J. Cell Sci. 19, 53i-54t (i975) 531Printed in Great Britain

MACRONUCLEAR DIFFERENTIATION

DURING ORAL REGENERATION IN

STENTOR COERULEUS

J.J.PAULIN AND A. SUSAN BROOKSDepartment of Zoology, University of Georgia, Athens, Georgia 30602, U.S.A.

SUMMARY

The moniliform macronucleus of Stentor coeruleus coalesces and renodulates during division,reorganization and regeneration. These nuclear events are spatially and temporally synchron-ized with oral primordium development occurring at stages six and seven of membranellarmorphogenesis. Coalesced, elongating and early renodulating macronuclei at stages six andseven contained microtubules within double membrane-bound channels, passing through thenucleus parallel to the long axis. The number of microtubules per channel varied between 4 and23. Microtubules were also found in the perinuclear cytoplasm at these stages, forming a loosenetwork around the nucleus. The microtubules and channels are absent in control cells andmacronuclei of regenerating cells prior to stage six. These transient microtubules and channelsappearing in late stage six and stage seven may provide the axial plane on which elongation ofthe macronucleus proceeds.

INTRODUCTION

During division the large heterotrich ciliate Stentor coeruleus produces a new mem-branellar band for the opisthe; the prater retains its feeding organelles. Eight well-defined stages of membranellar morphogenesis, each lasting about one hour, can beidentified from the onset of primordium formation to completion of the new mem-branellar band (Tartar, 1961; Paulin & Bussey, 1971). The developmental stagesobserved in the formation of the membranellar band in division are mimicked in re-organization and regeneration (Tartar, 1961; deTerra, 1969). Regeneration of oralmembranelles is inducible by surgical removal of existing membranelles (Tartar,1961) or by placement of stentors in various chemical agents (e.g. 12% sucrose, 4%urea; Tartar, 1957) causing them to shed their membranelles, providing populationsof regenerating stentors for critical analysis of cortical and cytoplasmic events.Excellent reviews have been published on these events based on morphogeneticstudies (Tartar, 1961; Margulis, 1973; deTerra, 1974) and electron microscopy(Paulin & Bussey, 1971). Concomitant with the cortical events (i.e. formation ofthe membranellar band) the moniliform macronucleus begins to coalesce at stage six,becoming unitary and ovoid in appearance by stage seven with subsequent elongationand renodulation by late stage seven; the membranellar band is completely differ-entiated at this time. Consequently oral replacement and nuclear events are spatiallyand temporally synchronized (Tartar, 1961; deTerra, 1969, 1974).

The present investigation explores the possible mechanism(s) responsible for the

532 J.J. Paulin and A. S. Brooks

coalescence and renodulation of the macronucleus during regeneration. A light andelectron-optical study of interphase, coalescing, clumped and renodulating macro-nuclei was initiated in an attempt to reveal the structural basis for the kinetic event.An abstract of this work has been published elsewhere (Paulin, 1973).

MATERIALS AND METHODS

Stentor cocruleus originally obtained from Carolina Biological Supply (Burlington, NorthCarolina) were maintained in a modified Peter's solution (Hetherington, 1934) and fed Euglenasp. every 3 days.

Stentors were induced to shed their oral membranelles in 12% sucrose (w/v, in distilledH2O), washed and resuspended in conditioned media. Regenerating stentors were removed ati-h intervals after shedding up to a period of 8 h and viewed in a Zeiss Photomicroscope IIwith phase optics to determine the specific stage of regeneration based on membranellar banddifferentiation and macronuclear configurations. Control cells (interphase cells) and experi-mentals were prepared by the Feulgen nuclear reaction (Galigher & Kozloff, 1964) and photo-graphed in the Zeiss Photomicroscope II utilizing Kodak Panatomic X, 35-mm film.

The procedures for fixation and embedding for electron microscopy have been describedelsewhere (Paulin & Bussey, 1971). Cells were prepared by these methods at each stage ofregeneration and flat-embedded. Thus, specific stages based on membranelle differentiationand macronuclear configurations could be selected and properly oriented before sectioning.Thin sections were obtained on a Porter Blum-MT. microtome utilizing diamond knives. Thesections were stained for 15 min with saturated uranyl acetate (w/v, in distilled water) followedby Reynolds' lead citrate (2 min) and viewed in a Siemens 101 electron microscope at 80 keVwith a 35-/im objective aperture.

RESULTS

Light microscopy

The interphase moniliform macronucleus of Stentor coeruleus consists of ovoidnodes (X 7-6 per cell, ea. X i6-2x 14-5 /tm, 53 cells counted) linked together andcontained by the nuclear membrane which often forms tenuous constrictions betweennodes (arrow, Fig. 1). If cells are induced to shed their oral membranelles and allowedto regenerate, the moniliform macronucleus remains quiescent until early stage 6 ofmembranellar regeneration; the membranellar band at this stage is well differentiated,and the buccal cavity is beginning to regress (see Paulin & Bussey, 1971). At thispoint in regeneration the macronuclear nodes begin to coalesce, nodes at the extremi-ties fusing first. Fig. 2 shows 2 macronuclear clumps formed by such fusions. Thisprocess continues until the once moniliform nucleus forms a single large mass(Fig. 3) by early stage 7. By late stage 7 the macronucleus has begun to elongate andrenodulate. Fig. 4 shows the initial nodes being formed by constrictions around theelongating macronucleus. Nodulation begins at both extremities and progresses tothe centre. All nodes are reconstituted by stage 8 in concert with completion of oralregeneration or just after cytokinesis in dividing stentors.

Electron microscopy

Control cell macronuclear nodes are encapsulated by 2 unit membranes (Figs. 5-8)perforated with numerous pores (p, Fig. 6). Portions of 3 nodes from a control cell

Macronuclear differentiation 533

are depicted in Fig. 5. The cytoplasm around the nucleus is often vacuolated and con-tains numerous mitochondria (m, Fig. 5). The karyoplasm contains dense amorphousmasses of chromatin (c, Fig. 5) and numerous nucleoli (n, Fig. 5).

Macronuclei of cells regenerating oral membranelles examined at i-h intervalsafter the stimulus to regenerate to mid-stage 6, when nodal fusion is near completion,appear ultrastructurally similar to control cell nuclei (see above). However, late stage6 and early stage 7 macronuclei, those fully clumped and beginning to elongate,possess microtubules either in double membrane-bound channels (Figs. 6-10) orforming a network around the nucleus (Figs. 8, 9). Channels containing the micro-tubules are found near the periphery of elongating nuclei, running parallel to thelong axis of the nucleus (Figs. 7, 9, 10). The number of microtubules per channelvaries between 4 and 23 (Fig. 6), and at high magnification they do not appear to becross-linked (Fig. 8). The microtubules are continuous through the channels andappear to radiate from the channels at the periphery of the nucleus (Fig. 9). Theirterminus in the cytoplasm cannot be ascertained.

After nodulation has begun in late stage 7 there is a noticeable reduction in thenumber of microtubule-bearing channels and microtubules in juxtaposition to thenuclear membrane. In Fig. 10 a node-forming furrow (small arrow) can be seenbisecting a portion of the nucleus. A portion of a channel (large arrow) is reflectedto the axis of the furrow which is at right angles to the channels. By stage 8 channelsand microtubules are nearly absent.

DISCUSSION

The presence of channels containing microtubules through elongating macro-nuclei of Stentor is surprising. Microtubules have been found in dividing macro-nuclei in a number of ciliates (e.g. Nassula, Tetrahymena and Diplodinium to name afew, see reviews by Falk, Wunderlich & Franke, 1968; Raikov, 1969). However, themicrotubules are contained within the karyoplasm, not membrane-bound, forminga compact bundle or 'pushing body' which has been postulated to be the kineticelement associated with elongation of the macronucleus in division (Tucker, 1967;Raikov, 1969). These bundles of microtubules are aligned parallel to the axis of elonga-tion. Jenkins (1969) has found in the heterotrich ciliate Blepharisma sp. microtubulesexternal and adjacent to the macronuclear membrane during elongation of the macro-nucleus in division. No microtubules were found in the karyoplasm or in coalescingnuclei. Colchicine (5 x io~3 M) blocks elongation of the macronucleus but not coales-cence or cell division (Jenkins, 1969).

Cytoplasmic channels containing microtubules have been found in a number ofdinoflagellates (Hollande, 1972). In Gyrodinium cohnii the channels are perpendicularto the division furrow, forming the axis on which the nucleus elongates (Kubai &Ris, 1969). The situation is similar in Amphidinium; however, Oakely & Dodge (1974)found the microtubules within the channels to be attached to kinetochore-like plaqueson the nuclear membrane serving as attachment sites for chromosomes and thusfunctioning as spindle microtubules. In the dinoflagellates as in regenerating stentors

534 J- J- Paulin and A. S. Brooks

the microtubules exiting from the channels terminate in the perinuclear cytoplasmand are not associated with kinetosomes or centrioles

What then is the function of the microtubules found within the channels and aroundthe macronucleus in regenerating cells? We can rule out a mitotic function since themacronucleus is polyploid, and no condensed chromosomes (polytene) have beenobserved in Stentor macronuclei (Tartar, 1961), and no chromatinic association hasbeen found. Whether they function as a pushing body as in other ciliates cannot beruled out, but in all other ciliates studied to date the pushing bodies have been intra-nuclear. The most plausible possibility is that the channels containing microtubulesand perinuclear microtubules form the axial plane for elongation and are the kineticelement responsible for elongation.

Tartar (1961) has indicated that some intrinsic impulse to nodulate resides in themacronucleus, due to the fact that clumped nuclei isolated in a small volume of cyto-plasm from dividing cells elongate and attempt to renodulate. The timing of thenuclear events (e.g. clumping, elongation and renodulation) has been shown to residein the cortex (Tartar, 1961; deTerra, 1969, 1974). Consequently, it may be hypo-thesized that the stimulus to form a primordium in regeneration (loss of corticalmembranelles) may at a specific time (stage 6) initiate macronuclear channel andmicrotubular formation triggering elongation and initial renodulation (intrinsicimpulse of Tartar, 1961) of the nucleus which is completed under continued corticalfeedback.

Whether these channels and microtubules are present in clumped and elongatingmacronuclei of dividing cells needs to be explored. By serial sectioning techniqueswe may be able to establish unequivocally whether there is a link between the nuclearmicrotubules and the cortex, a situation not resolvable in this study. Serial sectioningwill also demonstrate the continuity of the channels with the nuclear membrane,eliminating the remote possibility of the channels being intranuclear vesicles contain-ing microtubules. The continuity of the files of microtubules in the channels andperinuclear cytoplasm seen in Fig. 9 supports the hypothesis that they are indeedcontinuous through the nucleus.

The authors acknowledge the financial support of a Brown-Hazen Grant from ResearchCorporation and the technical assistance of Mr William Henk II.

REFERENCES

DETERRA, N. (1969), Differential growth in the cortical fibrillar system as the trigger for oraldifferentiation and cell division in Stentor. Expl Cell Res. 56, 142-153.

DETERRA, N. (1974). Cortical control of cell division. Science, N.Y. 184, 530-537.FALK, H., WUNDERLICH, F. & FRANKE, W. (1968). Microtubular structures in macronuclei of

synchronously dividing Tetrahymena pyrifonnis. J. Protozool. 15, 776-780.GALIGHER, A. & KOZLOFF, E. (1964). Essentials of Practical Microteclmique, pp. 394-396.

Philadelphia: Lea and Febiger.HETHERINGTON, A. (1934). The role of bacteria in the growth of Colpidium colpoda. Physiol.

Zool. 7, 618-641.HOLLANDE, A. (1972). Le d^roulement de la cryptomitose et les modalite's de la segregation des

chromatides dans quelques groupes de protozaires. Atmls Biol. 11, 427-466.

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JENKINS, R. (1969). The role of microtubules in macronuclear division in Blepharisma.jf. Protozool. I6J , 10 (Abstr.).

KUBAI, D. & Ris, H. (1969). Division of the dinoflagellate Gyrodinium cohnii (Schiller). J. CellBiol. 40, 508-528.

MARGULIS, L. (1973). Colchicine-sensitive microtubules. Int. Rev. Cytol. 34, 333-361.OAKELY, B. & DODGE, J. (1974). Kinetochores associated with the nuclear envelope in the

mitosis of a dinoflagellate. J. Cell Biol. 63, 322-325.PAULIN, J. (1973). Macronuclear reorganization during regeneration of oral membranelles in

Stentor coeruleus. In Progress in Protozoology: IVth Int. Congr. Protozool. (e&: P. de Puytorac),p. 314. France: Clermont-Ferrand.

PAULIN, J. & BUSSEY, J. (1971). Oral regeneration in the ciliate Stentor coeruleus: a scanning andtransmission electron optical study. Jf. Protozool. 18, 201-213.

RAIKOV, I. (1969). The macronucleus of ciliates. In Research in Protozoology (ed. T. Chen),pp. 1-128. New York: Pergamon.

TARTAR, V. (1957). Reactions of Stentor coeruleus to certain substances added to the medium.Expl Cell Res. 13, 317-332.

TARTAR, V. (1961). The Biology of Stentor. New York: Pergamon.TUCKER, J. (1967). Changes in nuclear structure during binary fission in the ciliate Nassula.

J. Cell Set. 3,481-498.(Received 15 May 1975)

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Figs. 1-4. Light micrographs of Feulgen preparations of successive stages of macro-nuclear reorganization.

Fig. 1. Control moniliform macronucleus with tenuous constriction between nodes(arrow) and pigment stripes of cortex are evident in this optical section, x 1075.

Fig. 2. Clumping macronucleus; 2 enlarged nodes formed by the fusion of othernodes are evident, x 1060.

Fig. 3. Clumped macronucleus. x 1350.Fig. 4. Elongating macronucleus; constriction furrow delineating new node can be

seen (arrow), x 2080.Fig. 5. Survey electron micrograph through 3 macronuclear nodes from control cell;nucleoli (n) and chromatin clumps (c) are dispersed throughout the nodes andnumerous mitochondria (in) are seen between the nodes, x 7600.

Macronuclear differentiation 537

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538 J. J. Paulin and A. S. Brooks

Fig. 6. Cross-section through a portion of an elongating nucleus (stage 7); numerousdouble membrane-bound channels containing microtubules are found in the nucleus,note pore (p) in the double nuclear membrane, x 38000.Fig. 7. Intranuclear channel cut in longitudinal section; note its close proximity to theperiphery of the nucleus, x 20400.Fig. 8. High magnification of intranuclear channel; microtubules are present in thelumen of the channel and outside the double nuclear membrane (arrow), x 76000.

Macronuclear differentiation

54° J. J. Paulin and A. S. Brooks

Fig. 9. Grazing section through nodulating macronucleus; note intranuclear channelscontaining microtubules and microtubules forming a network around the nuclearmembrane (arrows), x 36000.Fig. 10. Cytoplasmic furrow (small arrow) and channels (large arrow) containingmicrotubules are both seen in this micrograph, x 10400.

Macronuclear differentiation 54'