/. Embryol. exp. Morph. 82, 41-66 (1984)
Printed in Great Britain © The Company of Biologists Limited 1984
41
Mutational analysis of patterning of oral structures
in Tetrahymena
I. Effects of increased size on organization
By JOSEPH FRANKEL, LESLIE M. JENKINS, JULITA
BAKOWSKA 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 four
ordered arrays of ciliary units. In wild-type cells, these arrays are constant in spatial organization and vary little in size except during extreme starvation. Recessive mutations atfivegene
loci are known to increase the size of the OA. They do this by increasing the length of the
ciliary arrays, without affecting their width and often without increasing their number beyond
the usual four. Comparison of the oral arrays over a large range of sizes has revealed: (1) that
the lengths of the anterior two of three parallel arrays (membranelles) are rather tightly
coordinated; (2) that the specific basal body configurations resulting from remodelling of the
membranelles 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 largest
OAs in which the structure is lengthened but interrupted by a gap in the middle. This gap may
reveal the spatial extent of a putative zone of basal body regression. These phenomena are not
specific to any of the genotypes utilized in this investigation; the effect of the mutations is to
loosen quantitative restrictions and thus reveal underlying associations and constraints.
INTRODUCTION
The intracellular pattern of ciliary units in the oral apparatus of Tetrahymena
thermophila is useful for analysis because it is readily described quantitatively,
its development can easily be observed, and it is subject to environmentally and
genetically provoked variation. The organization of this system is at the same
time remarkably complex and surprisingly invariant in growing wild-type cells
(Williams & Bakowska, 1982; Bakowska, Frankel & Nelsen, 1982a). The nature
of this organization can be probed by provoking major changes in the size of the
system. Thus, severe starvation reduces the length of the ciliary arrays within the
oral apparatus without greatly affecting their organization; there is evidence for
alteration of organization only when the number as well as the length of these
arrays is reduced (Bakowska et al. 1982a). Substantial increase in the size of the
oral apparatus cannot be brought about reliably by any nutritional regimen of
which we are aware, but it is elicited by any of several genie mutations. This has
permitted an analysis of the response of oral patterns to an extended variation
in 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 the
absence of any increase in their number. This will also allow us to isolate analytically the unique effects of change in the number of these arrays, which is the
subject of the next paper. In both papers, phenotypic changes generated by
mutations are regarded as windows through which we may perceive underlying
associations and constraints that guide normal development.
MATERIALS AND METHODS
Stocks
All stocks used in this study were Tetrahymena thermophila of the inbred B
strain (Allen & Gibson, 1973, Table 2). The wild-type cells were of stock B-2079
(20th generation of inbreeding, established in 1979). Information about the
mutant stocks utilized in this and the companion investigation is summarized in
Table 1. The protocol for mutagenesis was the same as described earlier (Frankel, Jenkins, Doerder & Nelsen, 1976), with the substitution of 0-75 % ethylmethane sulfonate (EMS) for nitrosoguanidine in one case. Mutations were
brought to expression either as heterozygotes by macronuclear allelic assortment
(Carlson, 1971; Frankel et al. 1976) or as homozygotes by 'cytogamy', i.e. induced self-fertilization (Orias, Hamilton & Flacks, 1979; Sanford & Orias,
1981). Following either of these procedures, cells were isolated into microdrops
by the 'Poisson lottery' procedure (Orias & Bruns, 1976) and replicated by the
method of Roberts & Orias (1973) into microtitre plates. Screening was for
morphological abnormalities at 39-5°C as described by Frankel et al. (1976).
Subsequent genetic analysis was carried out following procedures originally
introduced by Nanney, as described in Frankel et al. (1976). Localization of
mutations to chromosome arms was carried out by crosses to germinal nullisomics
(Bruns & Brussard, 1981; Bruns, Brussard & Merriam, 1983), with the requisite
stocks (Bruns, Brussard & Merriam, 1982) kindly provided by Dr Peter Bruns.
Assessment of linkage among psm mutations was by the interrupted genomic
exclusion method of Ares & Bruns (1978).
Table 1. Mutant stocks employed in this investigation
Stock
number
Mutation
Mating type
Mutagen
Method
Year isolated
IA-309
IA-317
mpCl
mpC2
VII
VII
Cytogamy
Cytogamy
1981
1982
IA-305
IA-313
IA-223
IA-246
IA-157
IA-235
mpD
big
psmAl
psmA2
psmB
psmC
IV
II
IV
II
IV
II
Nitrosoguanidine
Ethyl-methanesulfonate (EMS)
Nitrosoguanidine
Nitrosoguanidine
Nitrosoguanidine
Nitrosoguanidine
Nitrosoguanidine
Nitrosoguanidine
Cytogamy
Cytogamy
Assortment
Cytogamy
Assortment
Cytogamy
1981
1981
1974
1979
1977
1979
Mutations affecting intracellular patterns in Tetrahymena
43
Media and growth conditions
Cells were grown in one of three different peptone-based culture media
described by Nelsen, Frankel & Martel (1981), with all of the analyses of mutants
carried out in the 2 % proteose peptone - 0-5 % yeast extract (PPY) medium.
Temperatures of culture growth ranged from 18 ° to 38 °C. Procedures for growth
of mass cultures were the same as described in Frankel, Mohler & Frankel
(1980), except that cultures for oral isolation were shaken continuously during
growth, 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 (see
Results), in which the flasks were not shaken and were maintained under conditions of poor temperature control, hence in these two cases the temperatures
indicated 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 mM
EDTA, 0-1% 2-mercaptoethanol, lOmM-Tris, final pH9-3) plus one to two
drops of a 10 % Triton-XlOO solution as an underlayer during centrifugal collection of oral apparatuses. All solutions were filtered carefully prior to use. Following centrifugation, the supernatant was aspirated, and the remaining pellet
suspended in about two drops of distilled water, and then fixed for 2-5 min in
1-2 ml of cold 1 % OsO4. This suspension was then diluted with 50 % ethanol,
centrifuged, and resuspended in 100 % ethanol. It was prepared for scanning
electron microscopy as described earlier (Bakowska et al. 1982a).
Chatton-Lwoff silver impregnation of whole fixed cells and their measurements 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 use
of regression procedures with data such as ours, which fit the conditions
described 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 around
ciliary units embedded within a triton-insoluble lamina (the epiplasm) located
directly underneath the cell membrane (Williams & Bakowska, 1982). Eighteen
to 21 longitudinal ciliary rows are uniformly spread over the cell surface. A
specialized feeding structure, the oral apparatus (OA), consisting in part of
44
J. FRANKEL AND OTHERS
modified ciliary units spaced close together, is situated near the anterior end of
the cell (Fig. 1A). The OA consists of four compound ciliary ensembles, namely
one undulating membrane (UM) and three membranelles (Fig. IB). The UM is
OA
UM QY
a-'
a-'
OA,
M2
M3
Oo
0
oooo
©
0
0
©
O° °OO- 0°^
OO1'"
O0° C
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0
PI
la
00
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on o o
O ° Q O 00
4a '"' °
lb
opPoo
' O '"-' OOOn'AO. - O rfi
o
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o
o
o,V
a'
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do
oo
oo
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88g
°6
OCA
'©00
% . .
5a-b
5d
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OO
4b o o o o'-'oo
5f-6
Mutations affecting intracellular patterns in Tetrahymena
45
made up of two parallel rows of basal bodies, the outer one ciliated and the inner
one unciliated. The anterior two membranelles (Ml and M2 respectively) consist
primarily of three rows of basal bodies. At the cell's right end of these rows the
basal bodies are displaced into a 'sculptured' pattern that is characteristic for
each membranelle, and is virtually invariant (Bakowska et al. 1982a). The third
membranelle (M3) appears to be sculptured in toto, so that the original parallel
arrangement of basal body triplets is obscured. Within the membranelles, all
basal bodies are ciliated except a few at the left end of Ml and the right-most
basal bodies in the sculptured region of M2 and M3.
The OA develops from an oral primordium, which in growing cells normally
appears 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 in
Fig. 1C. A field of basal bodies is produced (stage 1), from which oriented
couplets of basal bodies are generated (stage 2). These couplets line up side by
side to form two-rowed promembranelles (stages 3 and 4a). A third basal body
is then generated anterior to each couplet (stage 4b), converting these couplets
into columns of three basal bodies each. A fourth basal body is added anterior
to the one or two columns at the right end of each membranelle (stage 5). These
events create membranelle prototypes of nearly rectangular organization, with
row-and-column organization that becomes slightly tilted to generate a
hexagonal close packing. This simple form is modified near the end of stage 5 by
a combination of three processes, namely ciliary regression, basal body resorption, and ciliary unit displacement. These processes generate the characteristic
sculpturing of the right ends of the membranelles and a notch at the anterior-left
end of Ml. This remodelling during late oral development (described in detail
by Bakowska, Nelsen & Frankel, 1982b) endows each membranelle with a
unique pattern signature which is distinguishable at a glance when the final
structure 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 thermophila. (A) A sketch of the arrangement of basal bodies on the ventral surface of a cell
entering oral development. Each dot indicates a basal body; cilia are omitted. Seven
ciliary rows (CR) are shown, as well as the anterior oral apparatus (OA) and a
midbody oral primordium (OP). (B) A more detailed view of the arrangement of
basal bodies in the oral apparatus of wild-type cells. Closed circles indicate ciliated
basal bodies, while dashed circles show unciliated basal bodies. Ml, M2, and M3 are
membranelles 1,2, and 3 respectively; UM is the undulating membrane. (C) Stages
of midbody oral development. Each of the ten sketches shows a progressively later
stage of oral development. Symbols are as in (B), with a central dot within circles
indicating basal bodies of the right-postoral (stomatogenic) ciliary row, stippling of
circles indicating basal bodies soon to be resorbed. The dotted lines connecting
circles in the last two diagrams indicate the probable pathways of basal-body
displacement during the sculpturing process. The designation of substages of stages
4 and 5 follows Lansing et al. (1984). 'PI' signifies 'pre-1' (Nelsen et al. 1981). For
further explanation, see the text.
46
J. FRANKEL AND OTHERS
complex (see Nelsen, 1981; Bakowska etal. 1982ft; Lansing, Frankel & Jenkins,
1984), but the essential feature of importance here is that a pro-UM develops
during stage 4 by an alignment of single basal bodies at the right edge of the oral
primordium, virtually orthogonal to the developing membranelles. The staggered double-row organization of the completed UM is elaborated later.
The typical sequence of midbody oral development illustrated in Fig. 1C is a
prelude to cell division. The completed oral primordium becomes the OA of the
posterior division product, while the OA of the anterior division product is
derived from the pre-existing anterior OA. Tetrahymena also manifests an alternative mode of oral development, called oral replacement (Frankel & Williams,
1973). Here a stage-1 oral primordium is formed anteriorly, in part adjacent to
the anterior end of the right postoral ciliary row and in part from the UM of the
old OA (Frankel, 1969; Kaczanowski, 1976). These two basal body fields normally become fused into a single large field, from which membranelles and UM
develop in the same way as in predivision oral development. However, the OA
thus formed is not segregated into a posterior division product, but instead
replaces the old OA, whose membranelles are resorbed. Oral replacement can
function as a physiological substitute for cell division under conditions not permissive for division (Frankel, 1970; cf. Tartar, 1966; DeTerra, 1969), or as a part
of sequences of morphogenetic transformation to an elongated 'rapid swimmer'
form in Tetrahymena thermophila (Nelsen, 1978) or to 'macrostome' forms in
certain other Tetrahymena species (Williams, 1960; Stone, 1963; Buhse, 1966;
Metenier & Groliere, 1979). In the last-mentioned cases, the new oral apparatus
that 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 nonallelic, except for psmAl and psmA2. psmC, psmB, and psmAl have been
shown, using the methods of Ares & Bruns (1979), to be mutually unlinked.
psmB was previously localized to chromosome arm 4L by Bruns (1982). Using
the same methods and stocks, we have found that psmA and psmC are both on
chromosome 5, while mpD is on chromosome 3R.
(b) Phenotypes
The phenotypes considered here fall into two distinct classes, that of big (and
of 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 unusually large size of homozygous cells (Table 2). big is different from the fat mutations isolated earlier (Frankel et al. 1976; Jenkins & Frankel, unpublished)
because in big, unlike fat, cell length as well as width are substantially increased
Mutations affecting intracellular patterns in Tetrahymena
47
Table 2. Dimensions of cells and of OAs* in wild-type and big
Oral dimensions!
Cell dimensions!
Length
(/an)
Width
(/an)
LW
Gum)
LW
(Mm2)
dm3)
OALW-r
cell LWf
X100
A
A
(
r
Length
Gum)
Width
Genotype
n
WT$
50
49-6
±2-3
24-5
±1-9
1219
±115
9-8
±0-5
6-4
±0-5
62-7
±6-6
5-2%
±0-5%
bigX
44
62-5
+3-9
34-2
+3-5
2153
+308
11-2
+0-6
8-4
+0-6
94-3
+ 10-3
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 late
samples 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 oral
apparatus (Table 2, Figs 4 and 5). Cultures of big cells grow exponentially at
28 °C, with a doubling time 25 to 50% longer than that of parallel wild-type
cultures; in such cultures, about 90% of the cells that are undergoing oral
development are engaged in predivision development with midbody oral
primordia (Figs 2, 3), while the other 10% are carrying out oral replacement.
Roughly 90% of the OAs formed have 3 membranelles (Fig. 4), while the
remainder have short 'extra' membranelles either anterior to Ml or posterior to
M3. 3-membranelled OAs predominate even after a shift to 39-5 °C, a temperature that is near the upper limit for continuous exponential culture growth.
mpD (mp = 'membranellar pattern') is a conditional mutation selected on the
basis of increased cell length and some change in cell shape at 39-5 °C. Cultures
of mpD cells grow approximately as rapidly at 39-5 °C, and probably also at
28 °C, as do wild-type cultures. Oral development in such cultures, at either
temperature, is by the standard predivision mode. At elevated temperatures,
virtually all oral primordia and ensuing OAs possess four or five regular membranelles rather than the usual three (see the accompanying paper). Yet even at
28 °C, when all mpD OAs have the normal 3 membranelles, they are slightly but
significantly larger than in wild-type OAs (see Table 1 of Frankel, Nelsen,
Bakowska & Jenkins, 1984).
Thepsm ('pseudomacrostome') mutations also increase oral and cell size, but
in a different manner than big and mpD. In these mutations, which are all
conditional, the predominant phenotypic effect observed at the restrictive temperature is a change in longitudinal position (Frankel, 1979) and size of oral
primordia. The result is generally a switch, partial or complete, from predivision
to oral replacement development (Figs 6,7). The oral replacement primordia are
48
J. FRANKEL AND OTHERS
typically unusually long (Fig. 6), and often give rise to correspondingly large
OAs (Fig. 8). At the same time cell size increases, presumably because cell
growth 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 dividing
while most are undergoing repeated oral replacement, so that cell number increases slowly while many individual cells become large and sometimes misshapen.
As in big, the 'pseudomacrostome' OAs formed by the psm mutant cells
typically possess the normal complement of 3 membranelles. However, abnormalities are fairly common. These may involve an interrupted M3 (Fig. 8), short
supernumerary anterior membranelle fragments, or even a tandem subdivision
of all or part of the OA into two segments of similar organization but unequal
size, probably due to failure of union of the two oral replacement subfields [as
described 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 permissive temperature for this mutation is 23 °C, or below. By contrast, psmAl
cells show no expression at 28 °C, little at 36-5 °C, and delayed expression following transfer from 28 ° to 39-5 °C. psmB andpsmC (only one allele known for each
locus) are intermediate, with no expression at 28 °C and a rapid onset of
pseudomacrostome-type oral replacement in 20 to 30 % of the cells following a
shift to 36-5 °C. There is also an additional recessive, nitrosoguanidine-induced
mutation, named psmD, located on chromosome 3R, that resembles the other
psm mutations in sometimes bringing about formation of large OAs by oral
replacement at high temperature. This mutation, unlike the other psm mutations, may also express an mp-like phenotype, with extra membranelles present
even 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 oral
primordia (Figs 2,3,6 and 7) or membranelles of mature OAs (Figs 4, 5 and 8). All
of these photographs are printed at the same magnification, with the scale bar, shown
in Fig. 2, indicating lOjum.
Fig. 2. A big cell with a midbody oral primordium (OP) at stage 2. The anterior
oral apparatus (OA) is out of focus.
Fig. 3. A big cell with a midbody oral primordium at stage 5a. The three membranelles are not sculptured at their right ends.
Fig. 4. The anterior portion of a big cell, with a mature OA. The three membranelles (Ml, M2, M3) and the undulating membrane (UM) are labelled. Note the
sculptured right ends of the membranelles.
Fig. 5. The anterior portion of a wild-type cell, with a mature OA. Notice that
while 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 primordium (OP) at stage 3. The membranelles of the old OA are to the cell's left (viewer's
right) 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. Fragments
of regressing old membranelles (RM) are visible to the cell's anterior-left of the oral
replacement primordium.
Fig. 8. A psmAl cell with a mature OA. The three membranelles are labelled.
Note that M3 is split into two parts.
50
J. FRANKEL AND OTHERS
dissociated, they appear to be outcomes of the same mutational lesion. OAs of
this mutation have not been studied in detail because of relatively low penetrance
at 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 preparations
were used for assessment of the number and arrangement of ciliary units in
different parts of the OA. Cilia are mostly detached during preparation at a point
just distal to the basal body, leaving prominent, thick-walled stumps. Nonciliated 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 in
the epiplasm known as parasomal sacs (Williams & Bakowska, 1982). Their
visualization 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 such
preparations, but is fairly regularly seen, and is useful for distinguishing between
separate membranelles and interrupted portions of a single membranelle.
The preparations of isolated OAs allow assessment of the dimensions of membranelles by counting of basal bodies. The longer dimension of the membranelles
is termed 'length' and is tallied as number of basal body columns (indicated by
numerals in Fig. 11), while the shorter dimension is the 'width' and is counted
as the number of basal bodies per column, i.e. number of rows (indicated by the
letters a, b, and c in Fig. 11). Length assessed in this manner includes the
sculptured region, a convention different from that of Bakowska et al. (1982a),
in which the regions of unmodified and modified (sculptured) columns were
tallied separately. The basal bodies designated x and y in Fig. 11 are those of a
short additional row formed anterior to row a at the right ends of the membranelles (see Fig. 1C and accompanying text). Basal body y appears anterior to
column 1, while basal body x is the sole remnant of a column situated to the right
of 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, while
leaving their width at the standard value of 3 (Figs 13-20); very rarely, a short
ectopic fourth row of basal bodies is observed in OAs oipsmAl (Fig. 21). The
increase in length of M2 is modest and monomodal in 3-membranelled OAs of
mpD 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 column
numbered 1 in M2 and M3, in which case the column-designations for Ml should be increased
by one. This is not done here because of indications of short-lived basal body columns at the
right end of developing M2 and M3 as well (Bakowska et al. 1982b; Lansing et al. 1984).
Mutations affecting intracellular patterns in Tetrahymena
51
Table 3. Length of membranelle 2 in 3-membranelled OAs of wild-type and
mutant cells*
Lengthf of M2
TemperaGenotype ture (°G)
WT
mpD
big
psmAl
psmAl
psmB
psmC
r
2 1 - 26- 3 1 - 4 1 - 5 1 -
11 12 13 14 15 16 17 18 19 20 25 30 40 50 60
18-38°t
6 27 18
28-30°§
2
28°
23°
28°
28-+36-51
28^36-51
2
2
13 11
1 5
2
2
6
5
1
1
3
11
1
2
2
2
6
2
1
1
1
2
1
2
2
1
2
2
3
1
2
4
1
1
4
5
4
1
1
* All data are from OAs of cells grown in PPY medium and harvested in late exponential
phase of culture growth.
t The length is equal to the total number of basal body columns, including modified columns
in the sculptured region. For further explanation, see the text.
$ Combined data from OAs of cells fromfiveseparate 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 these
cultures are negligible.
§ Combined data from 3-membranelled OAs of cells from two cultures, one grown at 28 °C
and the other at approximately 30 °C.
1f Data from OAs of cells grown to late-exponential phase at 28 °C, followed by thefinal2 h
(psmB) or 2V2h (psmC) at 36-5°C.
variable in OAs oipsm cells (Table 3). In psmAl at 28 °C, the longest M2s are
fourfold 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 mutual
relationship can be evaluated. This is shown for 3-membranelled OAs of wildtype, mpD, and big cells in Fig. 9A, and for OAs oipsm mutants (on a compressed 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 genotype
of the cells: where values of M2 are the same, the values of Ml are similar
irrespective of genotype (which is why all the data points could not be seen on
a single plot). Third, no single linear regression fits the entire range of values of
Ml and M2; even within the wild-type group, analysis of covariance reveals a
significant difference of adjusted means between the distinct subset of small 3membranelled 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 through
both sub-groups (Fig. 6A). Reanalysis of the expanded data-set that is now available indicates
that a single straight line may not be justified.
52
J. FRANKEL AND OTHERS
I
I
I
I
I
I
1 _
\
I
i
|
|
I
I
I
L
00
I
I
I
Mutations affecting intracellular patterns in Tetrahymena
53
Table 4. Length of the undulating membrane in 3-membranelled OAs of wild-type
and mutant cells*
Lengtht of the UM
r
Genotype
WT
mpD
big
psmAl (23°)
psmAl (28°)
psmB
psmC
22
23
24
25
26
1
2
3
11
2
2
1
5
3
2
1
2
1
3
3
28
2
2
1
2
3
29
30
3135
3640
4145
1
1
2
2
27
2
2
1
1
* 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 curvilinear. This global curvilinearity is not inconsistent with a near-linear relationship within a restricted range of values of Ml and M2; for example, a linear
regression 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 when
extrapolated outside of this range.
(b) Imperfect coordination of the length of the undulating membrane and membranelles
The undulating membrane tends to be somewhat larger in mutant than in wildtype OAs (Table 4). However, 3-membranelled OAs of mpD and big cells have
a 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 only
in psmAl OAs, where they may be under-represented due to the tendency of the
longest UMs to be lost in preparation.
Fig. 9. Mutual relationship of length of Ml and M2, assessed in terms of number of
basal bodies, in 3-membranelled OAs from cells of (A) wild-type (•), mpD (•), and
big (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 of
equality of the two variates (Ml = M2). The dashed line in (A) gives the best-fit
linear regression computed for the mpD and big OAs. Note the extreme compression
of the scale in (B) compared to (A). The points of (A) wouldfitin the shaded region
of(B).
54
J. FRANKEL AND
OTHERS
O
30
28
B 9
26
A
B» $) •
s
ffl E
2 24
o
x:
I I
I
§
B
C» •
A A
A O
A
D
•
«1 A
20
a
18
D
B
•
B
a
16
i
i
i
i
12
10
i
14
16
Length of M2
i
i
18
i
i
20
i
i
22
i
i
24
UM
40
30
20
I
10
20
M2
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. The
symbols have the same meaning as in Fig. 9. The main graph (A) includes all of the
data points except two, which are added to a reduced version of the same graph with
extended ordinate and abscissa (B).
Mutations affecting intracellular patterns in Tetrahymena
55
The association between the length of the UM and that of M2 (Fig. 10) is clearly
much less close than that between Ml and M2; indeed, among 3-membranelled
OAs of mpD and big cells, the length of the UM is not significantly correlated
with that of M2. The scanty data for the OAs oipsm cells suggest a similar weak
relationship between UM and M2 lengths among moderately enlarged OAs, but
imply 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 row
identify 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 member
c, 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 the
right-most six columns are shown for M-l and M-2.
The diagrams on the left show the normal sculpturing patterns of the membranelles, while the central diagrams, prefixed with an 'e', indicate the corresponding
extended patterns, with posterior displacement of certain additional basal bodies
(shaded). 'eM-2' actually indicates three distinct patterns, since basal bodies 3b and
4c may be displaced separately or jointly (as shown). The 'cM-2' pattern is characterized by ciliation of the basal bodies of the first column plus basal body y (and
persistence of basal body x), but otherwise resembles the M-2 pattern.
56
J. FRANKEL AND OTHERS
points at the upper-right corner of Fig. 10B). The difficulty of obtaining unbiased
data 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 causes
unrelated to membranelle length.
(c) Limited variation in patterns of sculpturing of membranelles
Despite substantial increases in length of membranelles brought about by the
mutations under consideration, the distinctive sculptured patterns of the membranelles are affected only modestly. The normal patterns and the modifications
most commonly observed in 3-membranelled OAs are shown schematically in
Fig. 11 and are documented in Figs 12 to 21. In wild-type cells grown at temperatures 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 photographs
are 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 Ml
always most anterior (up) and M3 most posterior (down). Arrowheads refer to the
state of ciliation of basal bodies, straight arrows indicate basal bodies displaced less
than normal, while wavy arrows indicate basal bodies displaced more than normal.
The membranelles are individually labelled (Ml, Ml, M3) as is the anterior end of
the undulating membrane (UM). The posterior portion of the UM is frequently
displaced 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 basal
bodies are visible, except for the y basal body of Ml and a few basal bodies at the left
end 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 basal
bodies 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 row
b of Ml are seen in side view owing to the folding over of the epiplasmic border of
the OA. Note epiplasmic ridges (r) between membranelles. Compare with Figs IB
and 11 (left column).
Fig. 13. An OA from a big cell grown at 28 °C. Note the similarity to wild-type in
arrangement 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 on
a wedge of epiplasm, and its basal bodies are seen mostly in side view, obscuring most
of M3. Ml and M2 show extended sculpturing, with basal body 3c of Ml and 4c of
M2 (wavy arrows) displaced posteriorly; compare with the eM-1 and eM-2 diagrams
in 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 half
of the UM is elevated, obscuring most of M3. Ml is unsculptured, with the 1c and
2c basal bodies (arrows) not posteriorly displaced (basal bodies x and y are not
visible, probably covered by an epiplasmic flap). Sculpturing of M2 is reduced, with
basal bodies 2b, 2c, and 3c (arrows) displaced much less than normal (compare with
Figs 12, 13).
Fig. 16. An OA from apsmB cell maintained for 2h at 36-5 °C. The sculptured
pattern of M2 is highly abnormal, with simultaneously reduced displacement of basal
bodies 2b, 2c, and 3c (arrows) and extended sculpturing due to anomalous displacement of basal bodies 3b and 4c (wavy arrows). In addition, basal body la is
anomalously ciliated (arrowhead). The sculpturing of M3 is extended, probably due
to displacement of basal body 4c (wavy arrow, compare with the stippled basal body
in the eM-3 pattern in Fig. 11).
Mutations affecting intracellular patterns in Tetrahymena
57
58
J. FRANKEL AND OTHERS
psmAI
UM
M3
17.
psmAI
Mutations affecting intracellular patterns in Tetrahymena
59
(Frankel et al. 1984, Fig. 9) sculpturing is virtually invariant, manifesting the
standard patterns shown in Fig. IB and in the left column of Fig. 11. Such normal
patterns predominate in the other mutants as well (e.g. Figs 13,17), but modifications are fairly common (Table 5). The most frequent modifications, found in
3-membranelled OAs of all mutants except mpD, involve individual displacement 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 the
sculptured 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 displaced
as well (Fig. 19). More rarely, a subnormal displacement of basal bodies is
observed, which we call 'reduced' sculpturing (Fig. 15). Reduced and extended
sculpturing 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 of
column 1 of M2 (Fig. 11, right, and 18). Both of these abnormalities are most
common in the very large OAs oipsmAl cells.
Although the data are insufficient for meaningful statistical assessment, we
have a strong impression that the degree and type of abnormality are not independently determined within separate membranelles of each OA; cases of
extended sculpturing (Fig. 14) or reduced sculpturing (Fig. 15) in more than one
membranelle of a single OA are sufficiently frequent to suggest coordination of
sculpturing processes within OAs.
The frequency of abnormalities of sculpturing of M2 in 3-membranelled OAs
seems to be associated with membranelle length. This is clearest in comparisons
across 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 the
same meaning as in Figs 12-16; in addition, broad open arrows indicate places where
basal bodies are partially obscured. Scale bars indicate 1/itn.
Fig. 17. A psmAl OA with great elongation of Ml, M2 and UM combined with
a nearly normal membranelle sculpturing pattern. The sculptured ends of Ml and M2
are almost completely visible, and normal. M3 is of normal size but somewhat
abnormal pattern. The UM has been torn loose from the remainder of the preparation. The preparation is highly flattened, which may account for fading of some basal
bodies located in regions where basal bodies are normally situated in depressions of
the 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-2
sculpturing pattern (cf. Fig. 11). Basal bodies y, la, lb and lc of M2, invisible in
many preparations, are all ciliated (arrowheads). This preparation is extremely
flattened, with fading of basal bodies at the same positions as in Fig. 17 (single open
arrows) and also at the position usually occupied by the epiplasmic flap overhanging
Ml (double open arrows, also in Figs 19, 21).
60
J. FRANKEL AND OTHERS
. psmAI
Mutations affecting intracellular patterns in Tetrahymena
61
Table 5. Sculpturing of membranelle 2 in 3-membranelled OAs of wild-type and
mutant cells*
State of sculpturing of M2
K
r
Extendedf
A.
Genotype
WT
mpD
big
Normal Reduced
112
26
20
1
t
3b
4c
1
Otherwise
3b + 4c Ciliated^ modified§
2
2
4
1
2
4
1
Percent
modified
3
7
33
10
52
44
32
1
4
1
psmAl (23°;I
9
1
5
psmAl (28°;)
15
5
3
1
14
1
2
psmB
1
2
2
13
psmC
1
1
(1)
* From the same samples as the data in Tables 3 and 4, except that wild-type data are from
OAs 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 infig.11; l4c" refers
to posterior displacement of the basal body designated as 4c. l3b + 4c' refers to the posterior
displacement of both of these basal bodies in the same membranelle, as illustrated in the
eM-2 diagram in Fig. 11.
$The cM-2 pattern illustrated in Fig. 11. In psmAl OAs, all of the column-1 basal bodies
are ciliated, while in the psmB OAs, some are. The single psmC example is in an OA that is
otherwise highly abnormal (hence in parentheses).
§ Includes combinations of the tabulated modifications (including, for example, M2 of the
OA illustrated in Fig. 16).
and most frequent in psmAl at 28 °C (Table 5). However, assessment of correlations within mutant clones gives a more mixed picture: in psm clones there is a
definite 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 the
same meaning as in Figs 17-18; in addition, broad solid arrows indicate regions of
putative ciliary-unit resorption or a membranelle-fragment left over from such
resorption. Scale bars indicate ljum.
Fig. 19. A.psmAl OA with extended sculpturing of M2 (displaced basal bodies 3b
and 4c indicated by wavy arrows) and a split M3. The broad solid arrow indicates a
gap between a somewhat modified version of the typical M3 pattern, with basal body
la ciliated (arrowhead), and a membranelle-fragment with a regular row-andcolumn organization. There is no sign of fading of basal bodies on either side of the
region indicated by the broad solid arrow, suggesting that it is an area in which basal
bodies have been resorbed in vivo.
Fig. 20. A psmAl O A with normal sculpturing of Ml, extended sculpturing of M2
with displacement of basal body 4c (wavy arrow), and a partially interrupted M3. An
M3-like sculpturing pattern is discernible at the right end of M3, but the region of basal
body resorption (broad solid arrow) is well to its left. The portion of M3 to the left of this
region 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 sculpturing of M2 with a highly displaced 4c basal body (wavy arrow), and a large gap in
M3. A disorganized membranelle fragment (broad solid arrow) is seen to the cell's
left of the gap. The UM has been lost from this preparation. The 'e' indicates four
extra basal bodies, of an ectopic fourth-row segment of M2.
EMB82
62
J. FRANKEL AND OTHERS
of the O A, while within the big clone there is no such significant association (data
not 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 12
ciliated basal bodies (Figs 12,13) that bears scant testimony to its developmental
origin from a membranelle prototype resembling Ml and M2 (see Fig. 1C). As
the length of Ml and M2 in 3-membranelled OAs increases, M3 shows no parallel
increase: it either retains its standard size and pattern, as in the big OA depicted
in Fig. 13, or occasionally undergoes a modest increase in size that typically is
associated with extended sculpturing (Fig. 16).
Even in psmAl, very long Mis and M2s are commonly accompanied by M3s
of normal size and only slightly abnormal pattern (Figs 17, 18; also fig. 4k of
Frankel, 1983). However, in one half of the psmAl OAs with very long Ml and
M2, M3 is also strikingly elongated, but in an unusual manner: a gap, sometimes
partial (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 the
other side of the gap sometimes shows the well-defined row-and-column organization characteristic of Ml and M2 (Fig. 19), but more commonly is variably
disorganized (Figs 20, 21).
An interrupted M3 was commonly observed in silver-stained completed OAs
of psmAl (Fig. 8), indicating that the pattern observed in Figs 19 and 21 is not
an artifact of sample preparation for SEM. In contrast, M3 is almost invariably
long and continuous in stage 4 and 5 developing oral primordia seen on the same
silver preparations (Fig. 7). This indicates that the gap that appears within the
mature M3 of the large psmAl OAs must be a consequence of ciliary-unit
regression, rather than failure of initial development.
DISCUSSION
The increase in size of the oral apparatus (OA) of Tetrahymena thermophila
brought about by any of five mutations has differential effects on spatial organization of ciliary units that are comparable to the previously reported effects
of decrease of size brought about by starvation (Bakowska et al. 1982a). In both
situations, the lengths of the ciliary arrays (membranelles and undulating membrane) change while their widths remain the same; the lengths of Ml and M2 are
coordinately regulated while the length of the UM is less closely coordinated
with that of M2 and presumably Ml; finally, the patterns of sculpturing of the
right ends of the membranelles are not severely affected. The preservation of
these relationships when change is in opposite directions (increase of size in one
case, decrease in the other) strengthens the conclusions of the earlier study on
Mutations affecting intracellular patterns in Tetrahymena
63
starved cells (Bakowska et al. 1982a). These are, (1) the formation of membranelles is tightly integrated whereas development of the UM is a partially
independent process, (2) modification of membranelle size takes place primarily
through changes in number of basal-body couplets recruited into promembranelles, and (3) the spatial extent of sculpturing of these membranelles is
largely independent of the number of couplets thus recruited. These conclusions
are fully in accord with results of detailed studies on oral development in OAs
of wild-type cells (Bakowska et al. 1982b) and of a 'misaligned undulating membrane' mutant (Lansing et al. 1984), which show that the formation and the
sculpturing of membranelles take place at difference times during oral development, and that development of the undulating membrane not only differs greatly
from that of the membranelles (Bakowska et al. 19826) but also can be modified
C} O O O <3:m.w*MS>®-.:®:;:©:Q O O
o o o ommmmMMmmm, o o
Fig. 22. A model for control of the size of M3. The circles indicate the basal bodies
of M3 at stage 5d of oral development (see Fig. 1C), prior to sculpturing and resorption. 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 near
the end of stage 5. The bracketed lengths indicate the number of basal body columns
that may be formed during the initial development of M3 in OAs of (A) starved wildtype cells, (B) growing wild-type cells, (C) growing cells of mutants such as big and
most psms, and (D) psmAl cells with very large OAs. In each case a 'standard' M3
is formed despite continuous variation in the original number of basal bodies, except
when the original M3 is so long that it extends beyond the cell's left end of the zone
destined for resorption.
64
J. FRANKEL AND OTHERS
substantially while development of the membranelles remains virtually unaltered (Lansing etal. 1984).
The most novel observation in this study is of the enlarged and interrupted
M3 patterns observed in several of the largest OAs from psmAl cells. This
enlargement is an exception to the normal size- and pattern-constancy of M3,
observed both in enlarged OAs of mutants and in diminished OAs of starved
cells. The exception, however, sheds some light on how the normal constancy is
achieved. In normal oral development, there is 'evidence for occasional resorption 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 of
Ml or M2. Thus, during the initial development of M3 an excess of basal-body
columns is normally produced, which subsequently is pruned by a spatially
localized resorption activity. The final pattern of M3 presumably can remain
constant during starvation because what is diminished is the number of surplus
ciliary units otherwise destined for elimination. Increasing the length of M3
above normal increases the surplus, which can be eliminated completely — up
to a point. What is most revealing is the membranelle geometry that emerges
when the capacity for elimination of surplus ciliary units is finally exceeded. The
fact 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 left
as well as a right margin. This idea is illustrated schematically in Fig. 22. Its
essence is that the zone of resorption activity is positionally specified in some
reasonably precise manner, analogous to the localization of the 'posterior
necrotic zone' of cell death in the chick limb bud (Saunders, 1967). This idea will
be elaborated further, with additional experimental support, in the subsequent
paper (Frankel et al. 1984).
This study revealed relatively little novelty in the modifications of sculpturing
of the right ends of the membranelles. This was somewhat surprising, since
mutants were used, and other mutations affecting membranelle patterns, mpA
and mpB, bring about drastic and apparently random abnormalities of sculpturing (Frankel, 1983). Although abnormalities of sculpturing of membranelles are
certainly more common among the 3-membranelled OAs of the mutants considered in this study (mpD excepted) than they are in growing wild-type cells, the
difference is mostly one of frequency rather than kind. Thus, abnormalities
which here are given names have previously been documented in photographs
of OAs from wild-type cells: extended sculpturing through displacement of basal
body 4c (and 4b) of M2 in figure 5 of Bakowska et al. (1982a), displacement of
basal body 3b of M2 in figure 7, and reduced sculpturing of Ml in figures 8 and
9 of that paper. Only the cM-2 pattern of M2 (Fig. 11) and some variability in
shape of Ml are new to this study, and these were both found only in extremely
large OAs produced by oral replacement in psm cells.
This lack of novelty, however, obtains only when analysis is restricted to OAs
that possess the usual three membranelles. When there are four or more
Mutations affecting intracellular patterns in Tetrahymena
65
membranelles, new sculpturing patterns appear that are not found in 3-membranelled OAs of the same genotype (Frankel et al. 1984). Since these new patterns
are observed in mutant cells possessing OAs in which the length as well as the
number of membranelles is increased, it is imperative to dissect the specific
effects of increased number of membranelles from the background effects due
to the presence of a mutation and the increased size of the individual membranelles. The present study has shown that this background effect, especially for
the most informative mutant (mpD), is minimal.
The majority of the linkage analysis oipsmAl, psmB, and psmC was carried out by Elaine
Martel. 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 their
comments 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)