Mutational analysi osf patterning of oral structures in Tetrahymena · Department of Biology, University of Iowa, Iowa City, 52242, Iowa U.S.A. SUMMARY The oral apparatus (OA) of

/. Embryol. exp. Morph. 82, 41-66 (1984) 4 1Printed in Great Britain © The Company of Biologists Limited 1984

Mutational analysis of patterning of oral structuresin Tetrahymena

I. Effects of increased size on organization

By JOSEPH FRANKEL, LESLIE M. JENKINS, JULITABAKOWSKA AND E. MARLO NELSEN

Department of Biology, University of Iowa, Iowa City, Iowa 52242, U.S.A.

SUMMARY

The oral apparatus (OA) of the ciliated protozoan Tetrahymena thermophila consists of fourordered arrays of ciliary units. In wild-type cells, these arrays are constant in spatial organiza-tion and vary little in size except during extreme starvation. Recessive mutations at five geneloci are known to increase the size of the OA. They do this by increasing the length of theciliary arrays, without affecting their width and often without increasing their number beyondthe usual four. Comparison of the oral arrays over a large range of sizes has revealed: (1) thatthe lengths of the anterior two of three parallel arrays (membranelles) are rather tightlycoordinated; (2) that the specific basal body configurations resulting from remodelling of themembranelles are only slightly affected by large changes in lengths of membranelles; and (3)that the third membranelle is restricted to a nearly constant length, except in the very largestOAs in which the structure is lengthened but interrupted by a gap in the middle. This gap mayreveal the spatial extent of a putative zone of basal body regression. These phenomena are notspecific to any of the genotypes utilized in this investigation; the effect of the mutations is toloosen quantitative restrictions and thus reveal underlying associations and constraints.

INTRODUCTION

The intracellular pattern of ciliary units in the oral apparatus of Tetrahymenathermophila is useful for analysis because it is readily described quantitatively,its development can easily be observed, and it is subject to environmentally andgenetically provoked variation. The organization of this system is at the sametime remarkably complex and surprisingly invariant in growing wild-type cells(Williams & Bakowska, 1982; Bakowska, Frankel & Nelsen, 1982a). The natureof this organization can be probed by provoking major changes in the size of thesystem. Thus, severe starvation reduces the length of the ciliary arrays within theoral apparatus without greatly affecting their organization; there is evidence foralteration of organization only when the number as well as the length of thesearrays is reduced (Bakowska et al. 1982a). Substantial increase in the size of theoral apparatus cannot be brought about reliably by any nutritional regimen ofwhich we are aware, but it is elicited by any of several genie mutations. This haspermitted an analysis of the response of oral patterns to an extended variationin the size of the ciliary arrays that make up the oral apparatus. In this paper, we

42 J. FRANKEL AND OTHERS

will concentrate on the effects of increase in the size of these ciliary arrays in theabsence of any increase in their number. This will also allow us to isolate analytic-ally the unique effects of change in the number of these arrays, which is thesubject of the next paper. In both papers, phenotypic changes generated bymutations are regarded as windows through which we may perceive underlyingassociations and constraints that guide normal development.

MATERIALS AND METHODS

Stocks

All stocks used in this study were Tetrahymena thermophila of the inbred Bstrain (Allen & Gibson, 1973, Table 2). The wild-type cells were of stock B-2079(20th generation of inbreeding, established in 1979). Information about themutant stocks utilized in this and the companion investigation is summarized inTable 1. The protocol for mutagenesis was the same as described earlier (Fran-kel, Jenkins, Doerder & Nelsen, 1976), with the substitution of 0-75 % ethyl-methane sulfonate (EMS) for nitrosoguanidine in one case. Mutations werebrought to expression either as heterozygotes by macronuclear allelic assortment(Carlson, 1971; Frankel et al. 1976) or as homozygotes by 'cytogamy', i.e. in-duced self-fertilization (Orias, Hamilton & Flacks, 1979; Sanford & Orias,1981). Following either of these procedures, cells were isolated into microdropsby the 'Poisson lottery' procedure (Orias & Bruns, 1976) and replicated by themethod of Roberts & Orias (1973) into microtitre plates. Screening was formorphological abnormalities at 39-5°C as described by Frankel et al. (1976).Subsequent genetic analysis was carried out following procedures originallyintroduced by Nanney, as described in Frankel et al. (1976). Localization ofmutations to chromosome arms was carried out by crosses to germinal nullisomics(Bruns & Brussard, 1981; Bruns, Brussard & Merriam, 1983), with the requisitestocks (Bruns, Brussard & Merriam, 1982) kindly provided by Dr Peter Bruns.Assessment of linkage among psm mutations was by the interrupted genomicexclusion method of Ares & Bruns (1978).

Table 1. Mutant stocks employed in this investigation

Stocknumber

IA-309IA-317

IA-305IA-313IA-223IA-246IA-157IA-235

Mutation

mpClmpC2

mpDbigpsmAlpsmA2psmBpsmC

Mating type

VIIVII

IVIIIVIIIVII

Mutagen

NitrosoguanidineEthyl-methane-sulfonate (EMS)NitrosoguanidineNitrosoguanidineNitrosoguanidineNitrosoguanidineNitrosoguanidineNitrosoguanidine

Method

CytogamyCytogamy

CytogamyCytogamy

AssortmentCytogamy

AssortmentCytogamy

Year isolated

19811982

198119811974197919771979

Mutations affecting intracellular patterns in Tetrahymena 43

Media and growth conditions

Cells were grown in one of three different peptone-based culture mediadescribed by Nelsen, Frankel & Martel (1981), with all of the analyses of mutantscarried out in the 2 % proteose peptone - 0-5 % yeast extract (PPY) medium.Temperatures of culture growth ranged from 18 ° to 38 °C. Procedures for growthof mass cultures were the same as described in Frankel, Mohler & Frankel(1980), except that cultures for oral isolation were shaken continuously duringgrowth, and harvested at cell densities ranging from 4x 104 to 2x 105 cells per ml.Two exceptions are the 23° psmAl culture and the 30° mpD culture (seeResults), in which the flasks were not shaken and were maintained under con-ditions of poor temperature control, hence in these two cases the temperaturesindicated are only approximate.

Oral isolations and cytology

Oral isolations were carried out following the methods described by Williams& Bakowska (1982), with use of four to six drops of SEMT (1 M-sucrose, 1 mMEDTA, 0-1% 2-mercaptoethanol, lOmM-Tris, final pH9-3) plus one to twodrops of a 10 % Triton-XlOO solution as an underlayer during centrifugal collec-tion of oral apparatuses. All solutions were filtered carefully prior to use. Follow-ing centrifugation, the supernatant was aspirated, and the remaining pelletsuspended in about two drops of distilled water, and then fixed for 2-5 min in1-2 ml of cold 1 % OsO4. This suspension was then diluted with 50 % ethanol,centrifuged, and resuspended in 100 % ethanol. It was prepared for scanningelectron microscopy as described earlier (Bakowska et al. 1982a).

Chatton-Lwoff silver impregnation of whole fixed cells and their measure-ments were carried out as described previously (Bakowska et al. 1982a).

Statistical analysis

Statistical procedures were carried out as described by Sokal & Rohlf (1981).'Model IF regression was employed despite some reservations attaching to useof regression procedures with data such as ours, which fit the conditionsdescribed by that model (Sokal & Rohlf, 1981, section 14.13).

RESULTS

I. Oral organization and development in wild-type cells

The cell-surface pattern of Tetrahymena thermophila is organized aroundciliary units embedded within a triton-insoluble lamina (the epiplasm) locateddirectly underneath the cell membrane (Williams & Bakowska, 1982). Eighteento 21 longitudinal ciliary rows are uniformly spread over the cell surface. Aspecialized feeding structure, the oral apparatus (OA), consisting in part of


modified ciliary units spaced close together, is situated near the anterior end ofthe cell (Fig. 1A). The OA consists of four compound ciliary ensembles, namelyone undulating membrane (UM) and three membranelles (Fig. IB). The UM is

OA

UM QY

a-'a-'OA,

M3M2

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O ° Q O 00 OO

4b o o o o'-'oo

' O '"-' OOOn 'A O. - O rfioOo

5a-b

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made up of two parallel rows of basal bodies, the outer one ciliated and the innerone unciliated. The anterior two membranelles (Ml and M2 respectively) consistprimarily of three rows of basal bodies. At the cell's right end of these rows thebasal bodies are displaced into a 'sculptured' pattern that is characteristic foreach membranelle, and is virtually invariant (Bakowska et al. 1982a). The thirdmembranelle (M3) appears to be sculptured in toto, so that the original parallelarrangement of basal body triplets is obscured. Within the membranelles, allbasal bodies are ciliated except a few at the left end of Ml and the right-mostbasal bodies in the sculptured region of M2 and M3.

The OA develops from an oral primordium, which in growing cells normallyappears at the cell's left of the midregion of the right-postoral ciliary row (Fig.1A). Stages of development of this primordium are shown diagrammatically inFig. 1C. A field of basal bodies is produced (stage 1), from which orientedcouplets of basal bodies are generated (stage 2). These couplets line up side byside to form two-rowed promembranelles (stages 3 and 4a). A third basal bodyis then generated anterior to each couplet (stage 4b), converting these coupletsinto columns of three basal bodies each. A fourth basal body is added anteriorto the one or two columns at the right end of each membranelle (stage 5). Theseevents create membranelle prototypes of nearly rectangular organization, withrow-and-column organization that becomes slightly tilted to generate ahexagonal close packing. This simple form is modified near the end of stage 5 bya combination of three processes, namely ciliary regression, basal body resorp-tion, and ciliary unit displacement. These processes generate the characteristicsculpturing of the right ends of the membranelles and a notch at the anterior-leftend of Ml. This remodelling during late oral development (described in detailby Bakowska, Nelsen & Frankel, 1982b) endows each membranelle with aunique pattern signature which is distinguishable at a glance when the finalstructure is examined at sufficient resolution.

The UM develops quite differently from the membranelles. The details are

Fig. 1. Anatomy and development of the oral apparatus of Tetrahymena thermo-phila. (A) A sketch of the arrangement of basal bodies on the ventral surface of a cellentering oral development. Each dot indicates a basal body; cilia are omitted. Sevenciliary rows (CR) are shown, as well as the anterior oral apparatus (OA) and amidbody oral primordium (OP). (B) A more detailed view of the arrangement ofbasal bodies in the oral apparatus of wild-type cells. Closed circles indicate ciliatedbasal bodies, while dashed circles show unciliated basal bodies. Ml, M2, and M3 aremembranelles 1,2, and 3 respectively; UM is the undulating membrane. (C) Stagesof midbody oral development. Each of the ten sketches shows a progressively laterstage of oral development. Symbols are as in (B), with a central dot within circlesindicating basal bodies of the right-postoral (stomatogenic) ciliary row, stippling ofcircles indicating basal bodies soon to be resorbed. The dotted lines connectingcircles in the last two diagrams indicate the probable pathways of basal-bodydisplacement during the sculpturing process. The designation of substages of stages4 and 5 follows Lansing et al. (1984). 'PI' signifies 'pre-1' (Nelsen et al. 1981). Forfurther explanation, see the text.


complex (see Nelsen, 1981; Bakowska etal. 1982ft; Lansing, Frankel & Jenkins,1984), but the essential feature of importance here is that a pro-UM developsduring stage 4 by an alignment of single basal bodies at the right edge of the oralprimordium, virtually orthogonal to the developing membranelles. The stag-gered double-row organization of the completed UM is elaborated later.

The typical sequence of midbody oral development illustrated in Fig. 1C is aprelude to cell division. The completed oral primordium becomes the OA of theposterior division product, while the OA of the anterior division product isderived from the pre-existing anterior OA. Tetrahymena also manifests an alter-native mode of oral development, called oral replacement (Frankel & Williams,1973). Here a stage-1 oral primordium is formed anteriorly, in part adjacent tothe anterior end of the right postoral ciliary row and in part from the UM of theold OA (Frankel, 1969; Kaczanowski, 1976). These two basal body fields norm-ally become fused into a single large field, from which membranelles and UMdevelop in the same way as in predivision oral development. However, the OAthus formed is not segregated into a posterior division product, but insteadreplaces the old OA, whose membranelles are resorbed. Oral replacement canfunction as a physiological substitute for cell division under conditions not per-missive for division (Frankel, 1970; cf. Tartar, 1966; DeTerra, 1969), or as a partof sequences of morphogenetic transformation to an elongated 'rapid swimmer'form in Tetrahymena thermophila (Nelsen, 1978) or to 'macrostome' forms incertain other Tetrahymena species (Williams, 1960; Stone, 1963; Buhse, 1966;Metenier & Groliere, 1979). In the last-mentioned cases, the new oral apparatusthat develops is very much larger than the old one that it replaces.

II. Mutations that increase the size of the oral apparatus

(a) Genetics

The mutations considered here, mpD, big, psmAl, psmA2, psmB, andpsmC,are nitrosoguanidine-induced single locus recessives (Table 1). All are non-allelic, except for psmAl and psmA2. psmC, psmB, and psmAl have beenshown, using the methods of Ares & Bruns (1979), to be mutually unlinked.psmB was previously localized to chromosome arm 4L by Bruns (1982). Usingthe same methods and stocks, we have found that psmA and psmC are both onchromosome 5, while mpD is on chromosome 3R.

(b) Phenotypes

The phenotypes considered here fall into two distinct classes, that of big (andof mpD at a permissive temperature), and that of the psm family.

big is a non-conditional mutation that was selected on the basis of the unusu-ally large size of homozygous cells (Table 2). big is different from the fat muta-tions isolated earlier (Frankel et al. 1976; Jenkins & Frankel, unpublished)because in big, unlike fat, cell length as well as width are substantially increased


Table 2. Dimensions of cells and of OAs* in wild-type and big

Genotype

WT$

bigX

n

50

44

Cell dimensions!A

rLength

Gum)

49-6±2-3

62-5+3-9

WidthGum)

24-5±1-9

34-2+3-5

LW(Mm2)

1219±115

2153+308

Oral dimensions!A

(

Length(/an)

9-8±0-5

11-2+0-6

Width(/an)

6-4±0-5

8-4+0-6

LWdm3)

62-7±6-6

94-3+ 10-3

OALW-rcell LWf

X100

5-2%±0-5%

4-4%+0-7%

* 3-membranelled OAs of cells in stages 1-3 of oral development.t Means ± sample standard deviation(s).X Combined data from two samples taken at different times during exponential growth of

a single culture maintained in 2% PPY at 29 °C (Cell densities: WT, 13 600/ml and 37 700/ml;big, 6300/ml and 12300/ml). Significant differences were observed between early and latesamples in cell length and in OA length and LW.

(Table 2), as is the number of ciliary rows (not shown) and the size of the oralapparatus (Table 2, Figs 4 and 5). Cultures of big cells grow exponentially at28 °C, with a doubling time 25 to 50% longer than that of parallel wild-typecultures; in such cultures, about 90% of the cells that are undergoing oraldevelopment are engaged in predivision development with midbody oralprimordia (Figs 2, 3), while the other 10% are carrying out oral replacement.Roughly 90% of the OAs formed have 3 membranelles (Fig. 4), while theremainder have short 'extra' membranelles either anterior to Ml or posterior toM3. 3-membranelled OAs predominate even after a shift to 39-5 °C, a tem-perature that is near the upper limit for continuous exponential culture growth.

mpD (mp = 'membranellar pattern') is a conditional mutation selected on thebasis of increased cell length and some change in cell shape at 39-5 °C. Culturesof mpD cells grow approximately as rapidly at 39-5 °C, and probably also at28 °C, as do wild-type cultures. Oral development in such cultures, at eithertemperature, is by the standard predivision mode. At elevated temperatures,virtually all oral primordia and ensuing OAs possess four or five regular mem-branelles rather than the usual three (see the accompanying paper). Yet even at28 °C, when all mpD OAs have the normal 3 membranelles, they are slightly butsignificantly larger than in wild-type OAs (see Table 1 of Frankel, Nelsen,Bakowska & Jenkins, 1984).

Thepsm ('pseudomacrostome') mutations also increase oral and cell size, butin a different manner than big and mpD. In these mutations, which are allconditional, the predominant phenotypic effect observed at the restrictive tem-perature is a change in longitudinal position (Frankel, 1979) and size of oralprimordia. The result is generally a switch, partial or complete, from predivisionto oral replacement development (Figs 6,7). The oral replacement primordia are


typically unusually long (Fig. 6), and often give rise to correspondingly largeOAs (Fig. 8). At the same time cell size increases, presumably because cellgrowth is continuing without cell division. Under appropriate conditions

OAr

6

Mutations affecting intracellular patterns in Tetrahymena 49(e.g. psmAl at 28 °C) cultures can be maintained in which some cells are dividingwhile most are undergoing repeated oral replacement, so that cell number in-creases slowly while many individual cells become large and sometimes mis-shapen.

As in big, the 'pseudomacrostome' OAs formed by the psm mutant cellstypically possess the normal complement of 3 membranelles. However, abnor-malities are fairly common. These may involve an interrupted M3 (Fig. 8), shortsupernumerary anterior membranelle fragments, or even a tandem subdivisionof all or part of the OA into two segments of similar organization but unequalsize, probably due to failure of union of the two oral replacement subfields [asdescribed by Kaczanowski (1976) in another mutant].

Of the 'pseudomacrostome' mutations, psmAl is the most strongly expressed,with a majority of cells in the oral replacement mode even at 28 °C; the per-missive temperature for this mutation is 23 °C, or below. By contrast, psmAlcells show no expression at 28 °C, little at 36-5 °C, and delayed expression follow-ing transfer from 28 ° to 39-5 °C. psmB andpsmC (only one allele known for eachlocus) are intermediate, with no expression at 28 °C and a rapid onset ofpseudomacrostome-type oral replacement in 20 to 30 % of the cells following ashift to 36-5 °C. There is also an additional recessive, nitrosoguanidine-inducedmutation, named psmD, located on chromosome 3R, that resembles the otherpsm mutations in sometimes bringing about formation of large OAs by oralreplacement at high temperature. This mutation, unlike the other psm muta-tions, may also express an mp-like phenotype, with extra membranelles presenteven in OAs of normal size. Although expression of the two phenotypes can be

Figs 2-8. Photographs of silver-impregnated T. thermophila cells, focused an oralprimordia (Figs 2,3,6 and 7) or membranelles of mature OAs (Figs 4, 5 and 8). Allof these photographs are printed at the same magnification, with the scale bar, shownin Fig. 2, indicating lOjum.

Fig. 2. A big cell with a midbody oral primordium (OP) at stage 2. The anteriororal apparatus (OA) is out of focus.

Fig. 3. A big cell with a midbody oral primordium at stage 5a. The three mem-branelles are not sculptured at their right ends.

Fig. 4. The anterior portion of a big cell, with a mature OA. The three mem-branelles (Ml, M2, M3) and the undulating membrane (UM) are labelled. Note thesculptured right ends of the membranelles.

Fig. 5. The anterior portion of a wild-type cell, with a mature OA. Notice thatwhile Ml and M2 are shorter than in big, M3 is approximately the same size.

Fig. 6. A psmAl cell from a 28 ° culture with an anterior oral replacement primor-dium (OP) at stage 3. The membranelles of the old OA are to the cell's left (viewer'sright) of the oral-replacement primordium.

Fig. 7. A psmAl cell with an anterior oral replacement primordium at stage 5a.M3 is similar in form to Ml and M2, and only slightly shorter than M2. Fragmentsof regressing old membranelles (RM) are visible to the cell's anterior-left of the oralreplacement primordium.

Fig. 8. A psmAl cell with a mature OA. The three membranelles are labelled.Note that M3 is split into two parts.


dissociated, they appear to be outcomes of the same mutational lesion. OAs ofthis mutation have not been studied in detail because of relatively low penetranceat temperatures below 37 °C.

Ill. Analysis of oral patterns

(a) Coordinate regulation of the length of the first and second membranelles

Scanning electron micrographs of OAs from detergent-extracted preparationswere used for assessment of the number and arrangement of ciliary units indifferent parts of the OA. Cilia are mostly detached during preparation at a pointjust distal to the basal body, leaving prominent, thick-walled stumps. Non-ciliated basal bodies have distal terminations within the surface lamina(epiplasm) and can sometimes be seen (Fig. 12). The holes in the preparation,which form rows posterior to each membranelle, are openings of perforations inthe epiplasm known as parasomal sacs (Williams & Bakowska, 1982). Theirvisualization in these preparations is variable, even in OAs from wild-type cells.Epiplasmic ridges are located between membranelles, especially M2 and M3(Fig. 12, r; cf. Smith, 1982). This feature has not been noted before in suchpreparations, but is fairly regularly seen, and is useful for distinguishing betweenseparate membranelles and interrupted portions of a single membranelle.

The preparations of isolated OAs allow assessment of the dimensions of mem-branelles by counting of basal bodies. The longer dimension of the membranellesis termed 'length' and is tallied as number of basal body columns (indicated bynumerals in Fig. 11), while the shorter dimension is the 'width' and is countedas the number of basal bodies per column, i.e. number of rows (indicated by theletters a, b, and c in Fig. 11). Length assessed in this manner includes thesculptured region, a convention different from that of Bakowska et al. (1982a),in which the regions of unmodified and modified (sculptured) columns weretallied separately. The basal bodies designated x and y in Fig. 11 are those of ashort additional row formed anterior to row a at the right ends of the mem-branelles (see Fig. 1C and accompanying text). Basal body y appears anterior tocolumn 1, while basal body x is the sole remnant of a column situated to the rightof column 1 of Ml, which is resorbed before the end of oral development (Fig.1C).*

The mpD, big, and psm mutations all increase the length of Ml and M2, whileleaving their width at the standard value of 3 (Figs 13-20); very rarely, a shortectopic fourth row of basal bodies is observed in OAs oipsmAl (Fig. 21). Theincrease in length of M2 is modest and monomodal in 3-membranelled OAs ofmpD cells, greater and also monomodal in OAs of big cells, and much more

* It is possible that this transient basal body column of Ml corresponds to the columnnumbered 1 in M2 and M3, in which case the column-designations for Ml should be increasedby one. This is not done here because of indications of short-lived basal body columns at theright end of developing M2 and M3 as well (Bakowska et al. 1982b; Lansing et al. 1984).


Table 3. Length of membranelle 2 in 3-membranelled OAs of wild-type andmutant cells*

Genotype

WTmpDbigpsmAlpsmAlpsmBpsmC

rTempera-ture (°G) 11

18-38°t 628-30°§28°23°28°28-+36-5128^36-51

12

272

22

13

1813

12

65

14

1152

11

15

3111222

16

621

1

Lengthf of M2

17

1212

18

2

2

19

1

23

20

1

2

21-25

411

26-30

4

1

31-40

5

41-50

4

51-60

1

* All data are from OAs of cells grown in PPY medium and harvested in late exponentialphase of culture growth.

t The length is equal to the total number of basal body columns, including modified columnsin the sculptured region. For further explanation, see the text.

$ Combined data from OAs of cells from five separate cultures, grown at 18 °C, 28 °C, 36 °C,36-5°C, and 38°C respectively on PPY medium. Differences in size of OAs among thesecultures are negligible.

§ Combined data from 3-membranelled OAs of cells from two cultures, one grown at 28 °Cand the other at approximately 30 °C.

1f Data from OAs of cells grown to late-exponential phase at 28 °C, followed by the final 2 h(psmB) or 2V2h (psmC) at 36-5°C.

variable in OAs oipsm cells (Table 3). In psmAl at 28 °C, the longest M2s arefourfold the usual wild-type length. Similar relationships are observed for Ml(data not shown).

When lengths of both Ml and M2 are assessed in the same OA, their mutualrelationship can be evaluated. This is shown for 3-membranelled OAs of wild-type, mpD, and big cells in Fig. 9A, and for OAs oipsm mutants (on a com-pressed scale) in Fig. 9B. Three conclusions can be drawn from these plots. First,there is a clear-cut and strong association between the lengths of Ml and M2.Second, the relationship between Ml and M2 is not influenced by the genotypeof the cells: where values of M2 are the same, the values of Ml are similarirrespective of genotype (which is why all the data points could not be seen ona single plot). Third, no single linear regression fits the entire range of values ofMl and M2; even within the wild-type group, analysis of covariance reveals asignificant difference of adjusted means between the distinct subset of small 3-membranelled OAs from starved cells and the remaining normal-sized OAs.f

t In the preceding study (Bakowska et at. 1982a), a single regression line was drawn throughboth sub-groups (Fig. 6A). Reanalysis of the expanded data-set that is now available indicatesthat a single straight line may not be justified.


I I I I I I 1 _

\

I i | | I I I L I I I

00


Table 4. Length of the undulating membrane in 3-membranelled OAs of wild-typeand mutant cells*

Genotype

WTmpDbigpsmAl (23°)psmAl (28°)psmBpsmC

r

22

123

23

11

24

221

25

53212

2

Lengtht of the UM

26

133

2

27

221

1

28

23

1

29

1

30

1

31- 36- 41-35 40 45

2 2

* From the same cultures as the data in Table 3.t The length is equal to the number of sequential ciliated basal bodies from one end of the

UM to the other.

Comparison of the data points to an arbitrary reference line set at Ml = M2 (Fig.9A and 9B, dotted line) suggests that the overall M1-M2 relationship is cur-vilinear. This global curvilinearity is not inconsistent with a near-linear relation-ship within a restricted range of values of Ml and M2; for example, a linearregression computed for the combined mpD and big data (Fig. 9, dashed line)provides an excellent fit for values of Ml between 16 and 22, but fails whenextrapolated outside of this range.

(b) Imperfect coordination of the length of the undulating membrane and mem-branelles

The undulating membrane tends to be somewhat larger in mutant than in wild-type OAs (Table 4). However, 3-membranelled OAs of mpD and big cells havea similar range of UM lengths (Table 4) despite clearly different lengths of M2(Table 3) and Ml (Fig. 9A). UMs of length exceeding 30 have been found onlyin psmAl OAs, where they may be under-represented due to the tendency of thelongest UMs to be lost in preparation.

Fig. 9. Mutual relationship of length of Ml and M2, assessed in terms of number ofbasal bodies, in 3-membranelled OAs from cells of (A) wild-type (•) , mpD ( •) , andbig (A) genotypes, all at 28°C, and (B) psmAl at 23 °C (O), psmAl at 28°C (•) ,psmB 2 h after a shift from 28 ° to 36-5 °C (n), and psmC 2\ h after a shift from 28 °to 36-5°C (O). The dotted line in both graphs is an arbitrary reference line ofequality of the two variates (Ml = M2). The dashed line in (A) gives the best-fitlinear regression computed for the mpD and big OAs. Note the extreme compressionof the scale in (B) compared to (A). The points of (A) would fit in the shaded regionof(B).

54

30

28

26

s2 24ox:

I

20

18

1 6

J . F R A N K E L A N D O T H E R S

O

B 9 A A A

B» $) • A O

ffl E C» • A

I I D

§ B • «1 A

a

D B •

Ba

i i i i i i i i i i i i i

10 12 14 16

Length of M218 20 22 24

UM

40

30

20

10

I

20M2

30

Fig. 10. Mutual relationship of length, assessed in terms of number of basal bodies,of the UM and M2 in 3-membranelled OAs from wild-type and mutant cells. Thesymbols have the same meaning as in Fig. 9. The main graph (A) includes all of thedata points except two, which are added to a reduced version of the same graph withextended ordinate and abscissa (B).


The association between the length of the UM and that of M2 (Fig. 10) is clearlymuch less close than that between Ml and M2; indeed, among 3-membranelledOAs of mpD and big cells, the length of the UM is not significantly correlatedwith that of M2. The scanty data for the OAs oipsm cells suggest a similar weakrelationship between UM and M2 lengths among moderately enlarged OAs, butimply a substantial increase of UM lengths in extremely large OAs (note the two

*• «c

M-l

CMDM-2

cM-2

eM-2

M-3 eM-3

Fig. 11. Patterns of sculpturing of membranelles. Conventions, except for shading,are the same as in Fig. 1B,C. The numbers within the basal bodies of the top rowidentify the basal body columns, proceeding from the cell's right end (viewer's left)of each membranelle to its left. The anterior member of each set of three basal bodies(i.e. of each column) is designated a, the middle one b, and the posterior memberc, creating a coordinate system for identification of basal bodies, x and y are the'fourth row' basal bodies. Membranelle 3 (M-3) is shown complete, while only theright-most six columns are shown for M-l and M-2.

The diagrams on the left show the normal sculpturing patterns of the mem-branelles, while the central diagrams, prefixed with an 'e', indicate the correspondingextended patterns, with posterior displacement of certain additional basal bodies(shaded). 'eM-2' actually indicates three distinct patterns, since basal bodies 3b and4c may be displaced separately or jointly (as shown). The 'cM-2' pattern is charac-terized by ciliation of the basal bodies of the first column plus basal body y (andpersistence of basal body x), but otherwise resembles the M-2 pattern.


points at the upper-right corner of Fig. 10B). The difficulty of obtaining unbiaseddata on UM lengths makes it difficult to draw unequivocal conclusions; however,it appears as if much of the variation in UM length is generated by causesunrelated to membranelle length.

(c) Limited variation in patterns of sculpturing of membranelles

Despite substantial increases in length of membranelles brought about by themutations under consideration, the distinctive sculptured patterns of the mem-branelles are affected only modestly. The normal patterns and the modificationsmost commonly observed in 3-membranelled OAs are shown schematically inFig. 11 and are documented in Figs 12 to 21. In wild-type cells grown at tem-peratures between 18° and 37 °C (Fig. 12) and in 3-membranelled mpD cells

Figs 12-16. Isolated OAs from cells lysed following growth in PPY. All photographsare oriented so that the cell's left edge of the OA corresponds to the viewer's right.The UM is thus to the viewer's left, the membranelles to the viewer's right, with Mlalways most anterior (up) and M3 most posterior (down). Arrowheads refer to thestate of ciliation of basal bodies, straight arrows indicate basal bodies displaced lessthan normal, while wavy arrows indicate basal bodies displaced more than normal.The membranelles are individually labelled (Ml, Ml, M3) as is the anterior end ofthe undulating membrane (UM). The posterior portion of the UM is frequentlydisplaced or broken off in whole or part. Scale bars indicate 1 /mi.

Fig. 12. A typical OA from a wild-type cell grown at 36-5 °C. All ciliated basalbodies are visible, except for the y basal body of Ml and a few basal bodies at the leftend of Ml and possibly of M2, which are covered by folds of the surface lamina(epiplasm). Most of the preparation is seen in external view, with ciliated basalbodies visible as thick-walled rings. Unciliated basal bodies of column 1 of M2(arrowheads) are barely visible as much thinner rings. Row a and the left end of rowb of Ml are seen in side view owing to the folding over of the epiplasmic border ofthe OA. Note epiplasmic ridges (r) between membranelles. Compare with Figs IBand 11 (left column).

Fig. 13. An OA from a big cell grown at 28 °C. Note the similarity to wild-type inarrangement of basal bodies, despite the difference in length of Ml and M2.

Fig. 14. Another OA from a big cell grown at 28 °C in PPY. The UM is raised ona wedge of epiplasm, and its basal bodies are seen mostly in side view, obscuring mostof M3. Ml and M2 show extended sculpturing, with basal body 3c of Ml and 4c ofM2 (wavy arrows) displaced posteriorly; compare with the eM-1 and eM-2 diagramsin Fig. 11, in which these basal bodies are stippled.

Fig. 15. An OA from apsmB cell maintained for 2 h at 36-5 °C. The posterior halfof the UM is elevated, obscuring most of M3. Ml is unsculptured, with the 1c and2c basal bodies (arrows) not posteriorly displaced (basal bodies x and y are notvisible, probably covered by an epiplasmic flap). Sculpturing of M2 is reduced, withbasal bodies 2b, 2c, and 3c (arrows) displaced much less than normal (compare withFigs 12, 13).

Fig. 16. An OA from apsmB cell maintained for 2h at 36-5 °C. The sculpturedpattern of M2 is highly abnormal, with simultaneously reduced displacement of basalbodies 2b, 2c, and 3c (arrows) and extended sculpturing due to anomalous displace-ment of basal bodies 3b and 4c (wavy arrows). In addition, basal body la isanomalously ciliated (arrowhead). The sculpturing of M3 is extended, probably dueto displacement of basal body 4c (wavy arrow, compare with the stippled basal bodyin the eM-3 pattern in Fig. 11).



psmAI

UM

M3

17.

psmAI

Mutations affecting intracellular patterns in Tetrahymena 59(Frankel et al. 1984, Fig. 9) sculpturing is virtually invariant, manifesting thestandard patterns shown in Fig. IB and in the left column of Fig. 11. Such normalpatterns predominate in the other mutants as well (e.g. Figs 13,17), but modifi-cations are fairly common (Table 5). The most frequent modifications, found in3-membranelled OAs of all mutants except mpD, involve individual displace-ment of the 3c basal body in Ml and of the 4c basal body in M2 or M3 (Fig. 11,centre). The displacement of the 4c basal body has the effect of extending thesculptured region by one column to the cell's left, for which reason we call it'extended' sculpturing. Examples are shown in Fig. 14 for Ml, Figs 14, 19, 20,and 21 for M2, and Fig. 16 for M3. In M2, the 3b basal body may be displacedas well (Fig. 19). More rarely, a subnormal displacement of basal bodies isobserved, which we call 'reduced' sculpturing (Fig. 15). Reduced and extendedsculpturing may occur together (Fig. 16).

While expression of the extended sculpturing pattern is not mutation specific(Table 5), two other abnormalities appear to be peculiar to OAs oipsm mutants,in particular psmAl. One of these is a variable shape of the cell's right end of Ml(Fig. 21), while the other is ciliation of the normally unciliated basal bodies ofcolumn 1 of M2 (Fig. 11, right, and 18). Both of these abnormalities are mostcommon in the very large OAs oipsmAl cells.

Although the data are insufficient for meaningful statistical assessment, wehave a strong impression that the degree and type of abnormality are not in-dependently determined within separate membranelles of each OA; cases ofextended sculpturing (Fig. 14) or reduced sculpturing (Fig. 15) in more than onemembranelle of a single OA are sufficiently frequent to suggest coordination ofsculpturing processes within OAs.

The frequency of abnormalities of sculpturing of M2 in 3-membranelled OAsseems to be associated with membranelle length. This is clearest in comparisonsacross mutants: abnormalities are least frequent in mpD, more frequent in big,

Figs 17-18. Isolated OAs from psmAl cells lysed following growth in PPY at 28 °C.The orientation of these photographs is the same as in Figs 12-16. Symbols have thesame meaning as in Figs 12-16; in addition, broad open arrows indicate places wherebasal bodies are partially obscured. Scale bars indicate 1/itn.

Fig. 17. A psmAl OA with great elongation of Ml, M2 and UM combined witha nearly normal membranelle sculpturing pattern. The sculptured ends of Ml and M2are almost completely visible, and normal. M3 is of normal size but somewhatabnormal pattern. The UM has been torn loose from the remainder of the prepara-tion. The preparation is highly flattened, which may account for fading of some basalbodies located in regions where basal bodies are normally situated in depressions ofthe surface (open arrows). Cilia are retained at the left ends of Ml and M2.

Fig. 18. A psmAl OA with a normally sculptured Ml and an M2 with a cM-2sculpturing pattern (cf. Fig. 11). Basal bodies y, la, lb and lc of M2, invisible inmany preparations, are all ciliated (arrowheads). This preparation is extremelyflattened, with fading of basal bodies at the same positions as in Fig. 17 (single openarrows) and also at the position usually occupied by the epiplasmic flap overhangingMl (double open arrows, also in Figs 19, 21).


. psmAI

Mutations affecting intracellular patterns in Tetrahymena 61Table 5. Sculpturing of membranelle 2 in 3-membranelled OAs of wild-type and

mutant cells*

Genotype

WTmpDbigpsmAl (23°;psmAl (28°;psmBpsmC

r

Normal

1122620

I 9) 15

1413

Reduced

1

11

t3b

1

12

State of sculpturing of M2K

ExtendedfA.

4c 3b + 4c

1

4 1

5 31 21 1

Ciliated^

52

(1)

Otherwisemodified§

2241241

Percentmodified

37

3310524432

* From the same samples as the data in Tables 3 and 4, except that wild-type data are fromOAs of cells grown in PP and PPYGFe as well as PPY medium, at 36-5 °C or below.

t '5b' refers to posterior displacement of the basal body designated as 3b in fig. 11; l4c" refersto posterior displacement of the basal body designated as 4c. l3b + 4c' refers to the posteriordisplacement of both of these basal bodies in the same membranelle, as illustrated in theeM-2 diagram in Fig. 11.

$The cM-2 pattern illustrated in Fig. 11. In psmAl OAs, all of the column-1 basal bodiesare ciliated, while in the psmB OAs, some are. The single psmC example is in an OA that isotherwise highly abnormal (hence in parentheses).

§ Includes combinations of the tabulated modifications (including, for example, M2 of theOA illustrated in Fig. 16).

and most frequent in psmAl at 28 °C (Table 5). However, assessment of correla-tions within mutant clones gives a more mixed picture: in psm clones there is adefinite positive association of frequency of sculpturing abnormalities with size

Figs 19-21. Isolated OAs from psmAl cells lysed following growth in PPY at 28 °C.The orientation of these photographs is the same as in Figs 12-18. Symbols have thesame meaning as in Figs 17-18; in addition, broad solid arrows indicate regions ofputative ciliary-unit resorption or a membranelle-fragment left over from suchresorption. Scale bars indicate ljum.

Fig. 19. A.psmAl OA with extended sculpturing of M2 (displaced basal bodies 3band 4c indicated by wavy arrows) and a split M3. The broad solid arrow indicates agap between a somewhat modified version of the typical M3 pattern, with basal bodyla ciliated (arrowhead), and a membranelle-fragment with a regular row-and-column organization. There is no sign of fading of basal bodies on either side of theregion indicated by the broad solid arrow, suggesting that it is an area in which basalbodies have been resorbed in vivo.

Fig. 20. A psmAl O A with normal sculpturing of Ml, extended sculpturing of M2with displacement of basal body 4c (wavy arrow), and a partially interrupted M3. AnM3-like sculpturing pattern is discernible at the right end of M3, but the region of basalbody resorption (broad solid arrow) is well to its left. The portion of M3 to the left of thisregion is poorly organized, due only in part to tearing of the preparation in this region.

Fig. 21. A psmAl OA with modification of the right end of Ml, extended sculp-turing of M2 with a highly displaced 4c basal body (wavy arrow), and a large gap inM3. A disorganized membranelle fragment (broad solid arrow) is seen to the cell'sleft of the gap. The UM has been lost from this preparation. The 'e' indicates fourextra basal bodies, of an ectopic fourth-row segment of M2.

EMB82


of the O A, while within the big clone there is no such significant association (datanot shown).

(d) The role of ciliary-unit resorption in the patterning of the third membranelle

Membranelle 3 is characterized by a remarkably invariant arrangement of 12ciliated basal bodies (Figs 12,13) that bears scant testimony to its developmentalorigin from a membranelle prototype resembling Ml and M2 (see Fig. 1C). Asthe length of Ml and M2 in 3-membranelled OAs increases, M3 shows no parallelincrease: it either retains its standard size and pattern, as in the big OA depictedin Fig. 13, or occasionally undergoes a modest increase in size that typically isassociated with extended sculpturing (Fig. 16).

Even in psmAl, very long Mis and M2s are commonly accompanied by M3sof normal size and only slightly abnormal pattern (Figs 17, 18; also fig. 4k ofFrankel, 1983). However, in one half of the psmAl OAs with very long Ml andM2, M3 is also strikingly elongated, but in an unusual manner: a gap, sometimespartial (Fig. 20) but more usually complete (Figs 19, 21) is observed within M3.This gap is located adjacent to the typical M-3 configuration in all cases observed(e.g. Figs 19, 21), except the one shown in Fig. 20. The remainder of M3 on theother side of the gap sometimes shows the well-defined row-and-column or-ganization characteristic of Ml and M2 (Fig. 19), but more commonly is variablydisorganized (Figs 20, 21).

An interrupted M3 was commonly observed in silver-stained completed OAsof psmAl (Fig. 8), indicating that the pattern observed in Figs 19 and 21 is notan artifact of sample preparation for SEM. In contrast, M3 is almost invariablylong and continuous in stage 4 and 5 developing oral primordia seen on the samesilver preparations (Fig. 7). This indicates that the gap that appears within themature M3 of the large psmAl OAs must be a consequence of ciliary-unitregression, rather than failure of initial development.

DISCUSSION

The increase in size of the oral apparatus (OA) of Tetrahymena thermophilabrought about by any of five mutations has differential effects on spatial or-ganization of ciliary units that are comparable to the previously reported effectsof decrease of size brought about by starvation (Bakowska et al. 1982a). In bothsituations, the lengths of the ciliary arrays (membranelles and undulating mem-brane) change while their widths remain the same; the lengths of Ml and M2 arecoordinately regulated while the length of the UM is less closely coordinatedwith that of M2 and presumably Ml; finally, the patterns of sculpturing of theright ends of the membranelles are not severely affected. The preservation ofthese relationships when change is in opposite directions (increase of size in onecase, decrease in the other) strengthens the conclusions of the earlier study on


starved cells (Bakowska et al. 1982a). These are, (1) the formation of mem-branelles is tightly integrated whereas development of the UM is a partiallyindependent process, (2) modification of membranelle size takes place primarilythrough changes in number of basal-body couplets recruited into promem-branelles, and (3) the spatial extent of sculpturing of these membranelles islargely independent of the number of couplets thus recruited. These conclusionsare fully in accord with results of detailed studies on oral development in OAsof wild-type cells (Bakowska et al. 1982b) and of a 'misaligned undulating mem-brane' mutant (Lansing et al. 1984), which show that the formation and thesculpturing of membranelles take place at difference times during oral develop-ment, and that development of the undulating membrane not only differs greatlyfrom that of the membranelles (Bakowska et al. 19826) but also can be modified

C} O O O <3:m.w*MS>®-.:®:;:©:Q O Oo o o ommmmMMmmm, o o

Fig. 22. A model for control of the size of M3. The circles indicate the basal bodiesof M3 at stage 5d of oral development (see Fig. 1C), prior to sculpturing and resorp-tion. The basal-body columns are numbered following the same convention as in Fig.11. The stippled zone is the region within which all basal bodies are resorbed nearthe end of stage 5. The bracketed lengths indicate the number of basal body columnsthat may be formed during the initial development of M3 in OAs of (A) starved wild-type cells, (B) growing wild-type cells, (C) growing cells of mutants such as big andmost psms, and (D) psmAl cells with very large OAs. In each case a 'standard' M3is formed despite continuous variation in the original number of basal bodies, exceptwhen the original M3 is so long that it extends beyond the cell's left end of the zonedestined for resorption.


substantially while development of the membranelles remains virtually un-altered (Lansing etal. 1984).

The most novel observation in this study is of the enlarged and interruptedM3 patterns observed in several of the largest OAs from psmAl cells. Thisenlargement is an exception to the normal size- and pattern-constancy of M3,observed both in enlarged OAs of mutants and in diminished OAs of starvedcells. The exception, however, sheds some light on how the normal constancy isachieved. In normal oral development, there is 'evidence for occasional resorp-tion of one or two basal body columns at the left end of M3' (Bakowska et al.19826), while there is no indication of comparable resorption at the left end ofMl or M2. Thus, during the initial development of M3 an excess of basal-bodycolumns is normally produced, which subsequently is pruned by a spatiallylocalized resorption activity. The final pattern of M3 presumably can remainconstant during starvation because what is diminished is the number of surplusciliary units otherwise destined for elimination. Increasing the length of M3above normal increases the surplus, which can be eliminated completely — upto a point. What is most revealing is the membranelle geometry that emergeswhen the capacity for elimination of surplus ciliary units is finally exceeded. Thefact that M3 is then bipartite, with two surviving portions flanking a central gap,suggests that the zone of resorption activity is roughly wedge shaped, with a leftas well as a right margin. This idea is illustrated schematically in Fig. 22. Itsessence is that the zone of resorption activity is positionally specified in somereasonably precise manner, analogous to the localization of the 'posteriornecrotic zone' of cell death in the chick limb bud (Saunders, 1967). This idea willbe elaborated further, with additional experimental support, in the subsequentpaper (Frankel et al. 1984).

This study revealed relatively little novelty in the modifications of sculpturingof the right ends of the membranelles. This was somewhat surprising, sincemutants were used, and other mutations affecting membranelle patterns, mpAand mpB, bring about drastic and apparently random abnormalities of sculptur-ing (Frankel, 1983). Although abnormalities of sculpturing of membranelles arecertainly more common among the 3-membranelled OAs of the mutants con-sidered in this study (mpD excepted) than they are in growing wild-type cells, thedifference is mostly one of frequency rather than kind. Thus, abnormalitieswhich here are given names have previously been documented in photographsof OAs from wild-type cells: extended sculpturing through displacement of basalbody 4c (and 4b) of M2 in figure 5 of Bakowska et al. (1982a), displacement ofbasal body 3b of M2 in figure 7, and reduced sculpturing of Ml in figures 8 and9 of that paper. Only the cM-2 pattern of M2 (Fig. 11) and some variability inshape of Ml are new to this study, and these were both found only in extremelylarge OAs produced by oral replacement in psm cells.

This lack of novelty, however, obtains only when analysis is restricted to OAsthat possess the usual three membranelles. When there are four or more

Mutations affecting intracellular patterns in Tetrahymena 65membranelles, new sculpturing patterns appear that are not found in 3-membra-nelled OAs of the same genotype (Frankel et al. 1984). Since these new patternsare observed in mutant cells possessing OAs in which the length as well as thenumber of membranelles is increased, it is imperative to dissect the specificeffects of increased number of membranelles from the background effects dueto the presence of a mutation and the increased size of the individual mem-branelles. The present study has shown that this background effect, especially forthe most informative mutant (mpD), is minimal.

The majority of the linkage analysis oipsmAl, psmB, and psmC was carried out by ElaineMartel. Drawings were executed by Mary Thorson. The authors also thank Drs Anne W. K.Frankel, Stephen F. Ng, and Dennis Summerbell, as well as Mr Timothy Lansing, for theircomments and criticisms. This research was supported by grant HD-08485 from the U.S.National Institutes of Health.

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(Accepted 22 March 1984)

Documents

Mutational analysi osf patterning of oral structures in Tetrahymena · Department of Biology, University of Iowa, Iowa City, 52242, Iowa U.S.A. SUMMARY The oral apparatus (OA) of