/ . Embryo!, exp. Morph. Vol. 35, 3, pp. 499-505, 1976
Printed in Great Britain
499
Quantitation of the spatial distribution
of ' prespore vacuoles' in pseudoplasmodia of
Dictyostelium discoideum
By PAUL A. FARNSWORTH 1 AND WILLIAM F. LOOMIS 1
From the Department of Biology, University of California
SUMMARY
The axial distribution of an organelle, the prespore vacuole (PV), previously reported
absent from the prestalk region, was determined in pseudoplasmodia of varying sizes, under
differing conditions of photostimulation of migration.
The distribution of these organelles, determined quantitatively by electron microscopy of
sections from known axial locations, was found to have a spatial pattern which varied with
pseudoplasmodial size. The total complement of these organelles appeared constant for any
size of pseudoplasmodium under similar conditions of illumination. Increased illumination
decreased the total number of the organelles.
The spatial distribution of PV varies with total cell number, and the size of the region with
no PV bears no relationship to the proportion of the cell mass which would form stalk cells.
Similarly, the number of cells containing PV bears nofixedrelationship to the number of cells
which will form spores.
On these grounds, the reported role of PV, that of directing or reflecting spore differentiation, appears unlikely.
INTRODUCTION
Development of the cellular slime mold Dictyostelium
discoideum results in
the formation of multicellular pseudoplasmodia which culminate to give rise to
fruiting bodies in which about 80 % of the cells become spores and the rest form
thick-walled vacuolized stalk cells. Vital staining of pseudoplasmodial cells has
shown that the cells in the anterior of pseudoplasmodia differentiate into stalk
cells, and the cells in the posterior become spores (Raper, 1941). The only confirmed cytological difference reported between anterior and posterior pseudoplasmodial cells is the occurrence in the posterior cells of a unique organelle,
first described by George (1968) and subsequently studied by Hohl & Hamamoto
(1969), Maeda & Takeuchi (1969) and Gregg & Badman (1970). The organelle
was described as an irregular, membrane-bound structure, 0-4 to 1-0 ^m across,
with a fibrous matrix and a electron-dense lining 250 A wide separated from the
limiting membrane by an electron-transparent region of similar dimension.
The organelle was reported to be absent from growing cells and to appear
1
Authors' address: Department of Biology, University of California, San Diego, La Jolla,
California 92037, U.S.A.
500
P. A. FARNSWORTH AND W. F. LOOMIS
during late aggregation, the proportion of the cells which contained them and
the average number per cell increasing during migration and falling to zero after
the formation of mature spores.
The above authors suggested that the organelle was unique to cells which
were to form spores, and that they evaginated during culmination to form the
spore coat. They proposed the name prespore vacuole (PV) or prespore specific
vacuole (PSV).
Muller & Hohl (1973) have implied that possession of such organelles directs
cells towards spore differentiation and that the size independent pattern of
spores and stalks seen in the fruiting body is a reflection of a size-independent
distribution of these organelles. Their qualitative study of the spatial distribution
of these organelles describes, in general terms, anterior regions of pseudoplasmodia lacking any PV and posterior ones in which a variable proportion of
cells contain a variable number of PV. The axial distribution of these two cell
types was not, however, quantitated.
The precise quantitation of the axial distribution of these organelles is important in deciding the role of such organelles in the generation of the sizeinvariant pattern of differentiation. If, as many authors suggest, a boundary
between prestalk and prespore cells is set up in pseudoplasmodia, and if PV
are a prerequisite of spore differentiation, it seems essential that the pattern of
the spatial distribution of PV should be as independent of pseudoplasmodial
size as the final pattern of differentiation.
METHODS
Dictyostelium discoideum strain NC-4 was grown in association with Klebsiella aerogenes on SM agar (Sussman, 1966) or strain A3 was grown axenically
in liquid medium (Loomis, 1971). No differences were found in PV distribution
between these strains. Exponentially growing cells were harvested, washed and
5 x 107 cells deposited at one side of 9 cm Petri dishes containing 2 % agar. All
plates were incubated at 22°. Photostimulated migration was induced by
covering the dishes with black paper except for two small slits, allowing illumination with two orthogonal beams of light of controlled intensity.
Pseudoplasmodia were collected after 12 h of migration and prepared for
electron microscopy as follows:
(1) Fixation. 2 % glutaraldehyde + 0-1 % ruthenium red in 0-05 M phosphate
buffer pH 7-4, at 20 °C for 60 min.
(2) Wash. In 0-1 M phosphate buffer pH 7-4 at 20 °C.
(3) Osmication. 1 % OsO4 + 0-05 % ruthenium red in 0-05 M phosphate buffer
pH 7-4 at 5 °C for 60 min.
(4) Wash. Three changes in distilled water at 5 °C.
(5) Dehydration. Ten min each in 20 %, 50 %, 70 %, 90 % ethanol at 5 °C;
three changes of 20 min each in absolute ethanol at room temperature.
501
Prespore vacuole distribution
4
2
Strong illumination
(10 //W/cnr
0
. Weak illumination
(1//W/cnr)
8
6
»
2
13
4
0
-Dark8
I
o
E
a
6
0-5
10
0
0-5
10
0-5
10 0
Axial length (mm)
0-5
0
0-5
0-5
Fig. 1. The axial distribution of the mean number of PV per cell profile. The diameters of the data points indicate the standard errors of the means. The anterior end
is to the left in all cases. The values are plotted at the measured axial locations. The
pseudoplasmodia had migrated with illumination at 10/*W/cm2 (100ergs/cm2/sec),
1 /iW/cm2 (10 ergs/cm2/sec) or in the dark as indicated on the figure.
(6) Infiltration. In Spurrs low viscosity resin (standard hardness); 1 h in 50 %
resin/ethanol, 6 h in pure resin, or alternatively: overnight under vacuum in a
mixture of 50 % resin in alcohol followed by 3 h in fresh resin.
(7) Embedding. Individual pseudoplasmodia were flat embedded in silicone
rubber molds at 80 °C for 12 h.
Pseudoplasmodia were then aligned on a Riechert OMU2 microtome and
about 30 serial transverse sections taken at each measured 50 or 100 /*m interval
along their entire length. Sections were stained in saturated uranyl acetate in
50 % methanol for 2 min, followed by Reynolds lead citrate for 2 min.
The sections were examined at a magnification of x 15000. The image was
moved systematically over a square on the viewing screen which corresponded
to a section area of 50 /mi2, this representing approximately the average area of
random cross-sections through a 10 fim diameter cell. The number of PV contained in this 'average cell profile' was recorded using a foot-switch activated
tape recorder. The number of PV per cell profile was recorded for the total
area of each cross-section, rejecting only those positions at which less than
two-thirds of the measuring area was occupied by cells. This procedure was
applied to 2670 sections from 89 known axial locations on 18 pseudoplasmodia
of varying sizes and conditions of photostimulation of migration.
EMB 35
502
P. A. FARNSWORTH AND W. F. LOOMIS
48
40
r
> x 32
Dark
J
32
.
24
f
—
illumination
| |
16
o 16
/High illumination
i
i
i
i
0-5
i
i
10
i
i
i
•
i
Hi
i
i
1-5
i
0-5
Length (mm)
i
i
J-0
i
i
i
i
i
1-5
Length (mm)
Fig. 2
Fig. 3
Fig. 2. The variation of the total number of PV per pseudoplasmodium with axial
length. The area under the curve showing the axial distribution of the mean number
of PV/cell profile (Fig. 1), multiplied by 10, was used as an estimate of the number
of PV per pseudoplasmodium. The points indicate the number of PV in pseudoplasmodia of different lengths.
Fig. 3. The axial gradient in the average number of PV/cell. The area under the curve
describing the axial distribution of the number of PV/cell profile, multiplied by 10
and divided by the pseudoplasmodial length, generates a measure of the average
density of these organelles in pseudoplasmodia of various lengths. Though this may
not be an absolute measure, the distributions clearly show the relative variation of
PV density between pseudoplasmodia of various lengths migrating under varying
conditions of illumination.
RESULTS
The number of PV was determined in more than 3000 cell profiles at each
axial position in 18 pseudoplasmodia (Fig. 1). In order to assign statistical
significance to the data, frequency distributions of the number of PV per cell
profile were drawn for each axial location, and skewedness and kurtosis coefficients calculated for each distribution. Typically the distributions were
slightly platykurtic with a small amount of negative skewing; however, x2
'goodness of fit' tests showed that the distributions on average approximated
reasonably closely to the normal (P = 0-75) and the means and standard
deviations were used as representative of them (Geary, 1936). These values were
plotted against axial position as shown in Fig. 1.
The spatial pattern of the distribution of PV is obviously not the same in all
pseudoplasmodia. The organelles are not always absent from a fixed proportion
of the front and are not distributed homogeneously throughout the posterior
regions. General descriptive trends can, however, be seen. The number of
organelles per cell increases from a point somewhere in the front half and rises,
at a variable rate, with distance from the tip, falling again near to the posterior
end. It appears, from measurement of the area under the curves, that the total
number of these organelles is independent of pseudoplasmodial size (Fig. 2)
which, in turn, makes their density completely size dependent (Fig. 3). Figs.
2 and 3 also quite clearly demonstrate a dependence of the PV pattern on the
photostimulation of migration, the density of these organelles decreasing with
Prespore vacuole distribution
503
increasing light intensity. The smallest pseudoplasmodia under maximal photostimulation have almost no PV; however, populations of such pseudoplasmodia
culminate normally, forming fruiting bodies with the normal proportion of
spore and stalk cells. Members of such populations, fixed at hourly intervals
during the conversion of these pseudoplasmodia to fruiting bodies, and carefully examined in the electron microscope, have never been seen to have more
than 5 % of their cells containing PV.
Seven of the eleven pseudoplasmodia which had migrated in the dark or
under conditions of weak illumination contained about 1 PV per cell profile in
the most anterior sample. This indicates that cells within the anterior 5 % of
these pseudoplasmodia contained about 10 PV per cell. Cells within the anterior
5 % of the other four pseudoplasmodia contained an average between 0-1 and
1 PV per cell. Maximal photostimulation reduced the total complement of PV
such that cells in the anterior contained less than 1 PV per cell. Furthermore, in
the two smallest pseudoplasmodia only 1 in 30 cells was found to contain a PV.
No cell of these pseudoplasmodia was found to contain more than 1 PV even in
the most posterior sample.
DISCUSSION
Contrary to the assumptions of previous authors, it is apparent from these
results that there is no size-independent pattern of distribution of PV. These
results differ from those of Gregg & Badman (1970), who describe a uniform
distribution of PV in the rear 90 % of all pseudoplasmodia from late aggregation
onwards. This has already been disputed by Muller & Hohl (1973), who
reported that up to 36 % of the cells in the posterior have no PV. Both of these
studies report that cells in the anterior 10-15 % of pseudoplasmodia are devoid
of PV. However, we find the organelles in the anterior regions of some pseudoplasmodia, though always at a lower abundance than in the corresponding
posterior region. We do not find PV to be present in (or absent from) a fixed
proportion of the cells of pseudoplasmodia; however, it is well known that an
almost constant proportion of these cells will form spores (Bonner & Eldridge,
1945). The variation, between pseudoplasmodia, of the proportion of cells
containing PV is at least an order of magnitude greater than the variation in
the proportion of the cells of individual fruiting bodies which are spores, as
predicted from the quantitation of the pattern of differentiation in populations
(Farnsworth, 1975) or as measured in small numbers of individuals (Gregg
6 Bronsweig, 1956). Since a constant proportion of the cells form spores but an
extremely variable proportion of the cells contain PV, it is difficult to envisage a
direct, causal relationship between the possession of PV, and differentiation into
spores.
Phototaxis of pseudoplasmodia appears to dramatically affect the accumulation of PV. Strong unilateral illumination of migrating pseudoplasmodia
reduced the complement of PV to about a fifth of that in unilluminated
32-2
504
P. A. FARNSWORTH AND W. F. LOOMIS
pseudoplasmodia, while weak illumination results in an intermediate level of
PV. Since the conditions of phototaxis used result in an increase in the rate
of migration (Poff & Loomis, 1973), it is tempting to consider a relationship
between rate of migration and number of PV.
It is not at present clear what role PV may play in the development of
Dictyostelium. Muller & Hohl (1973) reported that, at late migration, only 58 %
of the cells had these organelles; nevertheless, 80% of the cells of all these
pseudoplasmodia differentiate into spores. Furthermore, we find that small
pseudoplasmodia, migrating under strong illumination, contain almost no PV
and appear not to make any during culmination, yet the majority of their cells
become viable, normal-looking spores. It thus seems unlikely that this variable
distribution of PV has any direct quantitative relationship to the generation of
the size-invariant pattern of differentiation.
Immunological studies (Takeuchi, 1963) have shown that antisera prepared
against spores of D. mucroides react specifically with PV of D. discoidewn
indicating a direct involvement of PV in the formation of an antigenic component of the spore surface. This has been interpreted as suggesting that the PV
form the spore wall. In the light of the large quantitative discrepancy reported
here between the number of cells containing PV and those which form spores,
this proposed role of PV might be further questioned. It may be, for example,
that at least part of the antigenic activity of spores is the result of components
other than the wall, and that PV contain the extracellular mucopolysaccharide
which holds the mature spores together in the sorus. A variation in the proportion of the cells containing PV would then not affect the proportion of the
cells which become spores, but rather would affect some component property
of the spore head of the fruiting body like spore cohesion, a property not easily
measured and, until now, overlooked.
This work was supported by a Grant from the National Science Foundation (GB 28959).
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{Received 14 October 1975)