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First published online 15 August 2007
doi: 10.1242/dev.000893

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Prolyl 4-hydroxylase-1 mediates O₂ signaling during development of Dictyostelium

Department of Biochemistry and Molecular Biology and the Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104 USA

View larger version (32K): [in this window] [in a new window]   Fig. 1. Hypoxia blocks culmination in the normal Dictyostelium strain Ax3. (A) Cells were developed for 25 hours on filters in an atmosphere of the indicated percentage of O2 with N2. Similar results were obtained in the presence of 1% CO2 (not shown). (B) Timecourse of effect of O2 shift on culmination visualized qualitatively at 20 hours. (C) Effect of hypoxia on cell proliferation. Ax3, P4H1oe (HW285) or P4H1- (HW288) cells (see later) were diluted to 106 cells/ml (indicated by dashed line) in growth medium (HL-5) and shaken in the indicated percent of O2 in N2. Cells were counted 24 hours later. (D) Effect of overexpressing PKAcat on the hypoxic blockade, quantitated by counting detergent-resistant spores after 36 hours. Spore numbers correlated with morphological culmination (not shown). Results are typical of three independent trials.

View larger version (42K): [in this window] [in a new window]   Fig. 2. P4H1-null Dictyostelium cells fail to culminate and form spores. (A) A normal strain containing randomly integrated P4H1 disruption DNA (bsr+), strain P4H1a- (HW288) and a pgtA- strain (HW260) were starved on non-nutrient agar under standard overhead light in ambient O2, and photographed after 19 and 23 hours. Normal cells culminate by 19 hours, but neither P4H1a- nor an independent P4H1- strain (HW289, not shown) culminated by 96 hours. Similar results were obtained in 24/30 trials (see text and panels C and E). (B) Culmination in different strains under ambient conditions was quantified by counting detergent-resistant spores. Spore numbers correlated with morphological appearance. Similar results were obtained in three or more trials, including P4H1- strains HW288 and HW289. (C) Effect of NH4Cl in MES buffer on strain Ax3 (normal) and P4H1a- in a trial under ambient conditions in which P4H1a- exhibited partial culmination breakthrough. (D) Effect of incubation in 40% O2. Similar effects were observed in ten independent trials. (E) Interplay of the effects of O2 and light on culmination, in Ax3 and P4H1a- cells, based on percent spore numbers relative to values at 21% O2, which were each within 20% of each other. P4H1a- required a lower of O2 than ambient (21%) to culminate in this trial, but nevertheless required 6% more than the parental strain Ax3.

View larger version (116K): [in this window] [in a new window]   Fig. 3. Effect of P4H1 deletion on developmental marker expression in Dictyostelium. (A) Western blot analysis for expression of the early pre-spore marker SP85 (mAb 16.1) and the late pre-spore marker SpiA (anti-SpiA) in normal (Ax3 and bsr+), P4H1a- (HW288) and pgtA- cells. Times indicate hours of starvation under ambient conditions. (B-E) Microscopic analysis of normal (Ax3) and P4H1a- cells transfected with various promoter::bacterial ß-galactosidase reporter constructs, developed under ambient conditions and processed for ß-galactosidase activity, which deposits a dark blue precipitate. Shown are representative examples of pre-culminants or slugs expressing (B) the pre-spore cell marker PspA-Gal (strain HW405), (C) the pre-stalk A and 0 cell marker ecmA0::lacZ (HW408), (D) the pre-stalk A cell marker ecmA::lacZ (strain HW406) or (E) the pre-stalk 0 cell marker ecm0::lacZ (HW409). Anterior tips point upward.

View larger version (52K): [in this window] [in a new window]   Fig. 4. P4H1 is expressed in Dictyostelium tip cells. (A-D) Normal (Ax3) cells, transfected with phyA::RFP as a reporter for P4H1 expression, were developed to the tipped aggregate (A,B), slug (C) or pre-culmination stage (D) under ambient conditions, placed under a coverslip and photographed for RFP fluorescence. A and B are different planes from the same aggregate. Although not all cells were fluorescent, fluorescence was detected throughout the structures including the tip (denoted by asterisk). (E,F) Tips (Pst) and central pre-spore (Psp) regions were dissected from normal (strain NC-4) slugs, and 67 µg of S17 fraction protein (lanes d,e) was analyzed by western blotting for P4H1 using anti-P4H1, or for Skp1 using mAb 4E10. Surrounding lanes contain 200 µg (b,g) or 67 µg (c,f) of S100 protein from stationary phase Ax3 (normal) cells (b,c) or P4H1a- cells (f,g), or Mr standards (a,h). Positions of hydroxylated/glycosylated and unmodified Skp1 are shown. Both panels are from the same blot, and are representative of two experiments.

View larger version (33K): [in this window] [in a new window]   Fig. 5. Rescue of Skp1 hydroxylation and culmination in mutant cells by P4H1 or PKAcat cDNAs. (A) Ax3, bsr+ (+, normal), P4H1- clones (-) and P4H1- clones complemented with P4H1-myc under control of its own promoter (P), the pre-stalk-specific ecmA(FL) promoter (E), the pre-spore-specific cotB promoter (C) or the discoidin promoter (D), were developed for 16 hours under ambient conditions and analyzed by western blotting with myc-specific mAb 9E10 for P4H1-myc accumulation. The position of P4H1-myc relative to the more intense background bands, which serve as loading controls, is indicated. (B) Parallel samples were analyzed by western blotting for Skp1 isoforms using mAb 4E1. Markers indicate high (glycosylated) and low Mr (non-hydroxylated/glycosylated) Skp1 isoforms, and a lower band of unknown identity. Results in A and B are typical of three experiments, including independently complemented HW289-derived cells (not shown). (C) Quantitation of culmination under ambient conditions by spore counts. No rescue was observed using an empty vector (not shown). P4H1- cells transfected with PKAcat (strain HW410) are also shown. (D,E) Complementation with P4H1-mutants. (D) Fifteen-hour slugs from Ax3, or P4H1a- expressing the P4H1-myc mutants D132N (DNa and DNb are independent clones) or R276A (RA) under control of the ecmA-promoter, were analyzed for P4H1-myc expression as in A, and for Skp1 electrophoretic mobility as in B. (E) Strains were developed at the indicated O2 level for the indicated time under strong overhead light. P4H1- cells exhibited breakthrough at 21% O2 in this trial, but were selectively inhibited relative to Ax3 at 15% O2. Spore numbers (shown) correlated with morphological appearance (not shown).

View larger version (42K): [in this window] [in a new window]   Fig. 6. Rescue of culmination of P4H1- slugs by P4H1-expressing cells, and accumulation of P4H1-expressing cells in the slug tip. (A) Various ratios of parental (Ax3) and P4H1a- cells were mixed at time 0, developed under ambient conditions, and harvested to count detergent-resistant spores after 48 hours. The straight line describes the calculated spore number if only Ax3 formed spores independent of an effect from P4H1a- cells. The vertical bar indicates that 20% of cells are normally pre-stalk cells. (B) RFP-expressing normal Ax3 cells were mixed with unlabeled cells, as indicated, in a 7:93 ratio at the start of development. Flattened 16 hour slugs were photographed by phase contrast and for RFP-fluorescence. (C) In the reverse experiment, RFP-expressing P4H1- cells were mixed with unlabeled cells, as indicated, in a 3:7 ratio. Numbers below image represent the average ratio of mean intensity in the pre-stalk region (demarcated by three white dots) to that of the pre-spore (remaining) region, for multiple slugs (n=3-6). (D) Cultures shown in panel B were allowed to culminate, and stalks were examined. Examples in B and D are typical (n>10). (E) Spore numbers were determined in the mixtures shown in D and for Ax3 and P4H1a- cells alone (left panel). Percent of spores that were red in the mixtures is shown in the right panel. Results are representative of two independent experiments.

View larger version (42K): [in this window] [in a new window]   Fig. 7. P4H1 overexpression reduces the O2 requirement for culmination in Dictyostelium. (A) Western blot analysis of P4H1-myc levels in clones of strain Ax3 transfected with the promoter expression constructs described in Fig. 5A. (B) Effects of overexpressing P4H1 in the Ax3 background on the hypoxic blockade, as determined by counting detergent-resistant spores. Spore numbers correlated with morphological culmination (not shown). Results are typical of three independent trials.

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