signal-induced reorganization of the microtubular ... · 1983; eichele et al. 1985) or growth...

Signal-induced reorganization of the microtubular cytoskeleton in the

ciliated protozoon Euplotes octocarinatus

MARIA JERKA-DZIADOSZ*

Department of Cell Biology, M. Nencki Institute of Experimental Biology, 3 Pasteur St, 02-093 Warsatc, Poland

CHRISTINE DOSCHE, HANS-WERNER KUHLMANN and KLAUS HECKMANN

Zoological Institute of the University of Munster, Schlofiplatz 5, D-4400 Munster, FRC

• Author for correspondence

Summary

A predator-released substance induces the fresh-water ciliate Euplotes octocarinatus to undergo,within a few hours, a drastic change in cell formthat makes engulfment by the predator moredifficult or even impossible. During this trans-formation, the outline of the cell changes fromovoid to circular and the size increases con-siderably. The cells cease dividing while theytransform, but later continue divisional morpho-genesis and maintain the circular form for manycell generations if the concentration of the pred-ator factor is maintained

The microtubular cytoskeleton of Euplotes wasstudied by transmission electron microscopy ofcells from -which the cytoplasm had beenextracted by mild treatment with Triton X-100.This procedure increased the visibility of micro-tubules, especially single microtubules located inthe endoplasm. In transformed cells, a consider-able increase in number of microtubular triads

on the dorsal and ventral surfaces and the ap-pearance of extra single microtubules betweenthe dorsal triads was observed. However, certaininterconnected groupings of microtubules locatedon the dorsal surface were greatly diminishedafter transformation. Intracytoplasmic micro-tubules were also more abundant in the enlargedcells than in the untreated ovoid ones. Thespacing and general pattern of microtubules,however, appears to be the same in untreated andtreated cells.

We conclude from these observations that thetransformation of Euplotes cells from their typi-cal ovoid form into the enlarged circular form isaccompanied by the mobilization and utilizationof microtubules already present in subcorticalregions and an assembly of new microtubulesneeded for support of the expanding cell cortex.

Key words: microtubules, cytoskeleton reorganization,Euplotes.

Introduction

A reorganization of cytoskeletal structures occurs inmany cell types. The process is precisely controlledboth temporally and spatially and is usually related tocell division and/or cell differentiation.

Studies on control mechanisms involved in theprocess of differentiation become of interest when theinvestigated organism has the choice between severalroutes of development and when the choice of route canbe experimentally controlled by signal substances.

Reorganization of actin- and tubulin-containingstructures in cultured mammalian cells has been ob-served after treating cells with tumour promoters

Journal of Cell Science 87, 555-564 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

(Schliwa et al. 1984), oncogene proteins (Bar-Sagi &Feramisco, 1985), and substances acting as morpho-gens (Stricland & Mahdavi, 1978; Lehtonen et al.1983; Eichele et al. 1985) or growth factors (Yanker &Shooter, 1982). In ciliates, reorganization of intracy-toplasmic microtubular structures has been observed incells undergoing shape transformation caused by lightstimulation (Matsuoka & Shigenaka, 1985) or overfeed-ing (Golifiska, 1986). Signal-mediated enlargement ofcells of Tetrahymena vorax (Buhse, 1967) and T.paravorax (M&e'nier, 1976) involves the production oflarge cortical ciliary structures.

In this study we present observations on a signal-induced cytoskeletal reorganization in a free-living

555

unicellular organism. The ciliate E. octocarinatus re-sponds to a water-soluble factor, released by thepredatory ciliate Lembadion lucens, by characteristicchanges in cell architecture that make engulfment bythe predator more difficult or even impossible. Cellsexposed to the Lembadion factor (LF) are transformedfrom their typical ovoid form into an enlarged circularform with dorsal and ventral protuberances. Theybecome about 60% wider and 25 % longer (Kuhlmann& Heckmann, 1985).

Ultrastructural analysis of the cytoskeletal frame-work, carried out primarily on extracted cells, revealedthat the change in size and shape of Euplotes cellsexposed to LF is accompanied by a substantial increasein microtubule polymerization of both intracellular andsubcortically localized microtubules. The general pat-tern of subcortical microtubules was found to be size-invariant.

Materials and methods

acetate and lead citrate, and examined with a transmissionelectron microscope (Siemens Elmiskop 101 and JEM 100 B).

Some preparations were made by extracting cells inextraction buffer containing Triton X-100, but lacking glu-taraldehyde (procedure B). After extraction cells were fixedin a mixture of glutaraldehyde and tannic acid, postfixed inOsO4 and then further processed as in procedure A.

By using the methods of extraction described above, thecytoskeleton of Euplotes cells was well preserved. Controlcells, not extracted, were fixed in an ice-cold mixture of twoparts of 0-05M-Pipes buffer (pH69) , one part of 4%aqueous osmium tetroxide and one part of 6 % bufferedglutaraldehyde. Cells were kept in this fixative on ice for 1 h,then they washed in cold buffer, dehydrated and embedded.

Light microscopyLiving cells, cytoskeletal frameworks and cells stained withProtargol were observed with a Zeiss photomicroscope and aLeitz Orthoplan microscope.

Staining with Protargol (silver proteinate, Merck) wasperformed according to methods described elsewhere (Jerka-Dziadosz, 1985; Goliriska, 1986).

Growth and shape transformationEuplotes octocarinatus strain 1(6)-VI was cultured inPringsheim solution and fed daily with Chlorogoniumelongatum grown on soil solution (Heckmann & Kuhlmann,1986).

Lembadion lucens was cultured in Pringsheim solution andfed every other day with Colpidium campylum grown eitheron bacterized hay infusion or on bacterized Pringsheimsolution supplemented with dried egg yolk suspension.Colpidium was washed out from culture medium by centrifu-gation and resuspended in Pringsheim solution. All cultureswere kept at room temperature.

In experiments involving size and shape transformation ofEuplotes we mixed relatively dense well-fed cultures ofEuplotes with dense well-fed cultures of Lembadion. Theratio of the two cultures was usually 1:2. In some exper-iments, Chlorogonium and/or Colpodium was added. KeepingEuplotes and Lembadion well fed is essential for obtaininggood transformation.

Isolation and fixation of cytoskeletal frameioorksWe used the procedure described for Tetrahymena by Wolfe(1985), with slight modifications. The extraction buffercontained 0-1 M-sucrose, 2-SmM-MgCl2, 0-lmM-KCl, 1 mM-EDTAand 10mM-Hepes (pH6-8). Up to 10 cells of Euploteswere pipetted into extraction buffer in depression slides kepton ice containing 0-5 % Triton X-100 and 0'25 % glutaralde-hyde. After 3-5 min, when cells became transparent, theywere quickly washed in cold extraction buffer withoutTriton. Next they were washed thoroughly in 0-05M-Pipesbuffer (pH 6-9) and then the fluid was replaced by Pipesbuffer containing 2 % tannic acid and 1 5 % glutaraldehydefor 1 h on ice (procedure A). After a wash in Pipes buffer thecells were postfixed in 1 % osmium tetroxide in 0-05 M-Pipesbuffer for 0'5 h on ice. The cells were then washed in bufferand water and transferred into agar blocks, dehydrated andembedded in Epon. Thin sections were stained with uranyl

Results

General body form and major structures of untreatedand LF-treated cells

E. octocarinatus is a dorsoventrally flattened ciliatedprotozoon with most of its locomotor and feedingstructures located on the ventral surface. The oralciliature consists of an adoral zone of membranellesthat surrounds the cell's left anterior side, and a paroralmembrane bordering the right side of the oral groove.The ventral ciliature consists of nine frontoventralcirri, five transverse cirri, and four to five caudal cirri.The dorsal surface is covered by eight (rarely nine)rows of dorsal bristles. The two external bristle rowsare located at the ventrolateral edges of the cell

(Fig- 1).Transformed cells are longer and wider than

untreated cells (Kuhlmann & Heckmann, 1985) but thenumber of ciliary structures on the ventral surface isthe same in both groups (Fig. 1). The cirri in trans-formed cells appear more massive; they probablycontain more cilia; 1-8% of transformed cells pos-sessed an additional dorsal bristle row located predomi-nantly close to rows 3-7 (row 1 is the one closest to theadoral zone of membranelles on the left dorsal side ofthe cell).

From earlier studies it is known (Kuhlmann &Heckmann, unpublished) that exposure of a growingculture of E. octocarinatus to LF causes a transientcessation of cell division. We stained samples fromwell-fed cultures of Euplotes with Protargol before andduring exposure to LF. Six hours after mixing Euploteswith Lembadion, only one cell in a sample of 18 cellswas found in an early stage of cell division. In a parallel

556 M. Jerka-Dziadosz et al.

1A

cc

B

Fig. 1. Ventral view of E. octocarinatus, stained with Protargol. A. Untreated ovoid form; B, LF-treated circular form.The broken lines indicate the area in the middle of the cell through which most of the cross-sections shown in subsequentfigures were made, azm, adoral zone of membranelles; rdb, row of dorsal bristles;fvc, frontoventral cirri; Ic, transversecirri; cc, caudal cirri; ma, macronucleus; mi, micronucleus. Bars, 10/im.

untreated sample, 41 % of the cells (n = 24) were foundwith primordia of ciliature in different stages of devel-opment. In samples fixed 24 h after exposure to LF,37 % (n = 62) of the cells possessed ciliary primordia.From observations of stained samples it appears that incultures exposed to LF the divisional morphogenesis isresumed after the cells have enlarged in size. Thisenlargement takes place during 3-12 h of exposure.

The ultrastructure of the cytoskeletal framework in E.octocarinatusThe cytoskeletal framework that remains after extract-ing cells with detergent retains the shape of the cells. Asdescribed by Wolfe (1985) for Tetrahymena, the ex-traction procedure yielded cell ghosts of Euplotes thatcontained demembranated cell cortices and nuclei. Theciliary axonemes of adoral membranelles, ventral cirriand dorsal bristles remained attached to their basalbodies.

In non-extracted cells single microtubules located inthe endoplasm were hardly distinguishable. In con-trast, detergent-extracted cells showed their cytoskel-etal framework but contained large spaces empty of cellstructures (Fig. 2). Membrane-bound organelles suchas the mitochondria and cortical ampules were absent.In mildly extracted cells, the remnants of partially

dissolved mitochondria and endosymbionts were vis-ible. Nuclei, both macronucleus and micronucleus,were well preserved.

The cortex of the cells appeared to be structurallywell preserved in cytoskeletal preparations. Theplasma membrane of ciliary axonemes and the cellsurface membranes (plasma membrane, inner andouter alveolar membrane) were removed in most prep-arations. In some preparations they were replaced byfragments of a single laminar sheet. The pieces oflamina were more prominent in preparations madeaccording to procedure B. The whole surface exceptthe buccal cavity was covered by the fibrillar epiplasm,which corresponds to the 'fibrous mat' described byRuffolo (1976). This was 30-40 nm thick.

The epiplasm in E. octocarinatus was underlain bysheets of microtubules arranged in a pattern verysimilar to that described in other species of Euplotes(Grim, 1967, 1982; Ruffolo, 1976).

On the dorsal surface in untreated cells of E. octo-carinatus, longitudinal microtubules were arranged intriads (Figs3A, 4). In cross-sections perpendicular tothe long axis of the cell it was possible to inspect allmicrotubules underlying the dorsal and ventral surface(Fig. 2). In growing cells, there were about 440 triadson the dorsal surface. The number of triads foundbetween two dorsal bristle rows ranged from 45 to 60.

Cytoskeleton reorganization in Euplotes 557

Immediately anterior and posterior to the pit in whichthe dorsal bristle unit is located (Ruffolo, 1976) four tofive single microtubules mark the dorsal bristle row onconsecutive sections (Fig. 5A).

Between triads, single microtubules were occasion-ally found. In a morphostatic cell, the number of singlemicrotubules (located outside a dorsal bristle row) onthe whole dorsal surface did not exceed 10. In contrast,in cells preparing for division the number of singlemicrotubules inserted between triads increased to 4—18in each sector between two dorsal bristle rows (onaverage, about 80 per whole dorsal surface).

In cells in the second half of the cell cycle (defined bythe presence of a replication band in the macronucleus)about 14—25 triads immediately to the right of each

dorsal bristle row appear to be more densely spacedthan the others. These triads are underlain by a largegroup of 30-80 single microtubules interconnectedwith each other by fibrillar links (Fig. 5). The role ofthese groupings of single microtubules underlyingsectors of more densely spaced triads is unknown.Perhaps these sectors represent locations where most ofthe triads are assembled while the cell enlarges duringdivisional morphogenesis. Single microtubules alsooccur on the left side of a dorsal bristle row, but theirnumber is much smaller.

The ventral epiplasm is underlain by regularlyspaced single microtubules (Fig. 3B). There wereseveral (3-4) sectors on cross-sections where micro-tubular triads similar to those existing on the dorsal

Fig. 2. Transverse sections through the middle part of: A. an untreated ovoid cell (cytoplasm extracted according toprocedure B); and B, an LF-treated circular cell (cytoplasm extracted according to procedure A), oa, oral apparatus;dr, dorsal ridge; le, lateral extensions; vr, ventral ridge; ma, macronucleus. Arrows designate sectors with ventralmicrotubular triads. Bars, 10^m.


Fig. 3. Transverse sections through the pellicle ofuntreated ovoid cells (cytoplasm extracted according toprocedure A). A. Dorsal triads. Note the laminarstructures (Is) covering the epiplasm. B. Ventral singlemicrotubules. ep, epiplasm; dt, dorsal triads; Is, laminarstructures. Bars, O'l^m.

surface were present (Fig. 2). The number of all triadson the ventral surface varied from 60 to 85. Thenumber was larger in dividing cells than in non-dividers. The left and right sides of the ventral surfacewere underlain by single microtubules only.

Immediately underneath the longitudinal micro-tubules lie interlinked transverse microtubules. Thespacing of these microtubules appears to be wider than,but as regular as, that of the longitudinal microtubules(Fig- 4).

In cross-sections of normal, non-dividing cells, sec-tions through microtubular structures other than theperipheral cortical microtubular arrays are also visible.These include sections through microtubular rootletsaccompanying each frontal, ventral and transversecirrus, the root microtubules accompanying the oralciliature and microtubules of the oral pouch. Manysections through microtubules are visible near the oralapparatus and the middle sector of the ventral part.The dorsal half of the cell (except in the vicinity of themacronucleus and the micronuclei) appears to bedevoid of intracytoplasmic microtubules (Fig. 2A).

The role of the intracytoplasmic microtubules is notknown. Whether they are purely cytoskeletal structuresor whether they play some role in the segregation ofcellular organelles remains to be discovered.

The cytoskeleton in LF-treated cellsIn a previous study (Kuhlmann & Heckmann, 1985), itwas shown that cells exposed to a factor released by thepredator Lembadion become about 60% wider andabout 25 % longer than unexposed cells. Enlargementis accompanied by the formation of about five dorsalprotuberances, among which the middle one becomesthe most prominent. Similarly, in the middle of theventral surface the ridge develops into a ventral pro-tuberance. Owing to these extensions the surface areaof the ciliate becomes considerably enlarged duringtransformation (Fig. 2B).

The main purpose of this study was to determinewhether the changes in the shape and size of Euplotestreated with LF involved reorganization of non-ciliarycytoskeletal structures.

The basic plan of the cytoskeletal structures intransformed cells was similar to that in untreated cells(Fig. 2B). The cell periphery was covered by a fibro-granular mat of epiplasm. The dorsal surface wassupported by microtubular triads. In the transformedcells, unlike untreated cells, the dorsal triad patterncontinued over the cell margins towards the ventralsurface for about 5-8 jJ.m up to the cell sector where thebristle row is located (Fig. 6A).

In transformed cells single microtubules occurredmore frequently among dorsal triads than in untreatedcells. They were more frequent in lateral parts of thecell. In cell f (see Table 1, section 8) there were 31sections of single microtubules on the left side betweendorsal bristle rows no. 1 and 2. In a comparable regionin section la of an untreated cell there was one singlemicrotubule. Single microtubules parallel to the triadslocated close to the cell surface and beneath theperipheral triads and singlets were found at the verymargins of the cell (Fig. 6A), where they probablysupport the lateral extensions.

The main quantitative difference between normaland transformed cells is that in transformed cells thereis a lack of or severe diminution in number of micro-tubules arranged as interconnected groupings underly-ing triads at the right side of the rows of dorsal bristles.In most sections studied, including the sectionsthrough cells in the process of cortical development,no significant gathering of microtubules close to thedorsal bristle meridians was found. In two transformedcells about 18 triads appeared more densely spaced,adjacent to dorsal rows no. 4-7, but there were veryfew single microtubules beneath them (Fig. 5B). Inone cell there were 15 cross-sections through single


microtubules adjacent to dorsal row no. 3 (comparedwith 80 in normal cells).

In order to analyse quantitatively the differencesbetween normal and transformed cells a morphometricanalysis was performed. Sections of cells cut perpen-dicularly to their long axis were photographed at amagnification of X6000-X12000, and printed. A sec-tion through the whole cell was then reconstructed andanalysed in each case. We measured the outline of thedorsal surface extending between the extreme bristlerows and counted the number of microtubular triadslocated in this region. We constructed altogether sevencomplete sections through five untreated cells and ninecomplete sections through four transformed cells. Thedata obtained from these sections are summarized inTable 1 and Fig. 7. In Table 1 the sections are

dt

designated by numbers and the cells by lower caseletters. The sections through a cell are arrangedconsecutively from anterior to posterior. Within thegroup of untreated cells, b and e were early dividers, asjudged by the position of the replication bands in themacronucleus and the morphology of the micronu-cleus. In cells c and d the measurements comprisedsectors reaching from dorsal bristle row no. 2 to 8. Thecontrol cells were oblong, therefore the differences inwidth of consecutive sections are rather small and therange of values in different cells is also limited.

In the group of LF-treated cells the width variedfrom 67-33 to 130jum as compared to 48-33 Jim to78-44 /J,m in the untreated cells. This widening of therange results not only from enlargement but also from a

dt db

5A

- 5B JFig. 4. Grazing section through an LF-treated circular cell (cytoplasm extracted according to procedure B). dt, dorsaltriads; tmt, transverse microtubules. Bar, 0 1 y.m.Fig. 5. Transverse sections through the dorsal pellicle near a dorsal bristle. A. Untreated ovoid cell (cytoplasm extractedaccording to procedure B). The dorsal triads on the cell's right side (viewer's left side) of the dorsal bristle are morecrowded than on the right side and underlain by a bundle of interlinked microtubules. The arrow points to singlemicrotubules located posterior to the dorsal bristle pit. B. LF-treated circular cell (cytoplasm extracted according toprocedure A), ma, macronucleus; db, dorsal bristle. Bars,


6A

' • »* • \

y VvFig. 6. Cross-sections through LF-treated cells (cytoplasm extracted according to procedure A). A. Left lateral extension.Note the arrangement of the triads around the cell edge and the location of the dorsal bnstle (db). Single microtubules arevisible among triads (arrows). Many cross-sections through intracytoplasmic microtubules (itnt) can be seen. B. Ventralridge. Note the microtubular triads (mtt) and the single microtubules (nit). Bars, 1 nm.

Table 1. Relationship between the width of the celland the number of microtubular triads on the dorsal

surface ofE. octocarinatus

Sectionnumber

Untreatedcells

Celldesignation Width

1234$I,:r

LF-treatedcells

&9

10111213141516

aa"bB

Ide

Mean±S.D.

1ff

IW1ti.i

Mean±s.D.

66-9365-3078-4456-1153-7748-3378-33

63-8811-81

130-00128-4094-4084-1881-4867-33

119-00120-0072-09

99-6524-84

No. of triads

439439521379324321405

40470-77

691691588544514392780801383

598-22154-25

difference in shape. The transformed cells were circu-lar rather than oblong, therefore sections through themiddle of the body were much wider than sectionsthrough the anterior or posterior parts of the cell. Cells

800

700

600

500

400

3001-

I

20 40 60 80Cell width (^

100 120 140

Fig. 7. The relation of number of dorsal triads to cellwidth. The line is given by the equation:m = 5-519w+49-608/. It fits a linear regression.(•) Untreated cells; (+) LF-treated cells.

f and h were sectioned near the middle of the body, cellg was sectioned in the posterior part, near the trans-verse cirri and posterior to the macronucleus. Thesections through cell i were made near the micronu-cleus in the anterior part of the body. Cells f and hpossessed replication bands, thus they were in the S-phase of the cell cycle.

The analysis of data presented in Table 1 shows thatthe mean cell width and the mean number of micro-tubular triads differ significantly in both groups (Dun-can test). Generally transformed cells possess about47 % more microtubular triads than untransformed


cells. The number of triads on the dorsal surface ishighly correlated with the cell width ( r= 0-9447).Analysis of linear regression (Fig. 7) revealed thatwidening of the cell by 1 ^m causes addition of about5-52 microtubular triads. This indicates that in thecircular forms the microtubular triads cannot extendover the whole cell length, but some must terminate orstart at different cellular locations.

The ventral surface of transformed Euplotes isunderlain by single longitudinal microtubules and isadditionally supported by single transverse micro-tubules. The sectors of ventral triads are more ex-panded compared to normal cells. The ventral ridgecontains about 100 triads in LF-treated cells as com-pared to about 67 in untreated cells (Fig. 6B). Thenumber of single microtubules underlying the ventralsurface was not counted. Since the spacing of thesestructures was found to be similar in normal andtransformed cells the absolute number of these micro-tubules is probably proportionately larger in trans-formed cells.

The third category of microtubules present in thecellular cytoskeleton of E. octocarinatus - the singleintracytoplasmic microtubules located inside the cell -is also more abundant in the transformed cells. Thelateral extensions as well as the dorsal and the ventralprotuberances are filled with single microtubules run-ning in many directions. Some microtubules are seen tobe attached to the surface and run perpendicularly orobliquely toward the cell interior (Fig. 6A). It seemsthat some of these microtubules may take part inshaping the lateral wings and dorsal and ventral projec-tions by supporting the extended form of these pro-tuberances.

In conclusion it can be stated that the exposure ofE. octocarinatus to the factor released by the predatorL. lucens causes an extensive increase in the amount ofmicrotubular structures. These additional microtubu-lar structures are located in the expanded corticalregions of the cell and support the intracellular cyto-skeleton.

Discussion

The main purpose of this study was to determinewhether the signal-induced cell transformation in E.octocarinatus involves a reorganization of the cytoskel-etal framework.

The general morphology of cytoskeletal structures inEuplotes is well known from detailed studies ofultrathin sections in the transmission electron micro-scope (Grim, 1967; Ruffolo, 1976) and from studies ofcytoskeletal preparations investigated by scanning elec-tron microscopy (Grim et al. 1980; Grim, 1982).

Our study confirmed previous descriptions of thecortical ultrastructure and revealed some novel features

of the extraciliary cytoskeleton of Euplotes. The novelfeatures are: (1) the presence of microtubular tripletsin the subcortical region, not only on the dorsal surfacebut also at defined regions of the ventral surface;(2) the occurrence of gatherings of single microtubuleslocated subjacent to the dorsal triplets neighbouringthe right side of the dorsal bristle rows in growing cellsin the second half of the cell cycle; (3) the presence ofan extensive and dispersed intracytoplasmic network ofmicrotubules.

The cellular locations of the nucleating centres of theintracytoplasmic microtubules are not known. Someof them seem to be attached to subcortical regions(Fig. 6A) or to plasma membrane structures aroundthe cytostome and the oral pouch, therefore theirmicrotubular organization centres may be located sub-cortically.

Exposure of growing cells of E. octocarinatus to thefactor released by the predator L. lucens provides aneasy-to-handle model system for the study of morpho-logical changes in cells. These changes are perhapsinitiated by binding of the factor to factor-specific cellreceptors, which then induce a chain of events culmi-nating in a change of cell morphology, which is not amere enlargement to a giant form but involves a changein cell shape.

The first visible step in the chain of transformationevents is the transient cessation of cell division in agrowing population of E. octocarinatus. Exposure ofEuplotes cells to a high concentration of LF could becompared with other experimentally applied shocks(e.g. heat-shock, starvation, transection, overfeeding),which are known in some ciliates to stop cells fromproceeding in their cycle and induce them to enter adifferent route (Jerka-Dziadosz & Frankel, 1970;Buhse & Rasmussen, 1974; Nelsen & DeBault, 1978;Golifiska, 1986). In Euplotes, this new route involvesmassive production of microtubules and their insertioninto expanding cortical structures. Transient cessationof cell division concomitant with transformation ofshape may suggest that tubulin and other macro-molecules that are assembled in preparation for celldivision are instead utilized for the assembly of cyto-skeletal microtubules required for the expanding cellsurface. It is highly tempting to speculate that thesubcortical groupings of microtubules found in normalgrowing cells (or their building blocks) could beused as an immediate source of microtubules for theexpanding cortex. The tubulin from 640 single micro-tubules present in the dorsal grouping may contributeto about 213 microtubular triads. This number ap-proximates the difference in the number of microtubu-lar triads between untransformed and transformed cells(Table 1). Such a mobilization of tubulin during thecell cycle is well known in other systems (Cohen et al.1982) and supports the idea of rapid redeployment of


microtubules via a disassembly-assembly steady state(Mitchison & Kirschner, 1984; Schulze & Kirschner,1986).

The general spacing of the dorsal triads in trans-formed cells is comparable to that in untransformedcells, and the relative distances between dorsal bristlerows are roughly proportional to the cell width (datanot shown). Therefore the addition of dorsal triads hasto be relatively uniform over the dorsal surface. Withineach dorsal sector (bordered by two adjacent bristlerows) new triads could be intercalated in a uniformmanner or else they could predominantly be assembledin a defined location, such as the right side of eachdorsal row where the triads appear more crowded. Thislast mode would resemble the mode found for theflagellate Euglena, where surface growth occurs byintussusception of new microtubular stripes betweenold ones at defined locations (Hofmann & Bouck,1976).

The general pattern of microtubular triads in normaland transformed cells appears to be size-invariant(Fig. 7). In transformed cells, in addition to a con-siderable increase of microtubular triads on the dorsalsurface, the number of triads on the ventral surfaceincreases correspondingly. Enlargement of Euplotesand a change from oblong to circular do not apparentlycause an increase in the number of other structurescontaining microtubules, such as the ventral cirri andmembranelles (except for the rare formation of an extradorsal bristle row). The cirri, however, appear to belarger and probably contain more cilia. Similarly ingiant forms of E. balteatus there is a relative constancyof the pattern of ventral ciliature (Tuffrau, 1964). Inother studies, it has been shown that although thenumber of large cirri does not change in a wide range ofcell sizes, the number of constituent kinetosomes infrontal cirri is related to the cell size (Bakowska, 1981).This relation is, however, not truly proportional. Insize-reduced cells the number of compound ciliarystructures and constituent microtubules diminishes(Golinska, 1984; Jerka-Dziadosz & Golinska, 1977).From this it follows that the size-dependent regulationof microtubular structures is different at differentlevels of structural hierarchy.

The intracytoplasmic network of microtubules ap-pears to be well developed in the transformed cells.Owing to the lack of defined markers of patternedarrays it is difficult to assess changes in their numberand distribution. From inspection of cross-sections oftransformed cells it is apparent, however, that theirnumber is significantly greater than in normal cells.

In conclusion, it can be stated that the cytoskeletalreorganization in transforming cells of Euplotes in-volves mobilization and utilization of microtubulesalready present in the subcortical regions, as well as

polymerization of new tubulin and other proteinsnecessary for building an expanded cell cortex.

The main part of this study was performed while the firstauthor (M.J.D.) was working at the Zoological Institute ofthe University of Miinster as an Alexander von Humboldtfellow. Investigations performed at the Nencki Institute inWarsaw were supported by grant CPBP, 04.01 from thePolish Academy of Sciences. Expert technical assistance ofMrs Lidia Wiernicka (Nencki Institute, Warsaw) and helpwith the electron microscope by Dr U. Mays (ZoologicalInstitute, Munster) is kindly acknowledged.

We thank Drs H. D. Gortz (Munster), J. Frankel (IowaCity) and K. Golinska (Warsaw) for comments on themanuscript, and Mrs B. Kriiger for secretarial help.

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{Received 15 December 1986-Accepted 9 February 1987)


signal-induced reorganization of the microtubular ... · 1983; eichele et al. 1985) or growth...

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