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First published online 15 August 2007
doi: 10.1242/dev.000893
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Department of Biochemistry and Molecular Biology and the Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104 USA
* Author for correspondence (e-mail: Cwest2{at}ouhsc.edu)
Accepted 5 July 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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12% to near or above ambient (21%)
levels. Overexpression of P4H1 reduces the O2 requirement to
<5%. The requirement for P4H1 can be met by forced expression of the active
enzyme in either pre-stalk (anterior) or pre-spore (posterior) cells, or
replaced by protein kinase A activation or addition of small numbers of
wild-type cells. P4H1-expressing cells accumulate at the anterior end,
suggesting that P4H1 enables transcellular signaling by the tip. The evidence
provides novel genetic support for the animal-derived O2-sensor
model of prolyl 4-hydroxylase function, in an organism that lacks the
canonical HIF
transcriptional factor subunit substrate target that is a
feature of animal hypoxic signaling.
Key words: Prolyl hydroxylase, Hypoxia, Oxygen, Dictyostelium, Cytoplasmic glycosylation, Skp1
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Solitary
amoebae aggregate to form migratory slugs consisting of multiple pre-stalk
cell types and pre-spore cells. Sensory mechanisms acting in the anterior
pre-stalk A cells guide the slug from its relatively dark, humid and cool
subterranean habitat to the soil surface, where it executes a slug-to-fruit
switch and undergoes culmination. This yields a sessile fruiting body
consisting of spores supported aerially on top of a vertical stalk, which
facilitates spore dispersal. The slug-to-fruit switch, thought to be mediated
by the anterior tip (Smith and Williams,
1980), is subject to regulation by NH3, light,
O2 and other external cues by mechanisms that involve at least ten
genes (Schindler and Sussman,
1977; Newell et al.,
1969; Sandona et al.,
1995; Newell and Ross,
1982). Genetic and biochemical studies suggest that an
NH3-sensor/transporter, 2-component His kinases (DhkA, DhkC and
DhkK), a cAMP phosphodiesterase (RegA), protein kinase A, an
E3(SCFFbxA)-Ub ligase, transcription factor-like proteins (STATa,
STATc, CudA, MybC), and cytoplasmic red-ox proteins are involved in
transducing the signals (Harwood et al.,
1992; Mohanty et al.,
2001; Kirsten et al.,
2005; Singleton et al.,
2006; Choi et al.,
2006; Thomason et al.,
2006).
Culmination appears to be exquisitely O2-dependent in
Dictyostelium, requiring O2 at a level of greater than 10%
(Sandona et al., 1995),
whereas growth and early development are relatively normal, down to 2.5%
(normoxia=21%). Many acute and chronic sensors and signaling pathways for
O2 availability have been identified in prokaryotes and eukaryotes.
Recent studies have revealed a major role for cytoplasmic/nuclear prolyl
4-hydroxylases (PHDs, HPHs or EGLNs) as mediators of hypoxic sensing in
animals (Kaelin, 2005b;
Dann and Bruick, 2005). PHDs,
encoded by three genes in humans, depend on physiological levels of
-ketoglutarate (KG) and ascorbate for optimal activity, and are
rate-limited atmospheric levels of O2. PHDs were discovered as
destabilizers of HIF(1-3), a family of basic helix-loop-helix-type
transcription factor subunits that mediate activation of hypoxia-associated
genes. The PHDs modify two Pro residues in HIF. The resulting 4-HyPro
residues render HIF a target for polyubiquitylation by the
E3VHLUb-ligase, resulting in its degradation by the 26S-proteasome.
Hypoxia limits PHD activity, which spares HIF, allowing it to dimerize
with HIFß and activate appropriate target gene transcription. PHD may
also have other hydroxylation substrates
(Kuznetsova et al., 2003).
Hypoxia might regulate PHD activity directly via O2 starvation, or
indirectly via reactive oxygen species
(Kaelin, 2005a) or effects on
other regulatory factors and substrates, such as Krebs cycle intermediates
(Briere et al., 2005;
Lee et al., 2005).
Dictyostelium possesses an ortholog of the animal HIF1-type
PHDs. P4H1 was discovered as a factor required for the glycosylation of Skp1
(van der Wel et al., 2005), a
small cytoplasmic protein known best as an adaptor of the SCF-class of
E3-Ub-ligases (Zheng et al.,
2002; Willems et al.,
2004) that is evolutionarily related to the VHL class described
above. The HyPro residue on Skp1 is subsequently capped by an GlcNAc
residue (van der Wel et al.,
2002b), which is then extended by a series of four more
glycosylation reactions (West et al.,
2004), resulting in the formation of a HyPro-linked
pentasaccharide (Teng-umnuay et al.,
1998). This modification appears to be conserved in selected other
protists (West et al., 2004;
West et al., 2006).
Dictyostelium lacks basic helix-loop-helix-type transcriptional
factors (Eichinger et al.,
2005) such as HIF, which are the best-known targets of PHDs
in mammals. A phenotypic analysis of the P4H1-null strain used to establish
the requirement of P4H1 for Skp1 glycosylation
(van der Wel et al., 2005)
revealed a specific effect on culmination, as occurs in hypoxia. Evidence
presented here suggests that PHDs mediate an unexpectedly ancient mechanism of
O2 sensing, predating animals, that in Dictyostelium
regulates culmination non-cell autonomously from the tip of the slug.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). For development, HL-5 grown cells were washed by
centrifugation and deposited on Millipore filters, non-nutrient agar or water
agar (for migrating slugs). Plates were incubated under overhead (ceiling)
fluorescent lighting in humid chambers at 22°C in ambient atmosphere, or
in sealed plastic boxes through which prepared mixtures of humidified
O2 and N2 (Airgas) flowed. Some trials were conducted
under strong overhead light (two 40 W fluorescent bulbs at 50 cm) or in the
dark. Strain NC-4 slugs and pre-culminants were dissected as described
previously (West and McMahon,
1979), lysed by freeze-thaw in 20 mM Tris-HCl (pH 8.0), and
centrifuged at 17,000 g for 30 minutes to yield a soluble S17
fraction. Cellulose stalk tubes and slime sheath were observed using 0.1%
Calcofluor White ST (American Cyanamid) in 10 mM potassium phosphate (pH 6.5).
lacZ expression was analyzed cytochemically as described
(Ferguson et al., 1994).
RFP-fluorescence was recorded from slugs partially flattened under a supported
coverslip. Spore number was determined in a hemacytometer after vortexing
filters in 15 ml polyethylene tubes containing 0.5% NP-40, 5 mM EDTA (pH 8.0).
Phase contrast and fluorescence images were photographed digitally and
processed in Adobe Photoshop to display the image over the full
28-step range, without changing contrast. Fluorescence intensity
was quantified using the Histogram function in Photoshop.
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promoter (pVS-P4H1)
(van der Wel et al., 2005) was
modified by removing its discoidin promoter using XhoI and
KpnI, and replacement by new promoter DNA amplified with primers
containing XhoI or KpnI sites in their 5' tails. P4H1
promoter DNA was amplified from genomic DNA using primers
5'-AACTCGAGAACTCTTGTTTCTGCTTAATTAAATTATTAAACT-3' and
5'-TTGGTACCTAATACCCGAAAGTAGTTCTTACCACACC-3' (restriction
sites in italics) in a PCR reaction, and cloned into pCR4TOPO (Invitrogen).
This includes the entire 740 nt of intergenic region between P4H1 and the stop
codon of the adjacent, upstream, forward-oriented gene. ecmA promoter
DNA, referred to as ecmA(FL), was amplified from genomic DNA using
5'-TTCTCGAG- TTTTTTTATTTTTTATTTTTTTAACTTTACAAAGC-3' and
5'-TTGGTACCTTTCAACGTTATAATTTTTAAACTAATGATTTTAG-3'. This
includes 1832 nt upstream of the ecmA start codon that matches the
full-length pDd63 promoter construct described previously
(Early et al., 1993).
cotB promoter DNA was amplified from cotBGal17
(Fosnaugh and Loomis, 1993)
using 5'-TTCTCGAGGACATTGTGTTATTATTTGTGTGAAAAAAA-3' and
5'-TTGGTACCTTTATTACTGGTACTTTTACTATATTAATGGTATATGTATA-3',
which includes 1714 nt upstream of the cotB start codon as described
previously. PCR-amplified DNA was identical to corresponding sequences at
dictyBase
(http://www.dictybase.org).
The resulting plasmids are pVSP-P4H1 (phyA), pVSE-P4H1
(ecmA) and pVSC-P4H1 (cotB).
Point mutations were introduced as previously described
(van der Wel et al., 2002a).
Primers for the D132N mutation, 5'-GGATCCATAGAA-
ATTCAAATAGTAGAATTCAAGATA-3' and 5'-CTATTTGAATT
TCTATGGATCCATTGAATATAATCAC-3', resulted in a silent
mutation, A387C, which creates a new BamH1 site, and G394A, the
desired missense mutation. Numbering starts at ATG of the start codon and
changed nts are in bold. Primers for the R276A mutation,
5'-TAATTTCGAA
CCAGCTATTGCAATTACAACTTGGATTTATAG-3' and
5'-TAATTGCAATAGCTGGTTCGAAATTACATTGTAAAACTTCAT-3',
resulted in a silent mutation, T819C, which creates a new BstB1 site,
and A826G/G827C/A828T, the desired missense mutation.
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) and
pTX-RFP, derived from pTX-GFP (Levi et
al., 2000) by replacement of GFP by RFP from mRFP-mars, were from
the Dictyostelium Stock Center
(http://dictybase.org/StockCenter/StockCenter.html).
EcmA(construct S)-Gal (Zhukovskaya et al.,
2006) was from J. G. Williams. The A7-Neo vector for
overexpression of the catalytic subunit of PKA
(Anjard et al., 1992) was from
C. Anjard.
The phyA::RFP expression plasmid was created by amplifying the RFP coding
region of mRFPmars (Fischer et al.,
2006) using
5'-AAGGTACCAAAATAAAATAAAAAAATGGCATCATCAGAAGATGTTATTAAAG-3'
and 5'-AAAGATCTTGCACCTGTTGAATGTCTACCTTCT-3', by PCR and
cloning into pCR4TOPO. The KpnI site in codon T127 of RFP was
eliminated by site-directed mutagenesis that did not alter the amino acid
sequence, using primers 5'-AAATTAAGAGGTACTAATTTTCCATCAGATGGT-3'
and 5'-TGATGGAAAATTAGTACCTCTTAATTTAACTTTATAAAT-3', as above. The
coding region was excised with KpnI and BglII, and cloned
into similarly digested pVSP, yielding pVSP-RFP. For expression of labile GFP,
the coding region was excised from pV18-I-S65T-GFP (from the Dictyostelium
Stock Center) and cloned into pVSP-RFP(T127T) digested with SacI and
BamHI, yielding pVSP-lGFP.
Plasmids were electroporated into growing Dictyostelium, and
G418-resistant cells were selected at 20 or 120 µg/ml G418 to enrich for
chromosomally integrated low- or high-level expressors, and cloned on bacteria
plates (van der Wel et al.,
2005). Strains are listed in
Table 1.
anti-P4H1 antiserum
Recombinant P4H1 was purified from Escherichia coli as described
(van der Wel et al., 2005),
mixed with Freund's complete/incomplete adjuvant, and injected into rabbits
(21st Century Biochemicals). Preimmune serum and antiserum from the seventh
bleed were used at 1:100.
Protein expression analysis
Whole cells (1-2x106) or soluble S17 or S100 fractions
were subjected to SDS-PAGE and western blotting
(van der Wel et al., 2005).
Skp1 was analyzed on 15-20% polyacrylamide gradient gels. Blots were blocked
in 3% bovine serum albumin or 5% nonfat dry milk in Tris-buffered saline, and
antibodies were prepared in the same solutions. Alexa 680-labeled secondary
antibodies (Molecular Probes) were used at 1:10,000, and blots were imaged in
a Li-Cor Odyssey infrared scanner.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). As
confirmed here (Fig. 1A),
incubation in the range of 2.5-10% O2 resulted in an arrest of
development before culmination. At the lower end of the range (2.5%),
development to the pre-culmination stage was also delayed by about 5 hours.
Development was blocked immediately in anoxia (100% N2), but
resumed after return to normoxia with a 4-8 hour delay (not shown).
Proliferation in axenic media was nearly normal down to 2.5% O2,
but less than one population doubling occurred at 1%
(Fig. 1C). Therefore
culmination was selectively dependent on O2 in the life cycle.
Hypoxic slugs induced pre-stalk and pre-spore promoter::lacZ markers
(see below) normally and did not initiate stalk formation (not shown). Hypoxic
shift studies showed that the sensitive period preceded culmination by 7-8
hours, i.e. a shift down from 21% to 5-10% O2 before 12 hours
blocked culmination, whereas a shift up from 5-10% to 21% before 12 hours
allowed normal developmental timing (Fig.
1B). To quantitate development, detergent-resistant spores were
counted and spore numbers were found to correlate with a visual assessment of
culmination (Fig. 1D), as in
all studies reported below.
Protein kinase A (PKA) has been implicated in regulation of the
slug-to-fruit switch at culmination
(Kirsten et al., 2005).
Activation of PKA by overexpression of the catalytic subunit of PKA under its
own promoter (PKAcat) partially rescued culmination in 10% O2
(Fig. 1D). Therefore, mild
hypoxia selectively blocks culmination with a crucial time that precedes
culmination by 7-8 hours, via a signaling pathway involving PKA.
P4H1-null cells require higher O2 levels to culminate
Examination of P4H1- cells, created previously to investigate
the requirement of P4H1 for Skp1 glycosylation, revealed a failure to
culminate in normoxia similar to the hypoxia arrest described above
(Fig. 2A). In most trials,
spore numbers ranged from <0.01-1% of normal strain number
(Fig. 2B), but variable
penetrance was observed (see below). Occasionally, aerial slugs consisting of
an elongate mass of undifferentiated cells supported by a delicate, collapsed
Calcofluor-positive slime sheath were seen (not shown), suggesting an abortive
attempt to bypass the culmination block. Other phases of the life cycle,
including proliferation in axenic media
(Fig. 1C) or on bacteria (not
shown), and development up to the slug stage, appeared normal. Culmination was
also relatively normal in a pgtA-null mutant
(Fig. 2A,B), which lacks a
glycosyltransferase that extends the HyPro-dependent glycan on Skp1 past the
first sugar, and the O2 threshold for culmination was
indistinguishable from that of normal cells (not shown). Thus the culmination
block was not attributable to inability to build HyPro-dependent glycan chains
beyond the GlcNAc core.
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20% of the trials (n=30), up to 80% of the normal number of
spores was produced with a delay of 3-8 hours. When breakthrough occurred,
fruiting bodies typically appeared around the perimeter of the filter or in
clusters, suggesting microenvironmental influence and possible positive
feedback regulation. Culmination is normally stimulated by light and
O2, and inhibited by NH3. In a trial in which
P4H1- cells spontaneously culminated, breakthrough was suppressed
by NH4Cl at concentrations that did not affect the parental strain
Ax3 (Fig. 2C). When developed
at 40-70% O2, culmination was consistently normal
(Fig. 2D) without restoring
Skp1 glycosylation (not shown). To investigate the mechanism of breakthrough,
cells were developed over a range of O2 levels. In the
dose-response study shown in Fig.
2E, under standard light conditions P4H1- cells
developed normally at 18 and 21% O2, but not at 15% O2.
In the same trial, parental Ax3 cells required 12% O2. Therefore
the proximity of the elevated O2 requirement to the ambient
O2 level (21%) may make culmination sensitive to variations in
other factors that also influence culmination. To test this idea, parallel
cultures were developed in the dark, which normally delays or suppresses
culmination. This resulted in a 3% increase in the level of O2
required for culmination of both parental and mutant cells
(Fig. 2E). Therefore, absence
of P4H1 increases the O2 level required for culmination by 6-10% to
a level that makes culmination at ambient conditions sensitive to variations
in NH3, light and other, unknown, factors, which probably explains
the variable penetrance. In an attempt to identify other factors that might
influence mutant culmination, P4H1- cells were grown on axenic
media or bacteria to different terminal cell densities, developed on different
substrata (filter or non-nutrient agar) at different pH values (5.5-7.4) and
ionic strength, or in PO4 or MES buffer. None of these variations
rescued culmination at ambient conditions (not shown). To minimize effects of
variable penetrance, which was not observed among replicates, all comparisons
were done within the same experimental trial. Altogether, the results suggest
that O2, P4H1, light, NH3 and probably other factors
contribute additive signals that influence the slug's decision to culminate
and, in the mild hypoxic range (10-21% O2), the role of
O2 depends on P4H1.
Cell differentiation in mutant slugs
Instead of culminating, mutant slugs appeared to continue migrating based
on accumulating slime sheath tracks visible on the filter surface. Slugs
consist of 80% pre-spore cells and 20% pre-stalk cells
(Early et al., 1993). Both
normal and P4H1- slugs accumulated an early pre-spore cell marker,
the spore coat protein precursor SP85/PsB
(Zhang et al., 1999), with
normal kinetics (Fig. 3A). By
contrast, SpiA, a late pre-spore marker expressed during culmination
(Richardson et al., 1994), was
barely detected in P4H1- cells but expressed normally in a control
strain containing randomly integrated P4H1-knockout DNA and in
pgtA- cells. Microscopic examination in the presence of
Calcofluor revealed that no cellulosic stalk tube was formed (not shown).
These results are consistent with mutant arrest before culmination.
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). P4H1- and Ax3 pre-culminants exhibited a similar
pattern of ß-galactosidase expression, confirming a normal pattern of
pre-spore cell differentiation (Fig.
3B). However, occasional blue cells in the anterior region of
normal pre-culminants, indicative of low-level pre-spore cell mixing, were not
observed in mutant slugs.
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), exhibited a similar pattern in both Ax3 and
P4H1- pre-culminants and slugs
(Fig. 3C). EcmA(construct
O)-Gal and EcmA(construct S)-Gal, which are similarly expressed in PstA cells,
located at the tip, were also expressed normally in the mutant
(Fig. 3D and not shown).
Ecm0-Gal, specific for pre-stalk 0 cells behind the PstA cells, was also
expressed normally (Fig. 3E),
although a somewhat higher level of background lacZ staining was
observed in the pre-spore region of mutant slugs. Therefore, the absence of
P4H1 interrupted development after the formation of an apparently normal
slug.
P4H1 and Skp1 are expressed in pre-stalk and pre-spore cells
Culmination is regulated in part by the action of anterior tip cells (see
Introduction). To address whether tip cells express P4H1, a phyA::RFP
promoter reporter was stably transfected into normal cells using a multi-copy
integrating DNA construct. Cells of clonal isolates exhibited varied
fluorescence during growth (not shown). During development, RFP fluorescence
was observed in the apical tip (Fig.
4A) and throughout the tipped aggregate (panel B), as well as
throughout the slug (panel C) and pre-culminant (panel D), suggesting that the
phyA/P4H1 gene is expressed in all developing cells. A labile GFP
reporter construct was examined to detect expression in a more restricted time
frame, but no GFP fluorescence was observed above background (data not shown).
To determine if the P4H1 protein was present in tip cells, dissected tips and
pre-spore regions were compared by western blot analysis using an anti-P4H1
antiserum developed against full-length recombinant P4H1 purified from E.
coli. This antiserum recognizes P4H1 in extracts of normal stationary
stage cells, but is not reactive, except for a faint presumably cross-reacting
band, in P4H1- cells (panel E). Preimmune serum was negative (data
not shown). Similar levels of P4H1 were detected in both regions of the slug,
suggesting that the P4H1 protein is constitutively expressed. To address
whether P4H1 is active, Skp1 was examined by western blotting. Skp1 was
present in similar levels in both regions of the slug, and exhibited a
mobility similar to that of normal Skp1 rather than that of Skp1 from
P4H1- cells, which is not hydroxylated and glycosylated (panel F).
Altogether, the data strongly suggest that P4H1 is expressed and active in the
tip and throughout the slug.
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promoter. Although below the level of
detection by western blotting using anti-myc Ab
(Fig. 5A, lane `D'), expression
was sufficient to fully modify Skp1 based on reduced electrophoretic mobility
as a result of restored hydroxylation-dependent glycosylation
(Fig. 5B). The complemented
strain formed fruiting bodies at the normal time and normal numbers of
detergent-resistant spores (not shown). Similar results
(Fig. 5C) were obtained when
P4H1 was expressed under its own promoter (phyA::P4H1; lanes `P' in
panels A,B). To determine if P4H1 is required in a specific cell type, P4H1 was expressed in pre-stalk cells under control of the pan-pre-stalk cell-specific full-length ecmA promoter [ecmA(FL)], which is similar to the ecmA0 promoter construct above, or in pre-spore cells using the pre-spore-cell-specific cotB promoter. P4H1-myc accumulated in slugs (Fig. 5A, lanes marked `E' and `C') at levels higher than achieved under the phyA or discoidin promoters, but not earlier in stationary stage cells as expected (not shown). In confirmation of activity, the majority of slug Skp1 glycosylation was rescued in ecmA(FL)::P4H1 and cotB::P4H1 slugs (Fig. 5B), and not in corresponding stationary stage cells as expected (not shown). The finding that most Skp1 was modified in either strain was surprising considering that these promoters are strictly cell-type specific; this may result from cell-type switching as discussed below. Expression of P4H1-myc in either pre-stalk or pre-spore cells rescued normal culmination and spore numbers (Fig. 5C), showing that ectopically expressed P4H1-myc functionally substitutes for endogenous P4H1.
To determine if complementation required enzyme activity, a point mutation
(R276A) was introduced into the ecmA(FL)::P4H1 plasmid at a conserved
Arg that coordinates the C-5 carboxyl group of KG and is required for
collagen prolyl 4-hydroxylase activity
(Myllyharju and Kivirikko,
1997). Both P4H1(R276A)-myc and another point mutant (D132N) at a
weakly conserved site were expressed at similar levels
(Fig. 5D). The R276A mutant
failed to modify the electrophoretic mobility of Skp1 consistent with the
expected absence of P4H1 activity. These cells did not culminate
(Fig. 5E), indicating that the
prolyl 4-hydroxylase activity of P4H1 is required for complementation. By
contrast, the D132N mutant, which partially restored Skp1
hydroxylation/glycosylation consistent with expression in only pre-stalk
cells, fully supported culmination. Therefore P4H1 requires its prolyl
4-hydroxylase activity to complement its deficiency.
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Chimera studies suggest that the culmination signal is provided by
pre-stalk cells at the anterior tip (Smith
and Williams, 1980). Pre-stalk cells represent about 20% of cells
in the slug, similar to the percentage of normal cells that rescue culmination
in chimeras. To determine their fate, normal cells were stably transfected
with actin15::RFP. Slugs formed from 7% Ax3-RFP and 93% unlabeled
P4H1- cells accumulated an excess of Ax3-RFP cells at the anterior
tip (Fig. 6B) compared with the
rest of the slug. Tip sorting was not an intrinsic property of the marked
strain, as sometimes occurs (Ferguson et
al., 1994), because Ax3-RFP cells did not preferentially sort to
the tip of unlabeled Ax3 or pgtA-null slugs. Similar tip accumulation
was observed when strain Ax2-expressing calnexin-GFP (gift of M. Clarke, OMRF,
Oklahoma City, OK) was mixed with P4H1- cells (not shown), showing
that sorting was dominant across strain types. Tip sorting was not as
pronounced as when Ax3-RFP cells were mixed with unlabeled fbxA-null
cells (not shown) (Ennis et al.,
2000).
To ensure that differential sorting of strain Ax3 was not the result of RFP expression, labeling was reversed. Mixtures of P4H1- cells transfected with actin15::RFP and unlabeled Ax3 cells at a ratio of 3:7 showed the reverse fluorescence pattern, i.e. less fluorescence in the pre-stalk region (Fig. 6C). This is consistent with Ax3 migrating to the tip and excluding labeled P4H1- cells. No sorting was observed when P4H1--RFP cells were mixed with unlabeled P4H1- cells, as expected. To determine if P4H1- cells complemented with P4H1 behaved as wild-type cells, they were also mixed with 30% P4H1--RFP cells. Like normal Ax3 cells, cotB::P4H1 and ecmA::P4H1 cells each appeared to exclude P4H1--RFP cells from the pre-stalk zone, as shown in Fig. 6C. P4H1- cells expressing cotB::P4H1 therefore must migrate anteriorly after first expressing P4H1 posteriorly in the pre-spore zone, where cotB is active. This behavior is consistent with their signaling culmination from the tip, as inferred from the similar behavior of wild-type cells and when P4H1 is expressed in pre-stalk cells by means of the ecmA promoter.
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Overexpression of PKAcat under its own promoter in P4H1- cells partially rescued culmination (Fig. 5C). As in O2 signaling (Fig. 1D), P4H1 therefore appears to function upstream of PKA, although the possibility that PKA functions as a bypass suppressor cannot be excluded.
P4H1 overexpression reduces the O2 requirement for culmination
If hypoxia inhibits culmination by limiting hydroxylation of one or more
P4H1 substrates, then culmination might be rescued by higher levels of the
enzyme protein. Stable transfection of normal Ax3 cells with the
pre-stalk-cell-specific ecmA(FL)::P4H1 overexpression constructs used
in the complementation studies above allowed development-specific expression
of P4H1 at a level (Fig. 7A)
comparable to that seen in P4H1- cells
(Fig. 5A). These cells
exhibited near full rescue of culmination and spore formation (75% of normal)
at 10% O2, and partial rescue (33% of normal) at the more stringent
5% O2 level (Fig.
7B). Stable transfection with pre-spore-cell-specific
cotB::P4H1, which also resulted in detectable P4H1 expression,
yielded partial rescue. Overexpression of P4H1 under its own promoter or the
discoidin promoter (not shown) led to negligible rescue, consistent with the
low level of expression achieved during development as detected by western
blotting (Fig. 7A). The related
overexpression strains formed in the P4H1- background, described in
Fig. 5, also did not culminate
in hypoxia (not shown), indicating an essential role for endogenous P4H1 in
rescue. In conclusion, overexpression of P4H1 enabled culmination at an
O2 level below that required by normal slugs.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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2FucT is inconsequential based on analysis of a
pgtA-null strain (Fig.
2). These data suggest a model that culmination is uniquely
regulated by O2 via a mechanism that involves the cytoplasmic
prolyl 4-hydroxylase P4H1.
Several lines of evidence reinforce the model that P4H1 mediates
O2 signaling of culmination in vivo. Hypoxia and disruption of
phyA (P4H1) interrupt development at the same stage
(Fig. 1A,
Fig. 2A). They are each
suppressed by activation of PKA (Fig.
1D, Fig. 5C), a
master regulator of culmination (Harwood
et al., 1992; Kirsten et al.,
2005), which suggests they function in the same pathway. P4H1 is
expressed throughout the slug before culmination
(Fig. 4). P4H1 activity is
rate-limited by O2 in vitro
(van der Wel et al., 2005),
with a Km of 40% O2, and the ability of
expressed P4H1 to complement the P4H1 deficiency is dependent on its
hydroxylase activity (Fig. 5E).
Extra copies of the P4H1 enzyme are expected to yield increased hydroxylation
in limiting O2. These findings offer direct genetic support for the
general model, derived from studies in animals, that P4H1 is a physiologically
important O2 sensor (Schofield
and Ratcliffe, 2004; Kaelin,
2005b). Further studies are required to differentiate whether P4H1
is regulated by O2 directly or indirectly, such as via
O2 radicals, -ketoglutarate (a co-substrate), and/or other
Krebs cycle intermediates that competitively inhibit the enzyme
(Lee et al., 2005;
Koivunen et al., 2007).
The P4H1- defect appears to involve a failure to send rather
than respond to a culmination signal, because culmination in ambient
O2 is rescued by co-development with small numbers of normal cells.
RFP-tagged normal cells accumulate in the slug tip and become PstA cells
(Fig. 6), which are thought to
normally regulate the slug-to-fruit switch. Forced expression of P4H1 in
either pre-stalk cells (under the ecmA-promoter) or pre-spore cells
(under the cotB promoter) of mutant slugs also rescues culmination.
Interestingly, both of the P4H1-expressing cell populations tend to accumulate
in the anterior region, as do normal cells
(Fig. 6C). In addition, forced
expression of P4H1 in pre-stalk cells is more effective than in pre-spore
cells for reducing the O2 dependence of normal cell culmination
(Fig. 7). Finally, pre-stalk
cell PKA can influence culmination
(Harwood et al., 1992).
Altogether, the data support the model that P4H1 signals culmination via
anterior cells. However, as sorting of pre-spore cells expressing P4H1 is not
absolute and P4H1 is expressed in all slug cells
(Fig. 4), a role for pre-spore
cell P4H1 in culmination is not excluded.
P4H1 appears to influence culmination jointly with other environmental
factors. O2 and P4H1 function synergistically with overhead light
(Newell et al., 1969) to
regulate culmination. The O2 requirement of both normal and mutant
cells is decreased by 2-3% when cells are developed in overhead light compared
with darkness (Fig. 2E).
Previous studies suggested that overhead light attracts the tip aerially,
which promotes culmination indirectly
(Bonner et al., 1982), although
in the present study of stalled pre-culminants, tips appeared to be elevated
from the substratum in either light or dark conditions (not shown). The
slug-to-fruit switch is also normally regulated by NH3
(Kirsten et al., 2005), a
catabolic by-product of slug metabolism. NH3 inhibits culmination
in P4H1- cells at lower concentrations than those required to
affect normal cells (Fig. 2C),
as described for other slugger mutants
(Gee et al., 1994), suggesting
that O2 and NH3 signaling interact. Evidence indicates
that NH3, O2 and P4H1 each influence culmination via
PKA. Genetic manipulations that affect superoxide levels and the cellular
redox balance also affect culmination
(Garcia et al., 2003;
Jeong et al., 2006;
Choi et al., 2006) and, as PHD
function depends on O2, reduced Fe+2 and ascorbate,
which may be affected by superoxides
(Kaelin, 2005a;
Kaelin, 2005b), these agents
might also signal via P4H1. In addition, hyperoxia rescues P4H1-
culmination (Fig. 2D), which
suggests a second mechanism by which O2 regulates culmination. This
might involve one of the other four PHD-like genes in the
Dictyostelium genome (West et
al., 2004). The slug-to-fruit switch is evidently regulated by a
complex pattern of extracellular signals, and subtle variations, in concert
with microenvironmental effects and potential positive feedback interactions
between slugs suggested by edge and group effects, may explain the 18-21+%
range of O2 required for culmination in different trials.
A candidate target of P4H1 action is Skp1, the glycosylation of which
requires P4H1 in Dictyostelium and which was used to monitor P4H1
activity in the mutants. However, Skp1 is glycosylated normally, within the
resolution of western blot analysis, in slugs of Ax3, and even HW403
(cotB::phyA in P4H1- background) or HW404
(corresponding ecmA::phyA expression strain), formed in
5-10% O2 (our unpublished data). In addition, Skp1 is not
glycosylated in 40% O2 (not shown), which overrides inhibition of
culmination in P4H1- cells. Although these results show that
hydroxylation of the bulk pool of Skp1 is not regulated in parallel with
culmination, a role for Skp1 is not excluded, as a critical subpool (e.g.
nascent, nuclear or pre-stalk) might be differentially hydroxylated. Skp1 is
the only substrate that accumulates in P4H1- cells based on an
indirect assay with rP4H1 and rGnT1 (our unpublished data). A role for Skp1 is
further supported by evidence that CulA and FbxA, proteins that physically
associate with Skp1 in SCF-type E3 Ub-ligase complexes
(Willems et al., 2004), are
also required for multiple developmental steps, including culmination
(Mohanty et al., 2001;
Nelson et al., 2000;
Ennis et al., 2000). However,
P4H1- slugs do not accumulate the E3(SCFFbxA)Ub-ligase
substrate RegA (not shown), as occurs in culA- and fbxA-null
cells (Mohanty et al., 2001).
Yet it is intriguing that the SCF class of E3-Ub-ligases, which contains
HyPro-Skp1, is evolutionarily related to the VHL class that recognizes
HyPro-HIF in animals. Additional studies are needed to evaluate the
O2 dependence of the hydroxylation of Skp1, which is encoded by two
genes, in the slug tip, and the role of GnT1, which mediates addition of the
GlcNAc cap to HyPro-Skp1 (van der Wel et
al., 2002b). The best-known substrate of animal PHDs, HIF1
and the E3VHLUb-ligase that recognizes HyPro-HIF1, are
apparently absent from the Dictyostelium genome based on
bioinformatics studies (not shown), but other potential targets
(Kuznetsova et al., 2003)
occur.
Hypoxic regulation of culmination may provide a selective advantage to
Dictyostelium in its normal habitat. Hypoxia encountered by
developing cells in confined or water-saturated microenvironments may signal
delay of culmination. In this scheme, O2 synergizes with
NH3 depletion and light, which can override a suboptimal level of
O2 (Fig. 2E). The
crucial period for O2 regulation is many hours in advance of
culmination (Fig. 1B), and
cells having the highest P4H1 activity tend to migrate to the slug tip
(Fig. 6), suggesting
complexities that remain to be explored. Another function for O2 in
Dictyostelium, cytochrome oxidase subunit VII switching
(Sandona et al., 1995), does
not involve P4H1 based on DNA microarray studies (L. Eichinger and C.M.W.,
unpublished). The role of P4H1 in O2-dependent slug polarization
and guidance of slug migration (Sternfeld
and Bonner, 1977; Sternfeld
and David, 1981) remains to be examined. Because hypoxic
regulation is connected with sporulation, Dictyostelium offers a
unique genetic opportunity to investigate the mechanism of P4H1 action,
including its role as a direct O2 sensor and the identification of
upstream and downstream regulatory genes.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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