Cell Division Laboratory, Temasek Life Sciences Laboratory, 1 Research Link, The National University of Singapore, Singapore 117604
* Author for correspondence (e-mail: mohan{at}tll.org.sg)
Accepted 2 December 2002
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Summary |
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Key words: Sterol, Plasma membrane, Polarity, Cytokinesis, Schizosaccharomyces pombe
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Introduction |
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S. pombe grows in a polarised manner at the cell tips and divides
by medial fission by the constriction of an actomyosin ring and concomitant
synthesis and deposition of septum material
(Chang, 2001). These processes
require the polarised localisation of proteins involved in cell wall and
septum synthesis, as well as the vectorial addition of new membrane material
to the existing plasma membrane. Therefore, we asked whether inherent
differences in the lipid composition of the plasma membrane might be important
for proper targeting of the growth and division machinery. To this end, we
decided to examine the localisation of sterols in S. pombe using the
fluorescent probe filipin, a polyene antibiotic that forms specific complexes
with free 3-ß-hydroxysterols. Excitation of filipin at 360 nm results in
strong fluorescence with an emission maximum at 480 nm
(Drabikowski et al.,
1973
).
In this study, we report that sterols are localised to distinct regions of the plasma membrane in a cell-cycle-dependent manner. Of particular importance is our finding that membrane sterols are detected at the division site and appear to play multiple roles in cytokinesis.
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Materials and Methods |
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Results |
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Sterols are enriched at the growing cell tips and at the site of
cytokinesis
To determine whether the localisation of sterols to the cell tips is
connected to cell growth, we used filipin to probe mutant strains or cells
grown under conditions that cause characteristically altered growth patterns.
Nitrogen starvation was induced by incubating for 24 hours a prototrophic
wild-type strain in medium lacking any source of nitrogen, which led to the
appearance of small, rounded, non-growing cells. These cells exhibited greatly
reduced levels of fluorescence when stained with filipin
(Fig. 2A). After release from
nitrogen starvation, cells resumed growth and exhibited wild-type fluorescence
intensity and pattern (Fig.
2B). Thus, the presence of sterols in the plasma membrane can be
correlated to sites of cell growth.
|
Using temperature-sensitive mutants we tested whether an altered growth
pattern could be reflected in the sterol distribution. Shift of
cdc10-V50 cells to the restrictive temperature causes a cell-cycle
block in G1-phase before growth at the new end is initiated,
leading to a monopolar growth pattern
(Nurse et al., 1976). Filipin
staining of arrested cells resulted in strong fluorescence at one end of the
cell only (Fig. 2C). Using GFP
fused to the actin-binding calponin homology domain of Rng2p (CHD-GFP; K. Eng
and M.K.B., unpublished), we confirmed that actin patches concentrate at the
same end as sterols (Fig. 2D),
suggesting that this is the growing old end of the cell. The new end showed
staining that was relatively weak in intensity and which covered a smaller
membrane area compared with the old end or both growing ends in a wild-type
strain. Hence, the distribution of sterols in the plasma membrane of cells
growing at the old end only is biased towards the growing tip. We assume that
the sterols detected at the new end stem from the previous cytokinesis that
occurred before the cell-cycle arrest.
Glucose-starved cells differ from cells starved from nitrogen in the cell-cycle stage at which they arrest (the majority are in G2 for glucose starvation and in G1 for nitrogen starvation) and are therefore not at the verge of initiating mating, which might require the reorganisation of the membrane. Glucose starvation was induced by incubating for 24 hours a prototrophic wild-type strain in medium lacking glucose. When stained with filipin, these cells exhibited greatly reduced levels of fluorescence (Fig. 2E) as compared with control cells grown in medium with glucose (Fig. 2F). Hence, the situation in glucose-starved cells resembles nitrogen-starved cells rather than the new end of arrested cdc10-V50 cells. However, it is possible that the uptake of sterols from the plasma membrane is a rather slow process and therefore, sterols are reduced, but still clearly detectable, at the new end of cdc10-V50 cells after the temperature shift, which is short lasting compared with the induction of starvation.
The shift of cdc25-22 cells to the restrictive temperature causes
a cell-cycle block at the G2/M-boundary a stage of the cell
cycle at which growth occurs at both tips
(Russell and Nurse, 1986).
Arrested cells showed filipin staining at both ends of the cell but never in a
medial band (Fig. 2G). Thus,
localisation of sterols in a medial band depends on entry into M-phase.
orb mutants that are shifted to the restrictive temperature have
been shown to exhibit unpolarised growth, leading to large, rounded cells
(Snell and Nurse, 1994).
Filipin-stained orb1-13 cells showed fluorescence covering the entire
circumference of the cell (Fig.
2H). We conclude that cells lacking growth polarity also lack
polarity in the distribution of sterols within the plasma membrane.
We then assessed whether sterols were detected at ectopic new ends
generated by treatment of cdc10-V50 cells with the
microtubule-depolymerising drug TBZ (Sawin
and Nurse, 1998). We probed branched cells with filipin and
observed that the ectopically growing tip was brightly stained, in many cells
with an even higher fluorescence intensity than at the endogenous tips
(Fig. 2I). Our results with
cells growing in monopolar, unpolarised or ectopical patterns indicate that
the distribution of sterols in the plasma membrane is correlated to the growth
pattern of the cell.
Mating projections constitute a specialised form of naturally occurring
polarised growth in S. pombe in which morphogenesis directed towards
pheromones overrides the usual antipodal growth observed in vegetative cells.
To examine whether sterols can be detected in mating projections, we induced
synchronous meiosis in a homothallic wild-type strain as described
(Beach et al., 1985). Filipin
staining showed that sterols in the plasma membrane were polarised towards the
projection (Fig. 2J),
indicating that enrichment of sterols in the plasma membrane also occurs
during mating projection formation. Our observations support recent studies in
S. cerevisiae that had shown that lipid rafts cluster at the mating
projection in budding yeast (Bagnat and
Simons, 2002
).
Sexual development and meiosis in S. pombe results in the
formation of four ascospores. As cell wall synthesis in ascospores requires
enzymes that are different from those required for cell wall formation during
vegetative growth (Liu et al.,
2000; Martin et al.,
2000
), we tested to see whether sterol localisation was also
evident in the plasma membrane underlying spore walls. Filipin uniformly
stained the plasma membrane of spores generated from a cross between wild-type
cells of opposite mating types (Fig.
2K). Interestingly, although the fluorescence intensity was
approximately the same as that at the growing tips of cells in vegetative
development, photobleaching of filipin occurred much more rapidly in spores
than in vegetative cells. It would be interesting to perform further
experiments to address the function of sterols in the plasma membrane of
ascospores.
A shift of mid1-18 cells to the restrictive temperature leads to
mispositioned actomyosin rings and septa
(Chang et al., 1996;
Sohrmann et al., 1996
;
Balasubramanian et al., 1998
).
We found that the plasma membrane invaginations associated with the site of
cell division in mid1-18 cells are stained by filipin even when these
sites are misplaced (Fig. 2L).
The medial band of sterols next to the invaginated membrane was found to
localise in a similar manner. Hence, we conclude that the sterol enrichment of
the plasma membrane at the site of cytokinesis is coupled to the localisation
of the division machinery.
The distribution of sterols is regulated in a cell-cycle-dependent
manner
To characterise the emergence of sterols in the medial region of the cell,
cdc25-22 cells expressing Hht2-GFP
(Wang et al., 2002) and
Rlc1-GFP (Naqvi et al., 2000
)
as nuclear and actomyosin ring markers were released from a cell-cycle block
at 36°C to 18°C, which allowed a better temporal resolution of mitotic
and cytokinetic events than release to 24°C. We found that the formation
of the actomyosin ring and nuclear division occurred before the concentration
of sterols in a medial band (Fig.
3; 0-45 minutes). However, constriction of the actomyosin ring was
observed after sterol targeting to the middle of the cell
(Fig. 3; 60-90 minutes).
Filipin also stained the plasma membrane invaginations that follow the
constricting ring (Fig. 3; 105
minutes). After constriction of the ring to a dot the plasma membrane
separating the two daughter cells exhibited intense fluorescence
(Fig. 3; 120 minutes). These
experiments establish that sterols are concentrated at the division site
following assembly of the actomyosin ring, and persist during ring
constriction and septum assembly. A medial band of sterols was observed only
in cells undergoing cell division. Hence, the localisation of sterols to the
middle of the cell is regulated in a cell-cycle-dependent manner.
|
Sterol localisation requires a functional secretory pathway
Our time-course studies led to the possibility that the formation of the
actomyosin ring might serve as a spatial and temporal landmark for sterol
localisation to the middle of the cell. To test this idea, we investigated
whether an intact F-actin cytoskeleton is required for sterol localisation to
the plasma membrane overlying the actomyosin ring. On release of
cdc25-22 cells from the cell-cycle arrest we added the
actin-polymerisation inhibitor LatA or DMSO as solvent control. The effective
disruption of the actin cytoskeleton in LatA-treated cells was confirmed by
rhodamin-phalloidin staining (data not shown). Sixty minutes after release we
found that similar fractions of LatA-treated cells and of control cells
exhibited medial sterol localisation (Fig.
4A; arrows). This localisation was observed at the centre of the
cell and symmetrically on both sides, suggesting that it forms a band
circumventing the cell. We conclude that the F-actin cytoskeleton is not
required for the localisation of sterols to the middle of the cell. In
addition to the medial band, many LatA-treated cells showed patches of filipin
staining at varying positions between the tips and the middle of the cell
(Fig. 4A; arrowheads). In
contrast to the medial band, these patches were frequently distributed in an
asymmetric manner, indicating that they did not form bands around the long
axis of the cell. The emergence of sterol patches at various sites of the
plasma membrane may be due to continued secretion of sterol-rich membrane
material with a simultaneous lack of endocytosis caused by disruption of the
F-actin cytoskeleton. Such disturbance of the equilibrium between secretion
and endocytosis of sterols may cause an excess of sterols at the plasma
membrane, leading to the formation of additional sterol patches.
|
To address the role of the microtubule cytoskeleton in sterol localisation
before cytokinesis, we used nda3-KM311, a cold-sensitive mutant that
does not form microtubules when shifted to 18°C
(Toda et al., 1983). We
counted cells with medial sterol localisation over a course of 6 hours after
downshift. The percentage of cells with a medial band increased with the time
of incubation at low temperature (Fig.
4B). We conclude that microtubules are not necessary to localise
sterols to the middle of the cell. Additionally, this result suggests that
arrest of cells at metaphase by the spindle assembly checkpoint does not
prevent the medial localisation. To test if this was the case, we
overexpressed mad2+ as an alternative means to activate the spindle
assembly checkpoint, thereby leading to metaphase arrest
(He et al., 1997
).
Overexpression of mad2+ led to a high percentage of cells
with a medial band of sterols compared with the control, confirming that cells
arrested at metaphase localise sterols in a medial band
(Fig. 4B). Although previous
experiments using cdc25-22 synchrony indicated that sterols are
detected at the division site at later stages of cytokinesis, the
mad2+ overexpression and nda3-KM311 experiments
indicate that sterols are detected there in metaphase-arrested cells. A
possible explanation for this inconsistency is that low levels of sterols may
accumulate in cycling cells at metaphase. Although these low levels may be
difficult to detect readily, this localisation may become more obvious in
metaphase-arrested cells due to the continued accumulation of sterols.
We then investigated whether a functional secretory pathway was necessary
for sterol localisation to the plasma membrane overlying the actomyosin ring.
On the release of cdc25-22 cells from the cell-cycle arrest we added
BFA or ethanol (EtOH) as solvent control. It has been shown that in yeast, BFA
inhibits transport from the endoplasmic reticulum (ER) to the Golgi apparatus
(Graham et al., 1993). Sixty
minutes after release we scored these cells for medial sterol localisation. A
high percentage of control cells exhibited bright medial filipin staining
(Fig. 4C), but almost all
BFA-treated cells showed none or only faint fluorescence in the middle of the
cell (Fig. 4C; arrow). This
indicates that a functional secretory pathway is required for efficient medial
sterol localisation. The faint band in BFA-treated cells may be caused by an
incomplete block of secretion. Alternatively, it is possible that a pool of
sterols may be present in a post-Golgi compartment that can still be targeted
to the plasma membrane when the ER-to-Golgi transport is blocked. Moreover, we
observed that BFA-treated cells exhibited a low level of fluorescence
throughout the plasma membrane whereby the tips no longer appeared brighter
than the rest of the plasma membrane. One explanation for this finding is that
endocytosis of sterol-rich membrane material may occur at the cell tips.
Studies in mammalian cells have shown that a pool of intracellular cholesterol
is present in the endocytic recycling compartment
(Mukherjee et al., 1998
;
Hao et al., 2002
). In case of
an intact secretory pathway, this membrane material may be recycled in a
polarised manner to the plasma membrane at the cell tips, whereas a block of
secretion may result in the accumulation of endocytic vesicles and subsequent
fusion with the plasma membrane in a locally unrestricted manner.
Because the strongest filipin fluorescence is observed during septum
deposition, we investigated whether the Septum Initiation Network (SIN) might
be involved in regulating the localisation of sterols to the site of
cytokinesis. To test this idea we used cdc7-24, a
temperature-sensitive mutant that renders the SIN pathway inactive
(Nurse et al., 1976). These
cells expressed Cdc4-GFP as an actomyosin ring marker (V. M. D'souza and
M.K.B., unpublished). After shift to 36°C for 4 hours, hardly any cells
showed medial staining as observed during cytokinesis
(Fig. 4D), regardless of the
presence or absence of an actomyosin ring (data not shown). However, the
majority of cells exhibited a faint filipin staining in the middle of the cell
(Fig. 4D; arrows). This
staining was often asymmetrical and extended over a varying length of the
cell. Many cells formed bulges at the site displaying faint staining, this
being reminiscent of cells beginning to form ectopic cell tips after TBZ
treatment. We therefore assume that the faint staining that we observe in
cdc7-24 is due to an attempted initiation of growth at a site where
the new cell end would be, had the cell undergone cytokinesis. The lack of
strong filipin staining in the middle of the cell in a cdc7-24 mutant
seems to conflict with our findings for cells arrested at metaphase, i.e. a
stage of the cell cycle that occurs before the SIN pathway is activated. A
possible explanation is the bypass of the post-anaphase array stage of the
cell cycle in SIN mutants. Hence, we propose that not an active SIN pathway
but the cell-cycle stage (between metaphase spindle stage and post-anaphase
array stage) is crucial for sterol localisation as a medial band. This would
correlate the staining with the timing of the orientation of the secretion
machinery towards the medial region.
Manipulating the integrity of sterol-rich membrane domains leads to
defects in cytokinesis
The localisation pattern of sterol-rich membrane domains suggests multiple
roles for sterols in polarity and/or growth on the one hand and in cytokinesis
on the other hand. To gain insight into the possible functions of sterols, we
attempted to alter the structure of sterol-rich membrane domains in the plasma
membrane of S. pombe cells. We have chosen to do so in two different
ways: by extended incubation in filipin and by overexpression of C-4 sterol
methyl oxidase.
In addition to the use of filipin as a probe for sterols in fluorescence
assays, incubation in high filipin concentrations and/or for an extended
time-span has been shown to induce deformations in sterol-containing membranes
that can be observed by freeze-fracture and electron microscopy techniques
(Friend and Bearer, 1981;
Severs and Robenek, 1983
).
Filipin also disrupts the structure and function of caveolae, non-coated
cholesterol-rich invaginations in the plasma membrane of mammalian cells
(Rothberg et al., 1990
). Cells
expressing Cdc4-GFP were grown to logarithmic phase. Filipin or DMSO as
solvent control were added and Cdc4-GFP epifluorescence was visualised after
60 minutes. In a high proportion of filipin-treated cells the localisation of
Cdc4-GFP was distorted (Fig.
5A), whereas control cells showed proper formation and positioning
of uniform Cdc4-GFP rings (Fig.
5B). The observed phenotypes ranged from occurrence of misshapen
ring-like structures in the medial region of the cell
(Fig. 5A; arrow) to the
accumulation of spots of various intensities
(Fig. 5A; arrowhead).
Occasionally, we observed rings that had drifted out of their original
position. The higher percentage of cells with Cdc4-GFP staining after filipin
treatment (33.9±5.7% compared with 19.7±1.6% of control cells)
indicates that filipin-treated cells divide inefficiently. Filipin staining in
the medial region of the cell was observed in some cells
(Fig. 5A,
Fig. 6A). However, we observed
numerous cells without medial staining in which distorted Cdc4-GFP rings were
approximately in their original position. The filipin signal at the cell tips
was visible in all examined cells.
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Using a screen for gene products which, on overexpression, lead to
cell-division abnormalities, we have isolated C-4 sterol methyl oxidase
encoded by an S. pombe homologue of S. cerevisiae ERG25
(Bard et al., 1996;
Li and Kaplan, 1996
), an
enzyme in the ergosterol biosynthetic pathway. Interestingly, when we
overexpressed C-4 sterol methyl oxidase, we observed a similar range of
Cdc4-GFP localisation phenotypes as with filipin treatment: Cdc4-GFP rings
were mis-shapen and mispositioned, and more frequently formed cables that ran
along the long axis of the cell (Fig.
5C). Control cells in which C-4 sterol methyl oxidase was
repressed by thiamine or which were transformed with an empty vector showed
wild-type-looking Cdc4-GFP rings and sterol distribution (data not shown).
After extended overexpression of C-4 sterol methyl oxidase, some cells became
elongated and branched (data not shown). The filipin staining was not
restricted to distinct areas of the plasma membrane though the cell tips, and
the middle of the cell showed the strongest fluorescence
(Fig. 5C). The overall
fluorescence intensity was higher than in control cells. Similar to
mid1-18 cells, septum material was deposited at aberrant positions
according to the misplaced actomyosin ring. Studies in S. cerevisiae
had reported that overexpression of ERG25 led to an overall pattern
of lipids and sterols similar to wild type
(Li and Kaplan, 1996
).
However, possible changes in the relative abundance of sterol intermediates
would not have been detected by this analysis. Because Erg25p is involved in
the C-4 demethylation of 4,4-dimethylzymosterol, it is possible that its
overexpression may lead to an accumulation of downstream intermediates in the
ergosterol biosynthetic pathway. We conclude that changes in the structure or
composition of sterol-rich membrane domains cause various defects in
actomyosin ring positioning and/or maintenance.
Manipulating the integrity of sterol-rich membrane domains
destabilises a colocalising plasma membrane protein
To examine the effects of extended filipin treatment on the plasma
membrane, we observed the intracellular distribution of the integral membrane
protein Bgs4p, a 1,3-ß-glucan synthase subunit that localises to the
plasma membrane at the growing cell tips and at the site of cytokinesis
(Liu and Balasubramanian,
2001). Cells expressing Bgs4-GFP were grown to logarithmic phase.
Filipin or DMSO as solvent control was added and Bgs4-GFP epifluorescence was
visualised after 60 minutes. In filipin-treated cells the localisation to
distinct sites of the plasma membrane was completely abolished
(Fig. 6A). Instead, a weak GFP
signal was distributed throughout the cell. By contrast, control cells showed
Bgs4-GFP localisation at the tips and the division site
(Fig. 6B). The loss of Bgs4-GFP
from the tips and the division site could be due to relocalisation from the
plasma membrane or to a reduction in Bgs4-GFP levels. To discriminate between
these possibilities we analysed equal amounts of total protein from
filipin-treated cells and from control cells treated with DMSO. On
immunoblots, anti-GFP antibodies recognised a single protein, with the
predicted molecular weight of 252 kDa. The band was easily detectable in the
control, but only a very weak signal was observed in the filipin-treated
sample. Quantitation showed that the signal strength in filipin-treated
samples was reduced to below 4% of the strength in control samples
(Fig. 6C). Our observations
show that the stability of a membrane protein that colocalises with
sterol-rich membrane domains is compromised when the structure of these
domains is altered.
These results suggest that sterol-rich membrane domains play an important role in positioning and/or maintenance of the actomyosin ring. One possible explanation is that proteins involved in ring maintenance may localise to intact sterol-rich membrane domains. The disruption of these membrane structures may affect the stability, localisation and/or function of these proteins. Another possibility is that sterol-rich membrane domains may be involved in anchoring the ring to the plasma membrane. The identification of proteins linking the actomyosin ring and the plasma membrane will provide further insight into the relevance of sterols in the regulation of cytokinesis.
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Discussion |
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It is crucial for the morphogenesis of S. pombe that it tightly
regulates where and when cell growth and division occur
(Verde, 1998). Therefore,
another possible role of sterol-rich membrane domains may be the spatial
limitation of the growth machinery and the division machinery to the growing
cell tips and to the site of cytokinesis, respectively.
Finally, recent work on cellulose synthesis in plants has implicated
sitosterol-ß-glucosides in 1,4-ß-glucan (cellulose) chain initiation
(Peng et al., 2002).
1,3-ß-glucan is an abundant cell-wall component in S. pombe, and
in growing cells 1,3-ß-glucan synthases localise to the cell tips and the
division site (Liu and Balasubramanian,
2001
; Liu et al.,
2002
). Hence, sterol-linked molecules may play a role in the
synthesis of the cell wall and septum as a primer or substrate. Being amenable
to molecular biology as well as to genetics and having a well-studied cell
cycle, S. pombe will be a suitable model organism to test the models
discussed above and to define the function of sterol-rich membrane domains in
cell polarity and cytokinesis.
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Acknowledgments |
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