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Vol. 14, Issue 5, 2163-2180, May 2003
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*Health Sector, Biotechnology Research Institute, National Research Council of
Canada, Montreal, Quebec, H4P 2R2, Canada; and Departments of Biochemistry and Department of Biology, McGill University, Montreal, Quebec, H3G 1Y6, Canada
Submitted May 5, 2002;
Revised November 15, 2002;
Accepted January 23, 2003
Monitoring Editor: Douglas Koshland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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).
The environmentally stimulated yeast to hyphal transition in Candida
is mediated by several signaling pathways
(Whiteway, 2000), the most
characterized of which involve MAP kinase and cAMP-based signaling. However,
several factors influence hyphal formation independent of the known pathways
in Candida (Whiteway,
2000; Liu, 2001),
and hyphal growth involves regulation beyond the level of transcription
(Torralba and Heath, 2001),
supporting the existence of additional regulatory networks and reinforcing the
complexity of the response systems.
In the model fungus Saccharomyces cerevisiae, morphogenesis is
tightly coordinated with cell cycle progression. Bud growth and the
corresponding polarization of actin, synthesis of DNA, and duplication of
spindle pole bodies occurs at the G1/S transition, whereas elongated or
pseudohyphal growth is associated with a block in G2 mediated by
Swe1p-dependent negative regulation of Cdc28p
(Kron and Gow, 1995;
Rua et al., 2001).
Polar or isometric growth of buds can be maintained by overexpression of G1 or
mitotic cyclins, respectively (Lew and
Reed, 1995). In filamentous and dimorphic fungi, the connection
between cell cycle factors and true hyphal growth is less clear. Hyphae
continue to grow whether their apical-most nuclei are in interphase or mitosis
(Kron and Gow, 1995), and it
was recently demonstrated that the duration of cell cycle stages was similar
in yeast, germlings, and apical hyphal cells of C. albicans
(Hazan et al., 2002).
However, the coordination between nuclear localization, division, and
septation with the initiation and maintenance of hyphal growth in
Candida requires a relationship between aspects of the cell cycle and
hyphal growth. For example, during the yeast to hyphal transition, the nucleus
and septins move to a hyphal specific position out in the developing germ tube
before the first mitosis, after which one daughter nucleus migrates with the
growing tip, while the other moves back into the mother yeast cell
(Gale et al., 2001;
Sudbery, 2001). Septation
follows each nuclear division in established hyphae, creating uni-nucleate
subapical compartments of similar length that remain arrested in G0 or G1
until branching takes place (Kron and Gow,
1995). A few cell cycle factors have been characterized in
Candida; these include the G1 cyclin CLN1, which is required
for maintaining hyphal growth (Loeb et
al., 1999) and the CDC2-related kinase
CRK1, which can promote hyphal formation
(Chen et al., 2000).
In addition, a forkhead transcription factor that regulates B-cyclin gene
expression is required for hyphal growth in Candida
(Bensen et al.,
2002).
The polo-like kinases (PLKs) comprise a family of cell cycle regulators
with the potential to influence hyphal morphogenesis in Candida,
because they function at various stages during the initiation and progression
through mitosis and are required for septation/cytokinesis (reviewed in
Nigg, 1998;
Alexandru et al.,
2001; Toyoshima-Morimoto
et al., 2001). Furthermore, the PLK homologue Cdc5p in
S. cerevisiae physically interacts with septins and Swe1p
(Bartholomew et al.,
2001; Song and Lee,
2001) and can alter cell morphology by generating elongated buds
upon overexpression (Song et al.,
2000).
To explore the relationship between cell cycle factors and hyphal morphogenesis in C. albicans, we investigated the role of a PLK homologue, CaCDC5. We demonstrate that CaCdc5p is required for spindle elongation and that gene repression under yeast growth conditions leads to dramatic hyphal-like growth. Similar inhibition of spindle elongation and a corresponding induction of filaments with hydroxyurea suggest there is an endogenous mechanism in place to connect aspects of the cell cycle and the hyphal signaling networks in C. albicans.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Control strains including RM1000 and CB102 were grown in
the same media supplemented with uridine and histidine as required. To repress
CaCDC5 expression from the PCK1 promotor, yeast cells were
grown for 24 h in SS medium, washed with dH2O, and then diluted
into SD medium to an OD600 of 0.25. SD medium with or without
methionine and cysteine was used to repress and induce expression,
respectively, from the Candida MET promotor
(Care et al., 1999).
Hyphal-inducing conditions involved the addition of 10% heat-inactivated fetal
calf serum (GIBCO/Invitrogen, Burlington, Ontario) to growth medium and
incubation at 37°C. For hydroxyurea (HU) treatment, cells of strain
SC5314, CB110, CR216, JKC19, HLC52, and HLC54 were grown overnight in yeast
extract/peptone/dextrose medium (YPD) or SD medium and then diluted to an
OD600 of 0.4 in fresh medium containing 200 mM HU (Sigma Chemical
Co., St. Louis, MO). Overexpression of CaCdc5p-GFP was performed in S medium
containing 2% casaminoacids (Leuker et
al., 1997).
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Transformation and Southern and Northern Analyses
Cells were transformed using lithium acetate
(Chen et al., 1992),
and DNA and RNA were extracted according to Rose et al.
(1990) and Köhrer and
Domdey (1991), respectively.
Southern analysis was performed using the DIG Hybridization System (Roche
Diagnostics, Mannheim, Germany). To determine the level of
PCK1::CaCDC5 expression, cells from strains RM1000, CB102, and CB104
were grown to an OD600 of 0.8 in SS media at 30°C or to an
OD600 of 0.2 followed by washing and transferring to SD media.
Strain CB104 was collected after 4, 7, or 24 h in SD medium, whereas strains
RM1000 and CB102 were collected at 4 h, because these dividing yeast cells
entered stationary phase with longer incubation periods. Total RNA, 20 µg,
was analyzed using a 32P-labeled (T7 Quick Prime Kit, Amersham
Pharmacia Biotech, Piscataway, NJ) PCR product of CaCDC5 and
hybridization conditions described by Srikantha and Soll
(1993). 32P-labeled
PCR fragments from HWP1, DDR48, ECE1, and ALS1 were used to
probe total RNA from strains CB102, CB104, SC5314 grown in SD medium, and
SC5314 grown in SD medium plus 200 mM HU. An ACT1 probe
(Rocha et al., 2001)
was used as a loading control. Northern blots were visualized with a
phosphoimager.
Cloning and Plasmid and Strain Production
To regulate expression of CaCDC5, a strain containing a single
copy of CaCDC5 under control of the PCK1 promotor was
created. Sequence data for C. albicans was obtained from the Stanford
Genome Technology Center website at
http://www-sequence.stanford.edu/group/candida.
Sequencing of C. albicans was accomplished with the support of the
NIDR and the Burroughs Wellcome Fund. The open reading frame plus 800 and 1500
base pairs of 5' and 3' flanking sequence, respectively, of
CaCDC5 was PCR amplified from genomic DNA derived from strain SC5314
using primers CB1F and CB1R and cloned into the KpnI/PstI
sites of vector pTZ18R, creating plasmid pCB100. Primers CB3F and CB3R
reverse-amplified the flanking and vector sequences from pCB100, into which
the BamHI/BglII URA3 blaster cassette
(Fonzi and Irwin, 1993) was
cloned, creating plasmid pCB101. Primers CB4F and CB4R were also used to
amplify the flanking DNA and vector sequence from pCB100, into which a
NotI/BamHI PCR-amplified CaHIS1 ORF was cloned,
creating plasmid pCB102. The URA3-containing deletion construct was
liberated from pCB101 with KpnI and PstI and transformed
into strain RM1000. Transformants were screened by PCR and Southern analyses
to confirm the complete elimination of one copy of CaCDC5 and
creation of strain CB100. CB100 was plated on 5-fluoorotic acid (5-FOA) to
select for the URA3-strain CB101. The ORF of CaCDC5 plus 300
base pairs of terminator sequence was PCR amplified from genomic DNA using
primers CB5F and CB5R and cloned into the KpnI site following the
PCK1 promotor in the URA3 blaster-containing plasmid pJA24
(a kind gift from J. Ash, derived from plasmid p5921 from
Fonzi and Irwin, 1993),
creating plasmid pCB103. pCB103 was cut at a unique XhoI site within
the PCK1 promotor sequence to direct integration at the PCK1
promotor locus in strain CB101, creating strain CB102. The empty plasmid was
transformed as a control, creating control strain CB103. The
HIS1-containing deletion construct was liberated from pCB102 with
SphI and SacI and transformed into strain CB102 to replace
the second endogenous copy of CaCDC5 while in the presence of
succinate, creating strain CB104. Removal of the URA3 marker by
plating onto 5-FOA resulted in strain CB105. All strains were analyzed by PCR
and Southern analyses to confirm correct integration of transforming DNA and
replacement of both endogenous copies of CaCDC5 (our unpublished
results), and all PCR-derived clones were sequenced. For additional controls,
strain RM1000 was transformed with plasmid pRM100, which contains Candida
URA3 and HIS1, producing strain CB400. Strain CB401 consisted of
one of several transformants from strain CB102 which did not correctly
integrate the HIS1 knockout cassette, remaining heterozygous for
CaCDC5 but HIS1+. These transformants behaved identically to
strains RM1000 and CB102 (supplemented with uridine and histidine) when
switching between glucose and succinate-containing medium (our unpublished
results).
To regulate expression of CaCDC5 in another strain using a
different promotor, strain CAI4 was transformed with the URA3-based
deletion construct from plasmid pCB101, to create strain CB106. CB107 was
created by plating CB106 on 5-FOA. A 3'-truncated copy of
CaCDC5, lacking 1000 base pairs upstream from the stop codon, was PCR
amplified with primers CB6F and CB6R and cloned into the
PstI/BamHI sites following the MET promotor in
plasmid pCa-DIS (Care et al.,
1999), creating plasmid pCB106. The plasmid was cut with
ClaI to direct integration at the remaining endogenous copy of
CaCDC5 in strain CB107. The resulting strain, CB108, contained a
full-length copy of CaCDC5 under control of the MET promotor
and a 3' truncated copy with no terminal processing sequence. As a
control, strain CB107 was transformed with empty pCaDIS plasmid, creating
strain CB109.
To regulate expression of CaCDC5 in
cacdc35
/cacdc35 and
efg1/efg1,
cph1/cph1 mutant backgrounds, strains CR276 and
HLC69, respectively, were transformed with the URA3-containing
knockout cassette liberated from plasmid pCB101. Colonies heterozygous for
CaCDC5 were grown on 5-FOA and then transformed with
ClaI-cut plasmid pCB106 to allow integration at the remaining
endogenous copy of CaCDC5, resulting in strains CB303 and CB305.
Analysis by Southern and Northern analyses confirmed the correct integration
of constructs and proper regulation of the remaining copy of CaCDC5
under control of the MET promotor (our unpublished results).
A GFP-tagged 
-tubulin strain was created by PCR-amplifying a
5'-truncated copy of CaTUB1, containing 1000 base pairs
upstream from the STOP codon and 300 base pairs of terminator sequence, from
genomic DNA isolated from strain SC5314 with primers CB50F and CB50R. The PCR
fragment was cloned into the BglII/SacI sites of plasmid
p5921 (Fonzi and Irwin, 1993),
creating plasmid pCB104. Primers CB51F and CB51R were designed to bind
immediately up and downstream of the STOP codon, respectively, and to
reverse-amplify the plasmid sequence, into which a XhoI/SpeI
PCR fragment (produced with primers CB44F and CB44R) of GFP
(Morschhauser et al.,
1998) was cloned, creating plasmid pCB105. pCB105 was digested
with BstEII for directed integration at the endogenous TUB1
locus, creating full-length 3'-tagged TUB1-GFP, under control
of its own promotor, and a 5'-truncated copy. pCB105 was integrated into
strains RM1000, CB101, and CB105 to create strains CB110, CB111, and CB112,
respectively.
CaCdc5p was tagged with GFP according to the same protocol. A 5'-truncated BamHI/PstI fragment of CaCDC5, containing 1000 base pairs upstream of the STOP codon and 300 base pairs of terminator sequence, was cut from plasmid pCB100 and cloned into plasmid p5921, creating plasmid pCB107. Primers CB43F and CB43R annealed immediately upstream and downstream of the STOP codon of 5'-truncated CaCDC5 in plasmid pCB107 and reverse amplified the plasmid, into which the XhoI/SpeI-containing GFP PCR fragment was cloned, creating plasmid pCB108. pCB108 was cut at BstEII for site-directed integration at the endogenous CaCDC5 locus in strain RM1000, creating a 3'-tagged copy of CaCDC5-GFP and a 5'-truncated copy in strain CB114. PCK1::CaCDC5-GFP was constructed in a similar way, using primers CB43F and CB43R to reverse amplify the gene plus vector sequence from plasmid pCB103, into which the XhoI/SpeI-containing GFP PCR fragment was cloned, creating plasmid pCB109. pCB109 was integrated at the PCK1 promotor in strain RM1000, creating strain CB115. CDC12-GFP in plasmid pVEC, a kind gift from Dr. Ursula Oberholzer, was transformed into strain CB105 to create strain CB113.
Cell Staining and Microscopy
Nuclei and septa were visualized by fixing cells in 70% ethanol for 1 h,
followed by incubation in 1 µg/ml 4',6'diamidino-2-phenylindole
dihydrochloride (DAPI, Sigma) for 20 min and 1 µg/ml calcofluor white
(Sigma) for 10 min. Immunofluorescence was performed by fixing cells in an
equal volume of double-strength fixative solution, containing 8%
paraformaldehyde (Sigma) in 80 mM PIPES buffer, pH 6.8, 5% DMSO (Sigma), 10
µg/ml leupeptin (Sigma), 4 mM AEBSF (Roche Diagnostics), and 1 µM
aprotinin (Roche Diagnostics), for 30 min, followed by washing with 1x
PIPES buffer, pH 6.8 (40 mM). For immunolocalization of -tubulin, cell
walls were then digested for 10 min at 37°C with 10 µg/ml zymolase (ICN
Biomedicals, Aurora, OH) in 40 mM PIPES buffer, pH 6.8, 5% BSA (Sigma), and
protease inhibitors described above. Membranes were permeabilized with 0.1%
Nonidet P-40 (BDH, Poole, England) in 40 mM PIPES, pH 6.8, for 5 min.
Immunolocalization of 16B1-F10
(Marot-Leblond et al.,
2000) omitted these two steps. Cells were incubated in 1/100
dilution of monoclonal anti– tubulin clone B-5–1-2 (Sigma),
or 1/500 dilution of Mab 16B-F10, in 1x PIPES buffer, 0.05% sodium azide
(Sigma), and 5% BSA overnight. After washing in PIPES buffer, cells were
incubated in 1/100 dilution of donkey anti-mouse FITC-coupled secondary
antibody (Sigma) for 2 h, washed, and stained with DAPI and calcofluor white
as described above. Nonspecific binding of the secondary antibody was
investigated by preparing a paired sample without primary antibody. A signal
was not detected (our unpublished results). To determine cell viability,
unfixed cells were stained with 10 µg/ml propidium iodide (Sigma) and
immediately visualized.
Cells were examined on a Leitz Aristoplan microscope using 10x, 40x, or 100x (1.32 NA) objectives with Nomarski differential interference contrast (DIC) or epifluorescence optics and on a Leica DMIRE2 inverted microscope using a 100x (1.32 NA) objective with phase contrast and fluorescence optics, using the appropriate filter sets.
Flow Cytometry
Cells were prepared for FACS analysis according to Lew et al.
(1992), with some
modifications. Cells (5 x 106–1 x 107
cells/ml) were fixed overnight in 70% ethanol, washed with 0.2 M Tris buffer,
pH 7.5, treated with 0.8 mg/ml RNaseA (Pharmacia, Piscataway, NJ) for 2 h at
37°C, washed, and incubated in 50 µg/ml propidium iodide (Sigma)
overnight. Cells were analyzed with a Becton-Dickinson FAC-Scan. Results are
based on 10,000–20,000 nongated events.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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).
Repression of CaCDC5 Induces Yeast Cells to Grow into Filaments
A strain containing a single copy of CaCDC5 under control of the
regulatable PCK1 promotor (Leuker
et al., 1997) was created to manipulate CaCDC5
expression (see MATERIALS AND METHODS). Northern analysis demonstrated that
CaCDC5 was overexpressed in SS-inducing medium and repressed in SD
repressing medium (Figure 1).
To confirm that the effects of repressing CaCDC5 were not due to
changes in carbon source, the heterozygote strain CB102 and parental strain
RM1000 were subjected to identical changes in medium. In addition, a second
strain containing a single copy of CaCDC5 under control of the
CaMET promotor (Care et
al., 1999) in strain CAI4 was created for comparison of
phenotype.
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Under yeast growth conditions at 30°C, overexpressing CaCDC5 in the PCK1-regulated strain (CB104) did not result in any gross changes in phenotype, because the morphologies of yeast cells and colonies were normal (Figure 2A). In contrast, shutting off CaCDC5 expression by streaking onto solid SD medium induced dramatic changes in morphology and proliferation. Yeast cells changed shape into filaments, which became highly elongate but did not branch out to form mycelial colonies. Repressing CaCDC5 expression in liquid medium at 30°C also induced the formation of filaments. By 3 h, the majority of cells contained an elongated extension, which continued to grow in a polarized manner, creating hyphal-like filaments (Figure 2B; Table 2). The filaments were initially wider in diameter (Figure 2, B and D) and grew at a mean rate of one third of that of serum-induced hyphae incubated at 37°C (0.13 ± 0.01 µm/min, n = 45, vs. 0.41 ± 0.01 µm/min, n = 32), although the presence of some longer filaments indicated an ability to grow at rates approaching that of serum-induced hyphae. Different environmental conditions and activation mechanisms could account for such differences in growth characteristics. In contrast to strain CB104, the heterozygote strain (our unpublished results) and parental strain (Figure 2B) grew as yeast under repressing conditions. The cells demonstrated a transient pseudohyphal growth stage upon switching from SS to SD medium but resumed normal yeast growth and morphology by 7 h (Figure 2B). The heterozygote strain appeared more pseudohyphal than the parental strain and contained some elongated cells, suggesting a gene-dosage effect. Repressing CaCDC5 with the MET promotor also produced filaments (Figure 2D), supporting that the phenotype is due to manipulation of CaCDC5.
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The relative absence of branching and mycelial colony formation suggested that filament growth was determinate. In support of this, transferring filaments from repressing medium back to inducing medium after 24 h allowed some reversion to yeast growth, but many cells remained trapped in a filamentous state. Several CaCDC5-repressed filaments became highly vacuolated and approximately half of the cells stained with propidium iodide after 24 h of repression (our unpublished results), indicating that the cells were dying. Therefore yeast cells switch to a new active growth mode upon repression of CaCDC5, but the cells loose viability at a later time, suggesting that either CaCDC5 is essential, or maintenance of the growth mode and the signals that generate it eventually become toxic to the cells.
CaCdc5p Is Not a Direct Negative Regulator of Hyphal Growth like
Tup1p or Nrg1p
Because the depletion of CaCdc5p results in the production of hyphal-like
filaments, the possibility that CaCdc5p is a direct negative regulator of
hyphal growth comparable to Nrg1p or Tup1p
(Braun and Johnson, 1997;
Braun et al., 2001;
Munir et al., 2001)
was investigated by analyzing CaCDC5 mRNA levels in yeast and
serum-induced hyphae and determining whether overexpression of CaCdc5p could
inhibit serum-induced hyphal growth. In contrast to that observed with Nrg1p
or Tup1p, CaCDC5 mRNA expression did not change between yeast and
hyphal cells (Figure 1B), and
serum-induced hyphal growth was not inhibited by CaCdc5p overexpression in
strain CB115 (our unpublished results). Therefore CaCdc5p does not appear to
act like general repressors of serum-induced hyphal growth, suggesting that an
aspect of CaCdc5p function is involved in generating and/or transmitting a
signal to activate the hyphal-like growth mode.
Filament Initiation upon Repression of CaCDC5 Is Associated with an
Early Block in Nuclear Division
Because CaCdc5p did not appear to be a direct negative regulator of hyphal
growth, the filaments could be forming in response to a cell cycle defect
induced by repression of CaCDC5. PLKs are essential for nuclear
division, as compromised PLK function results in a block at G2/M in most
organisms (Llamazares et al.,
1991; Okhura et al.,
1995; Lane and Nigg,
1996), or at anaphase B in S. cerevisiae
(Kitada et al.,
1993). To determine whether defects in nuclear division
accompanied filament formation upon depletion of CaCdc5p, cells incubated in
SD medium were fixed at several time points and stained with DAPI. At 3 h in
repressing medium, control yeast cells were budding, dividing, and undergoing
mitosis (Figures 2 and
3). In contrast, nuclei in the
majority of CaCDC5-repressed cells did not divide during the
formation of filaments up to 5 h, after which nuclei in 
45% of the cell
population escaped the division block
(Table 3; Figure 3). The ability of
nuclei to divide could reflect leakiness in the regulated expression of
CaCDC5. At 8 h, when the control yeast strains CB102 (our unpublished
results) and RM1000 had undergone four or five rounds of nuclear division, 65%
of CaCDC5 repressed cells contained two nuclei, whereas 28% of the
cells contained fragmented DNA (Table
3; Figure 3). Only
3.5% of the cells contained three nuclei. Fragmentation of DNA progressed up
to 24 h of repression, preventing an accurate quantification of nuclei at the
later time point. The filaments were 28.9 ± 1.5 µm (SEM; n = 45) in
length at 5 h of repression, when approximately half of the cells had escaped
the block in nuclear division, and 62.1 ± 2.6 µm (SEM; n = 45) after
8 h, when the majority of cells contained two nuclei. In contrast, the nucleus
in serum-induced hyphae at 37°C divided approximately every 1.5 h,
corresponding to hyphal lengths of 37.3 ± 1.1 µm (SEM; n = 32) and
92.8 ± 2.4 µm (SEM; n = 32) for the first and second rounds of
division, respectively (Figure
2B; Table 3). The
lack of similar coordination between filament length and nuclear division in
CaCDC5-repressed cells suggests that the block in nuclear division is
due to repression of CaCDC5 and not the initiation of a hyphal-like
growth mode. Although the nucleus eventually escapes the block in division,
normal rates of mitosis are not resumed and DNA fragmentation occurs over time
while filamentous growth continues. These results suggest CaCDC5 is
required for the early stages of nuclear division and chromatin separation,
similar to its counterparts in Schizosaccharomyces pombe and higher
organisms, but in contrast to that in S. cerevisiae.
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Staining the CaCDC5-repressed cells with calcofluor white demonstrated that septa or chitin deposition did not occur until later time points, consistent with when nuclei started to escape the block in division (Figure 3; Table 4). Taken together with the inhibition of chromatin separation, this observation suggests that CaCDC5-repressed cells were blocked at an early stage in the cell cycle, preceding septation, and that filament formation correlated with this early block.
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CaCdc5p Is Required for Spindle Elongation
Tub1p-GFP spindle patterns were analyzed to determine the specific stage in
nuclear division at which CaCdc5p is required. Tub1p was tagged with GFP in
strains RM1000, CB102, and CB104, and the resulting strains (CB110, CB111, and
CB112, respectively) responded to repressing medium in a manner similar to the
nontagged strains. The GFP-tagged CaCDC5-regulated strain (CB112),
however, was somewhat more sensitive to the absence of CaCdc5p than strain
CB104, as the filaments were shorter in length at the various time points. The
majority of cells from strain CB112 depleted of CaCdc5p for 3 h were elongated
or large doublets (Figure 4A; Table 5) and contained spindles
in the form of distinct spots or two spots side by side, corresponding to
unseparated spindle pole bodies or very short spindles in S-G2 phases of the
cell cycle (Barton and Gull,
1988; Hazan et al.,
2002). Despite the elongated and large doublet morphology of cells
at 3 h, only 2.2% of the population contained an extended mitotic spindle.
After 4–7 h, the majority of filamentous cells still contained spot or
short bar-like spindles, but an increasing proportion of cells contained a
slightly longer bar-like spindle, probably representative of G2/M, and 6% of
the filaments contained an extended mitotic spindle
(Figure 4A;
Table 5). Spindle orientation
was also disturbed in several cells, regardless of whether the nucleus was in
the mother yeast cell or in the filament. Filaments that survived after 24 h
of CaCDC5 repression contained extensive cytoplasmic microtubule
arrays resembling those in serum-induced hyphae of Candida
(Figure 4C) and hyphae of other
filamentous fungi (Han et al.,
2001; Hazan et al.,
2002), although the intensity of the Tub1p-GFP signal was greater
in CaCdc5p-depleted cells at 30°C compared with serum-induced hyphae at
37°C. The remaining dead cells either did not stain or contained a diffuse
signal. Similar patterns of spindles and cytoplasmic microtubules were
observed using immunofluorescence with anti– tubulin antibody in
the nontagged strains incubated in repressing medium for 3 h
(Figure 4B) or 24 h (our
unpublished results), respectively, indicating that the microtubule patterns
were not artifacts of GFP-tagged tubulin. Immunofluorescence demonstrated that
cytoplasmic microtubules similar to those in established hyphae were present
in the CaCdc5p-depleted filaments as early as 3 h of repression
(Figure 4B). Their relative
absence in the GFP-tagged strain at early time points is likely a reflection
of the amplified signal with immunofluorescence. In contrast, control yeast
cells of strain CB110 grown at 30°C were cycling at the different time
points and demonstrated the expected spindle patterns
(Figure 4, A and B;
Table 5). For example, of the
proportion of cells demonstrating a large budded morphology, the majority
contained a mitotic spindle (Table
5). Slightly elongated cells were present at early time points but
were pseudohyphal intermediates since they eventually budded. When the control
strain CB110 was grown in serum at 37°C for 2–3 h to induce hyphal
growth, nuclei in the apical regions of hyphae were cycling, with 8%
containing mitotic spindles (Table
5). The decrease in the proportion of CaCdc5p-depleted cells
containing spot-like spindles and the corresponding increase in cells
containing slightly longer bar spindles at a later time point (28% at 7 h
compared with 13% in serum-induced hyphae) supports the notion that the
majority of CaCDC5-repressed cells were inhibited at an early stage
in spindle elongation but eventually began to leak through the block.
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CaCdc5p Localizes to the Spindle Pole Bodies, Spindle, DNA, and Bud
Neck in Yeast
PLKs localize to spindle pole bodies/centrosomes, the spindle, chromosomes,
and sites of cytokinesis in diverse organisms
(Glover et al., 1998;
Song et al., 2000).
The localization of CaCdc5p-GFP was analyzed by integrating
PCK1::CaCDC5-GFP into strain RM1000 and overexpressing the protein
with synthetic medium containing 2% casaminoacids. The spindle, spindle pole
bodies, DNA, and bud neck clearly demonstrated a signal
(Figure 5, A–C), consistent with known PLK localizations. Similarly, a strain containing
CaCdc5p-GFP under control of its endogenous promotor demonstrated identical
localizations but with a weaker signal
(Figure 5, D–I).
Intriguingly, CaCdc5p-GFP under control of its own promotor localized to
unseparated spindle pole bodies in cells with very small buds
(Figure 5, E and G), suggesting
a function for CaCdc5p in the early stages of spindle elongation.
|
The DNA Synthesis Inhibitor Hydroxyurea Induces Similar Filament
Formation and Impairs Spindle Elongation
We and other groups (Bai et
al., 2002; Hazan et
al., 2002) found that exposing yeast cells of C.
albicans to HU produced filaments under yeast growth conditions. Because
the filaments closely resembled those of CaCDC5-repressed cells, we
analyzed the filaments further to identify common perturbed features and
therefore possible mechanisms involved in triggering filament initiation.
After 2–3 h in 200 mM HU at 30°C, cells from strain SC5314 were
elongated and developed into filaments resembling those of
CaCDC5-repressed cells (Figure
6A). By 24 h, the filaments were shorter in length than the
CaCdc5p-depleted filaments and more uniformly vacuolated. The response was
cell density dependent, as yeast cells with an OD600 greater than
0.6 did not form filaments upon incubation in HU. DAPI staining
(Figure 6A) and Tub1p-GFP
(Figure 6B) patterns in cells
from strains SC5314 and CB110, respectively, exposed to HU demonstrated that
nuclear division and spindle elongation were blocked in a way similar to that
of CaCDC5-repressed cells, although the HU-induced filaments retained
the short spindle for a longer period
(Table 5). FACS analysis
demonstrated that HU-exposed cells were blocked in S phase but progressed to a
G2 (4n) content of DNA, whereas CaCDC5-repressed cells progressed
through S phase but were subsequently blocked in G2
(Figure 6C). Some fragmentation
of chromatin occurred in both conditions at later time points
(Figure 6C). These results
demonstrate that impaired spindle elongation is a common, early defect in
HU-treated and CaCDC5-repressed cells, and thus an aspect of spindle
function may be linked to filament formation.
|
CaCDC5-repressed and HU-exposed Filaments Demonstrate Hyphal
Characteristics and Express Factors Normally Regulated by Hyphal Transcription
Pathways
The filaments described here demonstrated several hyphal-like
characteristics. A distinguishing feature of hyphal development in C.
albicans is the migration of the nucleus into the germ tube before
mitosis (Sudbery, 2001).
Nuclei in both CaCDC5-repressed and HU-exposed cells demonstrated
this behavior (Figures 3 and
6;
Table 3), where the nucleus
moved out of the mother cell and into the filament. The filaments also
contained extensive cytoplasmic microtubules, comparable to serum-induced
hyphae.
Further evidence for similarity between the filaments and true hyphae was
obtained by investigating the expression of a protein that is specifically
induced by the hyphal-generating condition of serum and high temperature,
using the MAb 16B1-F10 (Marot-Leblond
et al., 2000). Immunolocalization with 16B1-F10
demonstrated that the antigen was produced by several filaments depleted of
CaCdc5p for 7 h or exposed to HU for 24 h
(Figure 7A). The filaments did
not stain along their entire length, as seen with serum-induced hyphae,
indicating the antigen was not expressed immediately upon filament
initiation.
|
Northern analysis of cells depleted of CaCdc5p for 4, 7, or 24 h or exposed
to HU for 6 h revealed the expression of factors normally induced by the
hyphal signaling pathways in response to serum, including HWP1
(Sharkey et al.,
1999) and DDR48 (Lane
et al., 2001). The factors were not all induced at
similar stages of filament development
(Figure 7B), and certain other
hyphal-specific factors, including ECE1 and ALS1, were not
expressed (our unpublished results). Therefore, repression of CaCDC5
and exposure to HU leads to the formation of filaments and the activation of
aspects of the hyphal transcription program, suggesting a link between spindle
function and hyphal development.
Filament Formation Requires CaCDC35, but not EFG1 or CPH1
Several hyphal signaling pathways in C. albicans converge on the
transcription factors Efg1p and Cph1p
(Ernst, 2000). The absence of
both these factors prevents hyphal formation under most conditions
(Lo et al., 1997),
whereas CaCdc35p is required for hyphal formation under all conditions tested
(Rocha et al., 2001).
To determine whether these factors are required for the formation of filaments
described here, strains lacking CPH1, EFG1, and CaCDC35 were
depleted of CaCdc5p or exposed to HU. After 7 h of CaCDC5 repression
or exposure to HU, filaments were able to form normally in the double mutant
lacking both Efg1p and Cph1p (Figure
8).Efg1p and Cph1p single mutants (strains JCK19 and HLC52) also
formed filaments in response to HU (our unpublished results). However, the
absence of CaCdc35p severely compromised filamentous growth in both
conditions. The cells treated with HU or depleted of CaCdc5p looked identical
and resembled large budded yeast with some isotropically enlarged daughter
cells containing a distal polarized evagination. After 24 h, filamentous
growth was still severely compromised, but more cells contained abnormal
shapes and a short polarized extension in the CaCDC5-repression vs.
HU-treated condition (our unpublished results). This difference likely
reflects the more toxic effects of hydroxyurea and inhibition of DNA
synthesis, causing cell death more quickly than when CaCDC5 is
repressed. The results suggest that absence of CaCdc5p and exposure to HU act
through a similar pathway to produce hyphal-like filaments and communicate
with the hyphal signaling networks at the level of CaCdc35p.
|
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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CaCDC5 Is Required for Spindle Formation during S Phase
CaCDC5, along with PLKA from Aspergillus
nidulans (Bachewich and Osmani, unpublished results) are the first
characterized polo-like kinase homologues in hyphal-producing fungi. The short
spindles and unseparated chromatin that result from repression of
CaCDC5 and the localization of CaCdc5p to unseparated spindle pole
bodies in cells with small buds support a role for CaCdc5p in the early stages
of spindle elongation. Such a role is consistent with known PLK functions in
most other organisms, but the demonstration that a PLK is required as early as
S phase has not been previously reported. In contrast to higher organisms and
S. pombe, spindle initiation occurs during S phase in both C.
albicans (Barton and Gull,
1988; Hazan et al.,
2002) and S. cerevisiae
(Winey and O'Toole, 2001).
Several lines of evidence suggest a requirement for Cdc5p in spindle formation
and DNA replication during S phase in S. cerevisiae
(Hardy and Pautz, 1996;
Cheng et al., 1997;
Bartholemew et al., 2001), but the cdc5–1 mutant
arrests with elongated spindles in mitosis with partially separated chromatin
(Kitada et al., 1993)
and early spindle defects associated with compromised Cdc5p function have yet
to be reported. Therefore CaCdc5p acts earlier in the cell cycle and/or has
some different functions than Cdc5p.
Depletion of CaCdc5p Induces a Dramatic Switch in Morphology from
Yeast Cells to Actively Growing Filaments with Hyphal Characteristics in the
Absence of Serum or High Temperature
The development of filaments upon repression of CaCDC5 is
consistent with a switch to a hyphal-like growth mode, as opposed to the
terminal phenotype of slowly expiring elongated buds, because the filaments
were actively extending at one third of the rate of serum-induced hyphae
incubated at the higher temperature of 37°C, and demonstrated several
hyphal characteristics, including movement of the nucleus out of the yeast
cell into the filament, development of an extensive organization of
cytoplasmic microtubules, and expression of factors lying downstream of the
hyphal signaling pathways. In filamentous fungi, mutations in a variety of
genes result in wide diameter hyphae
(Harris et al., 1997;
Kaminskyj and Hamer, 1998;
Momany et al., 1999),
supporting that the wide filaments described here are closely related to true
hyphae. The filaments are also not analogous to elongated buds of S.
cerevisiae that form in response to an inability to deposit a septum
(Jimenez et al.,
1998), because the early block in nuclear division and spindle
elongation, and ability to produce filaments with HU, support the notion that
filaments emerge during an early stage in the cell cycle, before the timing of
septation. Septin rings, visualized with Cdc12p-GFP, appeared normal in
CaCdc5p-depleted cells at 7 h and were localized at the yeast/filament
junction (our unpublished results), supporting that septin-related defects did
not generate the filamentous growth described here. The localization of septin
rings at the neck of the filaments is consistent with filament emergence
during late S/G2 of the cell cycle and is comparable to the localization of
the first septa in serum-induced hyphae, which initiated growth later than
G1/S (Hazan et al.,
2002).
The eventual death of the filaments suggests that CaCDC5 is
essential. This possibility exists despite the fact that active hyphal-like
growth occurs upon gene repression, because hyphae of filamentous fungi can
grow for a determinate period of time in the absence of nuclear division.
However, at a later point the nucleus must divide in order for growth to
continue. For example, mutations in some essential cell cycle genes such as
nimXcdc2p34 in A. nidulans do
not prevent spore germination and determinate hyphal growth
(d'Enfert, 1997), and blocking
nuclear division in established hyphae of C. albicans also does not
prevent hyphal growth (Yokoyama et
al., 1990). Therefore, depletion of CaCdc5p and the
associated block in nuclear division trigger a change in growth mode to a
hyphal-like state, which continues for some time, but eventually the cells
loose viability in the absence of proper nuclear division.
Intriguingly, several hyphal characteristics appeared at different stages
in the development of CaCDC5-repressed filaments. Increased
expression of DDR48, movement of the nucleus into the filament, and
initiation of a cytoplasmic microtubule network were observed as early as
3–4 h after repressing CaCDC5. HWP1 expression was delayed but
normally does not increase in serum-induced hyphae until later in development,
after 60 min (Nantel et al.,
2002). Microarray analysis of the filaments indicates the
expression of additional factors that are normally induced by serum and
regulated by the hyphal signaling pathways (our unpublished results). The
differences in timing of gene expression could be due to the fact that an
internal cue, as opposed to serum and high temperature, initiated the
hyphal-like growth process, and different forks in the signaling networks were
utilized. Indeed, there are some differences in the expression of transcripts
(Nantel et al., 2002)
and utilization of known components of the hyphal signaling pathways in hyphae
produced under different environmental conditions
(Giusani et al.,
2002). Delayed expression of certain genes, especially surface or
secreted factors that are not required for hyphal development but turn on as a
consequence of hyphal growth, could also reflect differences in growth rate,
feedback regulation from the developing hypha to the transcriptional pathways,
and/or the involvement of factors required for initiation vs. maintenance of
hyphal growth, examples of which have been identified
(Nantel et al.,
2002). These results suggest that internal signaling, as opposed
to external environmental cues such as serum, may activate some
transcriptional and other regulatory aspects governing hyphal initiation.
Defects in Spindle Elongation and the Corresponding Generation of
Filaments in CaCDC5-repressed and HU-exposed Cells Suggest a Link between
Spindle Function and Activation of Hyphal Growth
CaCdc5p did not act as a direct negative regulator of hyphal formation like
Tup1p (Braun and Johnson, 1997)
or Nrg1p (Braun et al.,
2001; Munir et al.,
2001), suggesting the induction of hyphal-like growth upon CaCdc5p
depletion could be due to a CaCdc5p-dependent cell cycle–related
function. The high degree of similarity between HU-induced and
CaCDC5-repressed filaments suggests that filamentous growth was
triggered by the same cue. A common feature of CaCDC5 repression and
HU treatment is impaired spindle elongation, suggesting a link between spindle
function and the hyphal regulatory program. Consistent with this, repression
of DpbIIp, a subunit of DNA polymerase, results in determinate filament
formation in C. albicans (Backen
et al., 2000), and defects in DNA synthesis can inhibit
spindle elongation. In addition, nocodazole induces similar filaments in
C. albicans that can be partially suppressed by deletion of the
spindle checkpoint factor MAD2
(Bai et al., 2002),
suggesting filamentous growth is partially, but not fully, dependent on the
kinetochore attachment/spindle assembly branch of the spindle checkpoint.
Activation of this branch of the spindle checkpoint alone, however, is not
sufficient to induce filament formation because deletion of a homologue of the
centromere protein CENP-A in C. albicans did not result in
filamentous growth (Sanyal and Carbon,
2002). Although the data support the idea that spindle function is
involved in the cue leading to activation of hyphal-like growth, we cannot
rule out other pathways or some additional role for CaCdc5p itself, because
polo-like kinases can be negatively regulated by spindle and DNA damage
checkpoints (Sanchez et al.,
1999; Smits et al.,
2000; Hu et al.,
2001) and nocodazole and HU activate spindle checkpoints
(Hu et al., 2001;
Garber and Rine, 2002). The
nature of the internal signal and mechanism of transmission to the hyphal
regulatory pathways is currently under investigation.
The morphogenic effects of inhibiting different stages in the cell cycle of
Candida are not known, but repression of another essential cell cycle
factor in C. albicans, CaCdc42p, resulted in isometric growth under
yeast growth conditions (Ushinsky et
al., 2002). In addition, applying different stresses to yeast
cells of Candida at 30°C did not elicit the filamentous response
described here (Martchenko and Whiteway, unpublished results;
Enjalbert et al.,
2003), indicating the phenotype is not a reaction to general cell
stress.
Activation of Filament Formation Is Dependent on CaCdc35p but not
Efg1p or Cph1p
The formation of both CaCDC5-repressed and HU-exposed filaments is
dependent on CaCdc35p but not Efg1p/Cph1p, suggesting that depletion of
CaCdc5p and exposure to HU may act through similar pathways leading to
hyphal-like formation, and communication with the hyphal signaling networks
occurs at the level of CaCdc35p. The facts that CaCdc35p is predicted to act
upstream of Efg1p (Rocha et al.,
2001), Efg1p in turn was not required for filament
formation, and the filaments expressed HWP1, a factor lying down-stream of
Efg1p (Sharkey et al.,
1999), suggest that additional pathways feed into and out of
CaCdc35p for filament formation. Transcript profiling of the
cacdc35/cacdc35 strain (Harcus, Nantel, and Whiteway, unpublished
results) also supports a role for CaCdc35p outside of Efg1p regulation.
Function of a Link between Spindle Elongation and Hyphal
Development
The recent demonstration that initiation of hyphal growth in C.
albicans is not limited to one cell cycle phase suggests that the cell
cycle is not a direct regulator of hyphal growth
(Hazan et al., 2002).
The ability to induce hyphal-like formation upon depletion of CaCdc5p and
perturbation of spindle function in yeast cells, however, suggests that there
is a connection between aspects of the cell cycle and the hyphal signaling
machinery. This regulatory relationship could exist as a type of checkpoint,
perhaps with the duration and/or extent of spindle formation being monitored.
Interestingly, blocks in S phase can prevent hyphal growth in A.
nidulans, whereas mutations in some other essential cell cycle factors do
not prevent short-term hyphal growth
(d'Enfert, 1997). Regardless
of variations in different organisms, the demonstration that lack of CaCdc5p
and perturbation of spindle function influences hyphal-like growth and
transcription in Candida indicates that hyphal-like growth can be
activated by internal, cell cycle–related cues, as opposed to external
signals like serum, and introduces a new level within the hyphal signaling
networks of C. albicans.
|
ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
Footnotes |
|---|
Corresponding author. E-mail address:
catherine.bachewich{at}nrc.ca.
|
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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