not easily accept the last hypothesis. On the contrary, it seems much more probable that the dome determines the form and the size of the daughter test. In fact, our micrographs, e.g., Fig. 8, clearly show that the expansion of the thecagenous bud pushes the daughter test wall until it meets the inner face of the pseudopodial dome, and then it spreads laterally. In these conditions, one can presume that the dome would limit the expansion of the thecagenous bud and that it constitutes a mould against which the growth of intralocular pseudopodia applies the future test wall. This process seems quite comparable to that used by the foraminifers when they construct their shell. In fact, Cesana [2] has shown that two types of pseudopodia, intralocular and extralocular ones, act in coordinated manner to build the test. The pseudopodial dome in Arcella would then represent the equivalent of the extralocular pseudopodia of the foraminifers. These peculiarities of the morphogenesis, implying the formation of a mould, are not very different from those observed during the formation of the shell in diatoms [29,30], of the chrysomonadid cyst [4, 31], or else of the scales in chrysomonadids [9,33]. In the last case, it is known that the spatial deployment of the membranes is itself controlled by cytoskeletal elements of the actin type which are associated with the membrane [1]. It is thus not sufficient to admit that the pseudopodial dome controls the morphology of the test to resolve entirely the problem, because one still has to find out what determines the spatial arrangement of the dome itself. The presence of cytoskeletal elements inside the dome cytoplasm is evident, but these are not different from those in usual pseudopodia. On the contrary, certain structures like those in Fig. 9 are perhaps more important. This is a thin layer of mucous appearance that, likely enough, cannot be pierced by the irregular extensions of the outer side of the cytoplasmic dome. It seems to be too thin to be a physical obstacle, but it could represent a signal or a suitable surface recognized by the membrane receptors. Its similarity with the secretion material observed at the surface of the early thecagenous bud (cf., Fig. 5) makes us suppose that the thin layer is a derivative of this first secretion product observed still before the extrusion of the thecagenous granules. This larger somewhat resembles the thin "pellicle" (Hautchen) observed around the daughter test in Netzelia oviformis by Netzel [21]. The last point resulting from our observations of sections concerns the transitory fixation device between the mother and daughter tests. It is in fact surprising to see, on the published micrographs of both A. vulgaris [23] and A. dentata [22], that the two tests remain closely apposed to each other by their pseudostomes during the period of shuttle movements of the cytoplasm between the two tests. The finding of a mass of partially swollen thecagenous vesicles accumulated inside both pseudostomes forming a kind of sleeve between them, explains this phenomenon.
The sleeve is formed rather late in division, when the daughter test has acquired its definitive form and size. This is confirmed by the fact that it lies inside not only the old but also the new test aperture. We do not know how some thecagenous vesicles are preserved in this particular area, what makes them to extrude later than the others, and then what makes the fixation device to disrupt at the end of cell division. Our work, essentially based on TEM observations of sections, thus complements the data by Netzel on the morphogenesis of the test. It additionally shows, besides, that the thecagenous granules are perhaps more complex and multifunctional than supposed earlier. References 1 Brugerolle G. and Bricheux G. (1984): Actin microfilaments are involved in scale formation of the chrysomonad cell Synura. Protoplasma, 123, 203-212. 2 Cesana D. (1985): Recherches sur les Foraminifercs, ultrastructure, reproduction. These Univ. P. et M. Curie, Paris VI, 238 pp. 3 Harrison F. W., DunkelbergerD., Watable N. and Stump A. B. (1976): The cytologyof the testaceous rhizopod Lesquereusia spiralis (Ehrenberg) Penard. I. Ultrastructure and shell formation. J. Morphol., 150, 343-358. 4 Hibberd D. J. (1977): Ultrastructure of cyst formation in Ochromonas tuberculata (Chrysophyceae). J. Physol., 13, 309-320. 5 Kiermayer O. (1970): Elektronenmikroskopische UntersuchungenzumProblemder Cytomorphogenese von Micrasterias denticulata Breb. I. Allgemeiner Uberblick, Protoplasma, 69,97-132. 6 Kiermayer O. (1981): Cytomorphogenesis in plants. Springer Verlag, Wien-New York. Cell Biol. Monogr., 8, 439 pp. 7 Meindl U. (1983): Cytoskeletal control of nuclear migration and anchoringin developing cells of Micrasterias denticulata and the change caused by the anti-microtubular herbicide Amipophos-methyl (APM). Protoplasma, 118, 75-90. 8 Meindl U. (1985): Aberrant nuclear migration and microtubule arrangement in a defect mutant cell of Micrasterias thomasiana. Protoplasma, 126, 74-90. 9 Mignot J. P. and Brugerolle G. (1982): Scale formation in chrysomonad flagellates. J. Ultrast. Res., 81, 13-26. 10 Mignot J. P., Brugerolle G. and Bricheux G. (1987): Intercalary strip development and dividing cell morphogenesis in the euglenid Cyclidiopsis acus. Protoplasma, 139, 51-65. 11 Moraczewski J. (1969): Composition chimique, structure et formation de la coque d' Arcella. In: Progress in Protozool. IIIrd Intern. Congo Protozool., Leningrad, 32-33. 12 Moraczewski J. (1971): Structure et formation de la coque d'Arcella. Acta Protozool., 8 (27-33),423-438. 13 Netzel H. (1971): Die Schalenbildung bei der Thekamobengattung Arcella (Rhizopoda, Testacea). Cytobiologie, 3, 89-92. 14 Netzel H. (1972a): Die Bildung der Gehausewand bei der Thekamobe Centropyxis disco ides (Rhizopoda, Testacea).Z. ZellE. Mikrosk. Anat., 135, 45-54. 15 Netzel H. (1972b): Die Schalenbildung bei Difflugia ouiformis (Rhizopoda, Testacea). Z. Zellf. Mikrosk. Anat., 135, 55-61. 16 Netzel H. (1972c): Morphogenese des Gehauses von Euglypha rotunda (Rhizopoda,Testacea). Z. Zellf.Mikrosk. Anat., 135,63-69.
Test Morphogenesis in Arcella . 141 17 Netzel H. (1975a): Struktur und Ultrastruktur von Arcella vulgaris var. multinucleata (Rhizopoda, Testacea). Arch. Protistenk., 117, 219-245. 18 Netzel H. (1975b): Die Entstehung der hexagonalen Schalenstruktur bei der Thekamobe Arcella vulgaris var. multinucleata (Rhizopoda, Testacea). Arch. Protistenk., 117, 321-357. 19 Netzel H. (1975c): Morphologie und Ultrastruktur von Centropyxis discoides (Rhizopoda, Testacea). Arch. Protistenk., 117, 369-392. 20 Netzel H. (1976): Die Ausscheidung der Gehausewand bei Centropyxis discoides (Rhizopoda, Testacea). Arch. Protistenk., 118, 53-91. 21 Netzel H. (1983): Gehausewandbildung durch mehrphasige Sekretion bei der Thekamobe Netzelia oviformis (Rhizopoda, Testacea). Arch. Protistenk., 127, 351-381. 22 Netzel H. and Grunewald B. (1977): Morphogenesis in the shelled rhizopod Arcella dentata. Protistologica, 13, 299-319. 23 Netzel H. und Heunert H. H. (1971): Die Zellteilung bei Arcella vulgaris var. multinucleata (Rhizopoda, Testacea). Arch. Protistenk., 113, 285-292. 24 Ogden C. G. (1979): An ultrastructural study of division in Euglypha (Protozoa: Rhizopoda). Protistologica, 15, 541-556. 25 Ogden C. G. (1989): The agglutinate shell of Heleopera petricola (Protozoa, Rhizopoda), factors affecting its structure and composition. Arch. Protistenk., 137, 9-24. 26 Ogden C. G. and Couteaux M. M. (1987): The biology and ultrastructure of the soil testate amoeba Tracheleuglypha dentata (Rhizopoda Euglyphidae). Europ. J. Protisto!', 23, 28-42.
27 Ogden C. G. and Meisterfeld R. (1989): The taxonomy and systematics of some species of Cucurbitella, Difflugia and Netzelia (Protozoa: Rhizopoda) with an evaluation of diagnostic characters. Europ. J. Protisto!', 25, 109-128. 28 Ogden C. G. and Pitta P. (1989): Morphology and construction of the shell wall in an agglutinate soil testate amoeba Phryganella acropodia (Rhizopoda). J. Protozoo!., 36, 437-445. 29 Pickett-HeapsJ. D., Tippit D. H. and AndreozziJ. A. (1979): Cell division in the pennate diatom Pinnularia. IV. Valve morphogenesis. Bio!. Cel!., 35, 199-203. 30 Pickett-Heaps J. D. and Kowalski S. E. (1981): Valve morphogenesis and the microtubule center of the diatom Hantzschia amphioxys. Europ. J. Cell Bio!., 25, 150-170. 31 Sandgren C. D. (1980): Resting cyst formation in selected chrysophyte flagellates: an ultrastructural survey including a proposal for the phylogenetic significance of interspecific variations in the encystment process: Protistologica, 16, 289-303. 32 Saucin-Meulenberg M., Bussers J. c. et Jeuniaux C. (1973): Composition chimique de la theque de quelques Thecarnoebiens (Protozoaires). Bull. Bio!. Fr. Belg:, 107, 107-113. 33 SchnepfE. und Deichgraber G. (1969): Uber die Feinstruktur von Synura petersenii unter besonderer Beriicksichtigung der Morphogenese ihrcr Kieselschuppen. Protoplasma, 68, 85-106.
Key words: Arcella vulgaris - Test - Morphogenesis - Division - Ultrastructure Jean-Pierre Mignot, Groupe de Zoologie-Protistologie, Complexe Scientifique des Cezeaux, F-63177 Aubiere Cedex, France
European Journal of
PROTISTOLOGY
New Ultrastructural Data on the Morphogenesis of the Test in the Testacean Arcella vulgaris Jean-Pierre Mignot and Igor B. Raikov* Zoologie et Protistologie, Universite Blaise Pascal Clermont II, Aubiere Cedex, France
SUMMARY Transmission electron microscopic studies of dividing Arcella vulgaris Ehrbg. have shown that: - The growth of the thecagenous bud originates at a particular cytoplasmic area where the nuclei and the thecagenous granul es concentrate : - The extrusion of the thecagenous granules is preceded by secretion of a mucous substance on the surface of the bud, likely to take part, thereafter, in the control of the test form: - The morpho logy of the test results from the combined acrion of two types of pseudopodia: (a) the thecagenous bud that moves the thecal alveoli newly assembled at its surface in centrifugal direction ; (b) the extralocular pseudop odia which form a tran sitory dome which limits this expansion; - Some thecagenous granules are utilized to form a clasp system to maintain the position of the daughter test against the mother test during the time of the cytoplasmic movements between the two.
Abbreviations DT MF MU N PD TA TB TG TW
= daughter test = microfilaments = mucous substance = nuclei = pseudopodial dome
test alveoli thecagenou s bud = thecagenous granules = test wall = =
Introduction Due to the extraordinary variety of form and structure of their tests, the testaceans have for a long time interested biologists for either tax onomic, phylogenetic or ecological reasons, their organization being sometime s closely adapted to certain environm ental conditions. Ho wever, the mechanisms of morphogenesis of the test are still little * Permanent address: Institute of Cytology, 4 Tikh oretsky Avenue 194064 Leningrad, USSR 0932-4739/90/0026-0132$3.50/0
known, and that calls for investigations of how does the cell control the construction and shaping of the replica shell, which generally involves a special form of secretion: extrusion and arrangement of thecagenous granules. The use of either transmission or scanning electron microscopical techniqu es has contributed to the knowledge of this mechanism in genera which form a purely organic test as Arcella [11-13, 17, 18, 22, 23] and Centropyxis [14, 19, 20], or in genera which form a test made of idiosomes as Netzelia [21], Euglypha [16,24) and Tracheleuglypha [26], or else in genera with a test involving exogenous elements as Lesquereusia [3], Difflu-
gia, Heleospora, Cucurbitella, Netzelia, Phryganella [15, 25,27,28] . The Arcella model which has certain advantages, test easy to cut, and culture s easily grown, has been the most studied. The investigations of Netzel et al. have provided the essential dat a on morphogenesis of the test due to cinematographic recording s, which permitted then to establish a precise chronology of the pro cess, SEM observations of the forming test and TEM investigation of sections. However, some points remained obscure, for instance, the exact role of the transitory pseudop odi al dome. © 1990 by Gustav Fischer Verlag, Stuttgart
Test Morphogenesis in Arcella . 133 H aving sta rted an ult rastructural study of the nuclear division in Arcella, we had the cha nce to find images which allowed us to complete the description made by Netzel and co-workers and to reconsider some interpretation s. This is thus the aim of the present pu blication.
Material and Methods The species used was Arcella vulgaris Ehrbg., very common in freshwater of the Auvergne region and cultivated in the laboratory in small Petri dishesin filtered water from the sampling place and baker's yeast for food. Cultures were maintained by transfers into fresh medium every three weeks, and the cultures were fed once a week with a suspension of fresh yeast. To obtain cells in exponential growth, the food was added every two or three days. The duration of cell division, which comprisesthe building up of a new test, is very short: 26 min after Netzeland Heunert [23], and the period of formation of the test is only about ten minutes. Thus, theprobability to find favorablestagesin non-synchronized cultures isvery low. To increasethis probability, wehave fixed the cells at the time when light microscopic observations showed a maximum of dividing individuals. Individuals were sectioned continuously with a diamond knife, except for occasional intercalation of semi-thin sections. A light microscopical examination of the semi-thin sections permitted us to select the grids with division stages. As fixative, we used 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), followed by 1% osmium tetroxide in the same buffer. Both fixation and postfixation timeswere 30 min at room temperature. Embedding was done first into agar gel, then in Epon. The sections were conventionally stained with aqueous uranyl acetate and lead citrate, and studied with a Siemens Elmiskop IA or a JEOL 1200 electron microscopes.
Results
It is known that the test of Arcella has a cap-l ike form and that its wall s show a honeycomb-like texture. It is form ed by juxtaposition of polyhedrical alveoli (Fig. 1) between which sma ller wells int ercalat e, being remn ants of cyto plasmic finger-like protrusion s [17, 18]. At first con sidered to be composed by glycosylated compounds, the wa lls of the alveoli lat er proved to con sist of a keratin-like pr ot ein [11, 12, 17, 32]. Th e formation of a new test is the first mani festati on of cell division. The test is built fro m prefabri cat ed elements, the th ecagenous granules (Fig. 2), which are all of the same type and Golgi derivatives, as sho wn by N etzel [17, 18]. Prior to division , the thecagenou s granules concentrate in the median cytoplasmic territory, i.e. near th e pseudosto me (Fig. 3) . Th e tw o nuclei, which were situated at the oppos ite sides of the cell bod y during inte rphase, also join this territory which is easy to distingu ish fro m the rest of the cyto plasm du e to its hom ogeneous appear ance. Th e formation of the new test sta rts with the gro wth of the th ecagenou s bud, followed by th e thecagenou s gra nules being co ncent rated near the summit of the bud (Figs. 4 an d 5). According to Netzel [13, 17, 18], the y become ar ra nged in a single layer and thereafter extruded syn-
chronously; at the sa me time they swell to form alveoli. In each corner, cyto plasmic villosities prevent a complete fusion of adj acent alveoli, and these lat er produce the square or tri angular wells visible in Fig. 1. We shall not redescribe these stages perfectly interpreted by Netzel. We would only indicate that the extrus ion of th e th ecagenous alveoli is pre ceded by secretio n of a hom ogeneo us substance which is rather electron-tr an spar ent. This substa nce, of unknown chemica l composition, caps the thec agenous bud and also spreads aro und the pseud ostome of th e mother cell test on its outer side (Figs. 4 and 5). At a shomew ha t more adva nced stage (Fig. 6), the alveoli have alrea dy fused, p roducing a con tin uo us stratum typical of the test wall . At thi s time ho wever, the new test does not have either the definitive for m or size. Pseudopods push the newly secreted test wall in centrifugal dir ection. One can see bundles of microfil am ent s in the pseudopodial cyto plasm, with occas iona lly some thicker filam ents (Fig. 7). Th ese pattern s are likely to be acto myosin complexes which ar e for med wh en pseud opodial gro wth is very active. Another ph ase probabl y fund am ental for test formation is the appeara nce of a pseudopodial (cytoplas mic) dom e, first observed by N etzel [13 ] in Arcella vulgaris. It ha s been demon strated, by cinema tography, that thi s is a tr ansient formation whi ch lasts for only a few minutes [23]. Beautiful SEM micrographs, thereafter obtained with Arcella dentata [22], have con firmed the formati on of this peculiar circular pseudopodium which does not seem to be completely continuous around the thecagen ous bud. We had the oppo rtunity to observe these stages on both semithin and thin sections . On e almost sagittal section, pr esented as Fig. 8, clearl y shows that the dome pseudopodium ha s a relati vely homogeneous stru cture and th at it sta rts from the pseudopod ial trunk just after the latter emerges from the pseudostom e. The pseud op odium then curves to envelop the thecagenou s bud outside the newl y formed daughter cell test. Its inner surface is rather smoo th, but its outer surface is far less regular (Figs. 8 and 9 ). Fiber bundles (actomyos in complexes?) can be observed in the dom e pseud op odium in some sections (no t shown). Inside the pseud opodial dom e, the sur face of the thecagenous bud is rather irregular (Fig. 8). It forms fold s and outgrow ths. H owever, the new test wall always remai ns closely adjacent to th e sur face of th e cyto plasm and follow s all its sin uos ities. Certain prep arations (e.g. in Fig. 9) show a kind of thin pellicle stretched over the outer surface of the pseudopodial dome. The daughter test once form ed, the two tests remain applied to each other with th eir pseudostomes. During this time and before actual cell division , there ar e shuttling movements of the cyto plasm between cells. Th ese movement s were st udied in detail by Netzel and Heunert [23 ] who also suggested th at th e tran sfer of the dau ghter nucle i into the daughter cell occurs during the se movements . Th e study of sagittal sections of cells which have reach ed this stage shows th at the mother cell test is fastened to the da ughter cell test by a curi ou s clasping system. Thi s system has the shap e of tw o clasps applying to the mar gins of the
134 . Mignot and Raikov
•
. '
..
TW
Fig. 1. Tangential section of the test showing the alveoli (TA) and the intercalary wells (arrows), x 8800. - Fig. 2. Thecagenous granules (TG) in situ. Note the non-uniformity of their internal structure, X 16500. - Fig. 3. Sagittal section of the moth er cell sho rtly before the growth of the thecagenous bud, showing thecagenous granules (TG) around the two nuclei (N), X 2300.
Test Morphogenesis in Arcella . 135
,
MU
MU
Figs. 4 and 5. Growth of the thecagenous bud (TB), marked by a concentration of thecagenous granules (TG), emission of a mucu slike secretion product (MU), Fig. 4: x 2300; Fig. 5: x 10000.
136 . Mignot and Raikov
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DT
Fig. 6. Beginning of formation of the daughter test (DT); expansion of the test wall under the pressure of the pseudopod ia, x 1800.Fig. 7. Detail of the intraloc ular pseudopodia showing bundles of cytoskeletal microfilaments (MF), x 15500.
Test Mo rph ogenesis in Arcella . 137
Fig. 8. Sagitta l section thr ou gh th e da ughter cell at the time of development of the pseud opodial dome (PO) which seems to be inte rrupted at th e summit (arr ow ). N ote the difference between the smooth inner face an d the ru gged outer face of th e dome. Insid e the dom e, th e thecagcnous bud (TB) is covered by th e new daughter test wall (DT) whi ch begins to flatt en again st the int ern al sur face of the dom e; arrows show pr esum ed dir ections of cyto plasmic flow, X 3200.
138 . Mignot and Raikov
2JJm Fig. 9. Detail of one of the phases of test morphogenesis, showing that the external pseudopodial dome (PD) and the internal thecagenous bud (TB) constitute two complementary interfaces of a mould. Note the thin layer (arrows) against which the pseudopodium applies, x 9600.
two pseudostomes from the inside (Fig. 10), but in reality it must have the shape of a short sleeve inserted into the two pseudostomes; this is also suggested by views of transverse sections through the connecting region (not shown). The connecting sleeve is formed late in division by an accumulation of incompletely swollen thecagenous alveoli (Fig. 10) which probably originate from a final reserve of thecagenous granules. About the end of the division of the cytoplasmic body, this complex is disrupted (Fig. 11), which permits the separation of the two daughter cells. Discussion Following our observat ions, the first point to stress is the migration of the nuclei into the cytoplasmic zone destined to form the thecagenou s bud . It is generally admitted that this migration precedes nuclear division which end s by the transfer of half of the daughter nuclei into the future daughter cell. But one has to remember that the nucleu s is often associat ed to a morphogenetic territory during various processes of cytomorphogenesis. It is the case, e.g., in Euglena, where the nucleus approaches the reservoir at
the time when the replication of the cuticular bands begins [10]. It is also the case in unicellular green algae like Micrasterias, where it has been shown that a correct reconstruction of a hcrni-cell after cytokinesis, in other words, the restoration of the cellular symmetry, implies a migration of the nucleus which is controlled by microtubuies [5-8]. It rema ins to be found out wh y the participation of the nucleus is necessary. The second point concern s the transitory formation of a pseudopodial dome. Our micrographs generally confirm the observations of N etzel [13, 18] and Netzel and Grunewald [22]. The y supp ose that this dome of cytoplasmic origin may have var ious functions : - formation of a support against which the thecal alveoli would flatten; - isolation of the thecagenous bud preventing it from attaching to the substrate; - creation of a space which would prevent dispers al of thecagenous granule s and whose contents would induce the transformation of granule s into thecal alveoli. Netzel's later observ ations with SEM on A. dentata [22], as well as our TEM data (e.g., Fig. 8) demonstrate that the space delimin ated by the cup-shaped pseudopodi-
Test Morphogenesis in Arcella . 139
. 10
11
TW- ~~
Fig. 10. Cell in late division showing the clasping system (arrows) between the two tests, x 2300. - Fig. 11. Separation of the two daughter cells, showing an early cytoplasmic constriction (arrow) and the rupture of the clasping device between the tests (asterisk), x 2300.
140 . Mignot and Raikov um is not completely closed, at least most of the time of the existence of the cytoplasmic dome which is already very short. On the other hand, our micrographs show that the test alveoli are formed and fused before the development of the dome (Fig. 6). Therefore we should not easily accept the last hypothesis. On the contrary, it seems much more probable that the dome determines the form and the size of the daughter test. In fact, our micrographs, e.g., Fig. 8, clearly show that the expansion of the thecagenous bud pushes the daughter test wall until it meets the inner face of the pseudopodial dome, and then it spreads laterally. In these conditions, one can presume that the dome would limit the expansion of the thecagenous bud and that it constitutes a mould against which the growth of intralocular pseudopodia applies the future test wall. This process seems quite comparable to that used by the foraminifers when they construct their shell. In fact, Cesana [2] has shown that two types of pseudopodia, intralocular and extralocular ones, act in coordinated manner to build the test. The pseudopodial dome in Arcella would then represent the equivalent of the extralocular pseudopodia of the foraminifers. These peculiarities of the morphogenesis, implying the formation of a mould, are not very different from those observed during the formation of the shell in diatoms [29,30], of the chrysomonadid cyst [4, 31], or else of the scales in chrysomonadids [9,33]. In the last case, it is known that the spatial deployment of the membranes is itself controlled by cytoskeletal elements of the actin type which are associated with the membrane [1]. It is thus not sufficient to admit that the pseudopodial dome controls the morphology of the test to resolve entirely the problem, because one still has to find out what determines the spatial arrangement of the dome itself. The presence of cytoskeletal elements inside the dome cytoplasm is evident, but these are not different from those in usual pseudopodia. On the contrary, certain structures like those in Fig. 9 are perhaps more important. This is a thin layer of mucous appearance that, likely enough, cannot be pierced by the irregular extensions of the outer side of the cytoplasmic dome. It seems to be too thin to be a physical obstacle, but it could represent a signal or a suitable surface recognized by the membrane receptors. Its similarity with the secretion material observed at the surface of the early thecagenous bud (cf., Fig. 5) makes us suppose that the thin layer is a derivative of this first secretion product observed still before the extrusion of the thecagenous granules. This larger somewhat resembles the thin "pellicle" (Hautchen) observed around the daughter test in Netzelia oviformis by Netzel [21]. The last point resulting from our observations of sections concerns the transitory fixation device between the mother and daughter tests. It is in fact surprising to see, on the published micrographs of both A. vulgaris [23] and A. dentata [22], that the two tests remain closely apposed to each other by their pseudostomes during the period of shuttle movements of the cytoplasm between the two tests. The finding of a mass of partially swollen thecagenous vesicles accumulated inside both pseudostomes forming a kind of sleeve between them, explains this phenomenon.
The sleeve is formed rather late in division, when the daughter test has acquired its definitive form and size. This is confirmed by the fact that it lies inside not only the old but also the new test aperture. We do not know how some thecagenous vesicles are preserved in this particular area, what makes them to extrude later than the others, and then what makes the fixation device to disrupt at the end of cell division. Our work, essentially based on TEM observations of sections, thus complements the data by Netzel on the morphogenesis of the test. It additionally shows, besides, that the thecagenous granules are perhaps more complex and multifunctional than supposed earlier. References 1 Brugerolle G. and Bricheux G. (1984): Actin microfilaments are involved in scale formation of the chrysomonad cell Synura. Protoplasma, 123, 203-212. 2 Cesana D. (1985): Recherches sur les Foraminifercs, ultrastructure, reproduction. These Univ. P. et M. Curie, Paris VI, 238 pp. 3 Harrison F. W., DunkelbergerD., Watable N. and Stump A. B. (1976): The cytologyof the testaceous rhizopod Lesquereusia spiralis (Ehrenberg) Penard. I. Ultrastructure and shell formation. J. Morphol., 150, 343-358. 4 Hibberd D. J. (1977): Ultrastructure of cyst formation in Ochromonas tuberculata (Chrysophyceae). J. Physol., 13, 309-320. 5 Kiermayer O. (1970): Elektronenmikroskopische UntersuchungenzumProblemder Cytomorphogenese von Micrasterias denticulata Breb. I. Allgemeiner Uberblick, Protoplasma, 69,97-132. 6 Kiermayer O. (1981): Cytomorphogenesis in plants. Springer Verlag, Wien-New York. Cell Biol. Monogr., 8, 439 pp. 7 Meindl U. (1983): Cytoskeletal control of nuclear migration and anchoringin developing cells of Micrasterias denticulata and the change caused by the anti-microtubular herbicide Amipophos-methyl (APM). Protoplasma, 118, 75-90. 8 Meindl U. (1985): Aberrant nuclear migration and microtubule arrangement in a defect mutant cell of Micrasterias thomasiana. Protoplasma, 126, 74-90. 9 Mignot J. P. and Brugerolle G. (1982): Scale formation in chrysomonad flagellates. J. Ultrast. Res., 81, 13-26. 10 Mignot J. P., Brugerolle G. and Bricheux G. (1987): Intercalary strip development and dividing cell morphogenesis in the euglenid Cyclidiopsis acus. Protoplasma, 139, 51-65. 11 Moraczewski J. (1969): Composition chimique, structure et formation de la coque d' Arcella. In: Progress in Protozool. IIIrd Intern. Congo Protozool., Leningrad, 32-33. 12 Moraczewski J. (1971): Structure et formation de la coque d'Arcella. Acta Protozool., 8 (27-33),423-438. 13 Netzel H. (1971): Die Schalenbildung bei der Thekamobengattung Arcella (Rhizopoda, Testacea). Cytobiologie, 3, 89-92. 14 Netzel H. (1972a): Die Bildung der Gehausewand bei der Thekamobe Centropyxis disco ides (Rhizopoda, Testacea).Z. ZellE. Mikrosk. Anat., 135, 45-54. 15 Netzel H. (1972b): Die Schalenbildung bei Difflugia ouiformis (Rhizopoda, Testacea). Z. Zellf. Mikrosk. Anat., 135, 55-61. 16 Netzel H. (1972c): Morphogenese des Gehauses von Euglypha rotunda (Rhizopoda,Testacea). Z. Zellf.Mikrosk. Anat., 135,63-69.
Test Morphogenesis in Arcella . 141 17 Netzel H. (1975a): Struktur und Ultrastruktur von Arcella vulgaris var. multinucleata (Rhizopoda, Testacea). Arch. Protistenk., 117, 219-245. 18 Netzel H. (1975b): Die Entstehung der hexagonalen Schalenstruktur bei der Thekamobe Arcella vulgaris var. multinucleata (Rhizopoda, Testacea). Arch. Protistenk., 117, 321-357. 19 Netzel H. (1975c): Morphologie und Ultrastruktur von Centropyxis discoides (Rhizopoda, Testacea). Arch. Protistenk., 117, 369-392. 20 Netzel H. (1976): Die Ausscheidung der Gehausewand bei Centropyxis discoides (Rhizopoda, Testacea). Arch. Protistenk., 118, 53-91. 21 Netzel H. (1983): Gehausewandbildung durch mehrphasige Sekretion bei der Thekamobe Netzelia oviformis (Rhizopoda, Testacea). Arch. Protistenk., 127, 351-381. 22 Netzel H. and Grunewald B. (1977): Morphogenesis in the shelled rhizopod Arcella dentata. Protistologica, 13, 299-319. 23 Netzel H. und Heunert H. H. (1971): Die Zellteilung bei Arcella vulgaris var. multinucleata (Rhizopoda, Testacea). Arch. Protistenk., 113, 285-292. 24 Ogden C. G. (1979): An ultrastructural study of division in Euglypha (Protozoa: Rhizopoda). Protistologica, 15, 541-556. 25 Ogden C. G. (1989): The agglutinate shell of Heleopera petricola (Protozoa, Rhizopoda), factors affecting its structure and composition. Arch. Protistenk., 137, 9-24. 26 Ogden C. G. and Couteaux M. M. (1987): The biology and ultrastructure of the soil testate amoeba Tracheleuglypha dentata (Rhizopoda Euglyphidae). Europ. J. Protisto!', 23, 28-42.
27 Ogden C. G. and Meisterfeld R. (1989): The taxonomy and systematics of some species of Cucurbitella, Difflugia and Netzelia (Protozoa: Rhizopoda) with an evaluation of diagnostic characters. Europ. J. Protisto!', 25, 109-128. 28 Ogden C. G. and Pitta P. (1989): Morphology and construction of the shell wall in an agglutinate soil testate amoeba Phryganella acropodia (Rhizopoda). J. Protozoo!., 36, 437-445. 29 Pickett-HeapsJ. D., Tippit D. H. and AndreozziJ. A. (1979): Cell division in the pennate diatom Pinnularia. IV. Valve morphogenesis. Bio!. Cel!., 35, 199-203. 30 Pickett-Heaps J. D. and Kowalski S. E. (1981): Valve morphogenesis and the microtubule center of the diatom Hantzschia amphioxys. Europ. J. Cell Bio!., 25, 150-170. 31 Sandgren C. D. (1980): Resting cyst formation in selected chrysophyte flagellates: an ultrastructural survey including a proposal for the phylogenetic significance of interspecific variations in the encystment process: Protistologica, 16, 289-303. 32 Saucin-Meulenberg M., Bussers J. c. et Jeuniaux C. (1973): Composition chimique de la theque de quelques Thecarnoebiens (Protozoaires). Bull. Bio!. Fr. Belg:, 107, 107-113. 33 SchnepfE. und Deichgraber G. (1969): Uber die Feinstruktur von Synura petersenii unter besonderer Beriicksichtigung der Morphogenese ihrcr Kieselschuppen. Protoplasma, 68, 85-106.
Key words: Arcella vulgaris - Test - Morphogenesis - Division - Ultrastructure Jean-Pierre Mignot, Groupe de Zoologie-Protistologie, Complexe Scientifique des Cezeaux, F-63177 Aubiere Cedex, France