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Journal of Bacteriology, July 2000, p. 3877-3880, Vol. 182, No. 13
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Organization and Cell-Cell Interaction in Starved
Saccharomyces cerevisiae Colonies
Mazal
Varon and
Mordechai
Choder*
Department of Molecular Microbiology and
Biotechnology, Faculty of Life Sciences, Tel-Aviv University, Ramat
Aviv 69978, Israel
Received 16 February 2000/Accepted 20 April 2000
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ABSTRACT |
Cell growth in yeast colonies is a complex process, the control of
which is largely unknown. Here we present scanning electron micrographs
of Saccharomyces cerevisiae colonies, showing changes in
the pattern of cell organization and cell-cell interactions during
colony development. In young colonies (
36 h), cell density is
relatively low, and the cells seem to divide in a random orientation. However, as the colonies age, cell density increases and the cells seem
to be oriented in a more orderly fashion. Unexpectedly, cells in
starved colonies form connecting fibrils. A single connecting fibril
180 ± 50 nm wide is observed between any two neighboring cells,
and the fibrils appear to form a global network. The results suggest a
novel type of communication between cells within a colony that may
contribute to the ability of the community to cope with starvation.
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TEXT |
Evidence for cell-cell communication
in bacteria has been accumulating in recent years. Numerous cases of
coordinated activities of cells inside a colony have been described. It
has been found that bacteria form complex communities that behave
analogously to multicellular organisms (for a recent review, see
reference 11). Multicellularity in bacteria
regulates many aspects of bacterial physiology, such as self defense,
hunting for prey, and specialization. Nevertheless, little is known
about communal behavior in eukaryotic unicellular organisms.
Previously, we showed that the growth of well-separated
Saccharomyces cerevisiae colonies is biphasic
(3). In the first growth phase (~24 cell divisions), most,
if not all, cells divide rapidly at a rate similar to that observed in
liquid medium and exhibit morphological, biochemical, and genetic
characteristics of cells engaged in the cell cycle. At the end of this
exponential growth, a transition to a slower growth phase is observed
accompanied by a global change in the pattern of gene expression. The
cells in the center of the colony then gradually enter the stationary phase, whereas the cells at the periphery continue to grow. The transition from the first to the second growth phases is sharp, suggesting that most cells synchronously respond to some environmental cues (3). This realization has led us to postulate the
existence of some sort of intercellular communication, which might be
manifested morphologically.
Cell organization increases with colony age.
Cells in
mid-logarithmic phase were plated on a solid support containing rich
medium (yeast extract-peptone-dextrose [YPD]) and allowed to form
colonies. Cells were allowed to undergo 4 to 5 doublings (8 h) before
colonies were fixed, adapting a protocol used to fix Escherichia
coli colonies (11), and inspected by scanning electron
microscopy (SEM). Photographs (×5,000) were taken at 45°C unless
otherwise indicated. Micrographs of several colonies reveal a
heterogeneous population of cells with smooth walls (Fig.
1A 8 h and results not shown). Many
cells carry a bud, and some show bud scars. Micrographs of the young
colonies show no discernible organization. The cells are relatively
distant from each other, and the orientation of the buds with respect to the center of the colony varies from cell to cell. There is physical
contact between a given cell and some of its immediate neighbors, but
not with all of them.
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FIG. 1.
SEM micrographs of colonies at various growth stages
reveal an age-related increase in cell organization. (A) Colonies of
strain SUB62 (3, 6, 7, 9, 13), grown on YPD, were fixed at
the indicated time points postplating and visualized by SEM. Original
magnification, ×5,000. The bar at 8 h represents 10 µm; at
other time points, it represents 5 µm. (B) Top-view SEM micrographs
of colonies at the indicated time points were enlarged, and the cells
were counted. A total of 200 to 400 cells were counted per time
point.
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To determine whether cell organization changes during colony
development, SEM analysis was performed at various time points
after
plating. Figure
1A (36 to 72 h) shows micrographs taken
from the
center of the colonies. Several changes in cell morphology
and cell
organization are observed as the colonies age. (i) Whereas
the cell
walls are smooth by up to 24 h, at later stages of colony
development, they become wrinkled. (ii) Cells in the young colonies
display little organization (e.g., Fig.
1A [8 and 36 h]); as the
colonies develop, the distance between the cells and their neighbors
seems to decrease, and the arrangement of the cells seems to be
more
ordered (Fig.
1A [72 h]). (iii) Quantitative support for
the
age-related increase in cell organization is provided by determining
cell densities. Thus, in spite of the observation that the average
cell
size remains constant during the first 72 h of colony development
(results not shown), the cell density on the surface of the colonies
increases as the colonies age (Fig.
1B).
Level of cell organization increases from the edge to the center of
a starved colony.
Figure 2a shows
that, whereas a lack of noticeable organization is a characteristic of
both the center and the periphery of young colonies (A and B), in old
colonies, there is a clear difference in cell organization between the
colony center and its periphery (C to F). As a control, we examined
artificial colonies, which were formed by placing a packed pellet of
cells on agar and immediately fixing and processing it for SEM, in
exactly the same way as was done for the natural colonies. Cell
organization in such "colonies" was uniform and showed no
difference between the center and periphery (Fig. 2a, panels G and H),
ruling out the possibility that the fixation process is affected by the
location of the cells within the colony.
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FIG. 2.
Cell organization depends on colony age and on the
location within the colony. (a) (A to F). Top-view micrographs were
taken from the colony periphery (left panels) or from the colony center
(right panels). Bars represent 5 µm. (G to and H). Cells that had
been grown logarithmically in liquid culture were collected, and the
pellet was taken by a toothpick and placed on agar to form a
colony-like structure. The artificial "colony" was immediately
fixed and further processed according to the same protocol used in
panels A to F. Cont., control. Micrographs of the periphery (G) and
center (H) are shown. Bars represent 10 µm. (b) Top-view SEM
micrographs of various locations (200 µm apart) across the radius
were taken, and cell densities were determined as described for Fig. 1B
and plotted as a function of the distance from the edge. In addition,
cells were classified as unbudded or budded and the percent budding was
similarly plotted.
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In a more detailed study, in which micrographs of a 70-h-old colony
were taken systematically from various locations (200
µm apart)
across the colony radius, we found that cell density
increases
gradually from the edge to the center (Fig.
2b).
Starvation-related formation of intercellular connecting
fibrils.
When 3-week-old colonies that had been grown on a
synthetic complete (but not YPD) medium were inspected, an unexpected
phenomenon was revealed: connecting fibrils that seemed to bridge
neighboring cells became apparent. Because cells in old colonies are
starving (3), we suspected that the appearance of the
fibrils is associated with the starvation. Therefore, we challenged the
colonies with more stringent starvation conditions by plating our
leucine auxotroph cells on a synthetic medium with a limiting
concentration of leucine. This medium could support a slow growth of
tiny colonies of this strain, which appeared after a week. The colonies
reached a size of ~1 mm and could grow no further, probably because
the leucine was consumed and its concentration decreased below a
critical level. Under these conditions, fibrils appeared uniformly
throughout the colony. They appeared in two different strain
backgrounds (not shown). Since this strain is also a uracil auxotroph,
cells were plated on a synthetic medium containing a limiting
concentration of uracil (2 µg/ml). Again, small colonies appeared on
the plate and the cells produced connecting fibrils similar to those
observed in colonies starved for leucine. Thus, fibrils are produced
when colonies grow on at least two different kinds of starvation media. Examples are shown in Fig. 3. Remarkably,
only one fibril is detected between two neighboring cells. Thus, it
seems that the number of fibrils is controlled, and their existence is
likely to be biologically significant.
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FIG. 3.
Intercellular connecting fibrils are found in starved
colonies. Colonies were grown on plates containing synthetic medium
(SD) supplemented with the standard amounts of various amino acids
(14), except that the leucine concentration was limiting
(1/10 that of the standard). After 3 weeks, the colonies were fixed and
visualized by SEM. (A) Top view. (C) Higher magnification (originally
×10,000). Bars in panels A and B represent 10 µm, and the bar in
panel C represents 5 µm.
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Our work, which employs for the first time the SEM technique to
investigate
S. cerevisiae colonies, reveals several new
features
of their organization. (i) Cells in young colonies show little
organization. This is different from the high degree of order
found in
"newborn"
E. coli colonies, where communication between
a daughter and a granddaughter of the colony founder cell has
been
found (
12). (ii) As the colony develops, the average cell
size remains constant, whereas cell density at the colony surface
increases. These results are consistent with the observation that
cells
in older colonies are closer to each other and with the
overall
impression that the micrographs provide of increased order
as the
colony develops. (iii) Features characteristic of young
colonies also
characterize the periphery of old colonies. Note
that the periphery of
an old colony is the region where growth
occurs and where nutrients are
readily available (reference
3 and references
therein). Taken together, our results demonstrate
a direct correlation
between the nutritional conditions that cells
experience and the level
of cell organization: the presence of
less nutrients is correlated with
a higher order of
organization.
For the interpretation of the present data, it is important to consider
the following observations. First, high cell density
is not only a
characteristic of old and large colonies, but also
of tiny, yet
starved, colonies that grow on leucine or uracil
limiting media.
Second, micrographs taken from various locations
across these tiny and
starved colonies show similar organization
and similar cell density
(results not shown). Third, we have created
an artificial colony and
found no difference in cell density or
organization at various
locations of the "colony" (see Fig.
2).
Taken together, our results
led us to propose that a high level
of organization is a feature of
starvation experienced by the
colonies prior to fixation and is not a
consequence of several
parameters that could artifactually affect the
results, such as
colony size or the actual location within the
colony.
The mechanism responsible for the increased organization in starved
colonies is unknown. Because there is no evidence for
cell motility in
S. cerevisiae, it is hard to conceive how cells
in the
colony can be rearranged. Therefore, it is likely that
a higher level
of organization is achieved by some coordination
of cell division.
According to this possibility, the organization
must be initiated
before the cells enter the stationary phase,
probably when they start
to sense starvation. Indeed, cell density
increases gradually during
development, beginning before the cells
enter the stationary phase
(Fig.
1B).
What adaptive advantages do yeast derive from the high degree of cell
organization? At present, the answer to this question
can only be
speculative and teleological. When a single cell is
plated on a solid
and rich medium, it encounters optimal conditions
for growth. It then
divides at its maximal capacity and starts
to form a colony. As long as
nutrients are not present at limiting
concentrations and there is no
accumulation of toxic metabolites,
the cells divide independently of
each other and no coordination
or communication is necessary for
optimal proliferation. Hence,
during this phase of colony growth, no
order is required. We hypothesize
that order is a burden on the
individual cell because it requires
energy-consuming communication.
Thus, order is established only
when it provides a significant
biological advantage. As the colony
develops, nutrients become less
available to cells that are distant
from the nutritious agar
(
3). We speculate that order is established
to cope with the
starvation itself, as well as with other stresses,
such as dehydration,
that are usually associated with long-term
starvation periods.
Moreover, order may also facilitate a passive
capillary movement of
nutrients from the agar to the top cell
layers through the pores in the
cell walls (see Discussion in
reference
3). Another
parameter that might affect organization
of colony cells is the
accumulation of metabolic by-products or
other molecules
(pheromone-like) secreted by the cells. Two well-known
examples of
by-products are (i) ethanol, a product of fermentation,
and (ii)
ammonia, a product of amino acid metabolism. Indeed,
ammonia is
involved in long distance communication between colonies
(
4). However, whether or not ammonia or other secreted
materials
play a role in cell-cell communication within a single colony
remains to be
determined.
Organization of old yeast colonies is more complex than is evident from
the SEM micrographs. Inspection of
lacZ-containing
colonies,
grown on 5-bromo-4-chloro-3-indolyl-
-
D-glucopyranoside
(X-Gal)-containing agar, revealed a complex pattern of staining
in
which clonal minisectors are detected. When sublethal concentrations
of
OsO
4 were used for fixation, a differential staining of the
colony cells was observed, which led to the appearance of concentric
rings of dark and light stains (results not shown), similar to
the
patterns observed in
E. coli (
10,
11). However,
our SEM
analyses failed to detect sectors or concentric rings. It is
likely
that in
S. cerevisiae, the differential staining is
based on biochemical
differences between cells rather than differences
in cell organization
within the
colony.
In addition to the increase in the overall organization during colony
development, suggesting some sort of communication between
cells, we
observed a possible new type of communication mediated
by connecting
fibrils. Under our experimental conditions, a single
fibril connected
two neighboring cells. This observation favors
the possibility that
fibril number is controlled. Their limited
number suggests that they
play a regulatory role rather than simply
a mechanical function. The
function of the fibrils remains to
be determined. Two alternative, but
not mutually exclusive, functions
can be envisaged. (i) Fibrils may
play a role in signal sensing
and transduction. The signals are likely
to be transmitted from
one cell to its immediate neighbors. However,
theoretically, the
fibrils can transmit the signal from one location in
the colony
to a far distant location, because the fibrils and the cell
walls
seem to form a continuous network that connects all the cells.
(ii) Fibrils serve as connecting channels for the transport of
molecules from one cell to the other. Their diameter, 180 ± 50
nm, is theoretically large enough to permit the passage of
macromolecules.
By way of comparison, the outer diameter of the nuclear
pore complex
in yeast (which includes the building proteins), which
serves
as the only channel for nucleocytoplasmic traffic, is 100 nm
(
15).
Cell-cell interaction and colony organization have been observed in a
number of unicellular organisms. The most thoroughly
investigated
examples are
Dictyostelium discoideum and myxobacteria.
In
both organisms, organization is by far more complex than that
observed
in
S. cerevisiae, ending in the formation of a
quasidifferentiated
organ, the fruiting body (
1,
5).
Development of the fruiting
body is induced by starvation and involves
cell-cell interaction.
In myxobacteria, this interaction is apparently
mediated by fibrils
30 nm wide, which are composed of polysaccharides
and proteins
(
1). Extracellular appendages have also been
found in other
prokaryotic organisms, where they usually serve for
attachment
to some matrix. For example, a role in the internalization
into
epithelial cells has been attributed to surface appendages of
Salmonella enterica serovar Typhimurium (
2).
Fibrilar structures
have also been found in the yeast
Candida
albicans (
8,
16).
The published SEM micrographs show an
irregular appearance of
the fibrils. It is not clear whether these
fibrils are present
constitutively or are synthesized in response to
some environmental
cues. Nevertheless, they were found in mature
colonies of the
O smooth morphology (
8), but there is no
documentation of their
presence in
C. albicans colonies of
other
morphologies.
The fibrils found in
S. cerevisiae differ from those
observed in bacteria. First, only a single fibril was found between two
neighboring
S. cerevisiae cells, whereas in bacteria, the
number
is much greater (
1,
2). Second, the diameter of the
S. cerevisiae fibrils is significantly larger than those
found in bacteria.
These differences imply that the
S. cerevisiae fibrils fulfill
a novel
function.
The picture that emerges from many studies is that colonies can be
regarded as well-organized multicellular organisms rather
than lines of
cells that coexist in close proximity (
11). According
to the
present study, this notion also holds true for
S. cerevisiae colonies, whose level of organization has not been appreciated
in the
past. One distinction that can be made between unicellular
organisms
and "true" multicellular organisms is that organization
of the
former is not an obligatory feature of the cell community
under all
conditions, but rather an induced feature occurring
in response to
environmental
signals.
| |
ACKNOWLEDGMENTS |
We thank Naomi Bahat for excellent technical assistance in taking
the SEM photomicrographs and James A. Shapiro for valuable advice.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Microbiology and Biotechnology, Faculty of Life Sciences,
Tel-Aviv University, Ramat Aviv 69978, Israel. Phone: (972)-36409030.
Fax: (972)-36409407. E-mail:
lcchoder{at}post.tau.ac.il.
| |
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Journal of Bacteriology, July 2000, p. 3877-3880, Vol. 182, No. 13
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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