The Drosophila mitochondrial ribosomal protein mRpL12 is required for Cyclin D/Cdk4-driven growth

Abstract

The Drosophila melanogaster cyclin-dependent protein kinase complex CycD/Cdk4 stimulates both cell cycle progression and cell growth (accumulation of mass). CycD/Cdk4 promotes cell cycle progression via the well-characterized RBF/E2F pathway, but our understanding of how growth is stimulated is still limited. To identify growth regulatory targets of CycD/Cdk4, we performed a loss-of-function screen for modifiers of CycD/Cdk4-induced overgrowth of the Drosophila eye. One mutation that suppressed CycD/Cdk4 was in a gene encoding the mitochondrial ribosomal protein, mRpL12. We show here that mRpL12 is required for CycD/Cdk4-induced cell growth. Cells homozygous mutant for mRpL12 have reduced mitochondrial activity, and exhibit growth defects that are very similar to those of cdk4 null cells. CycD/Cdk4 stimulates mitochondrial activity, and this is mRpL12 dependent. Hif-1 prolyl hydroxylase (Hph), another effector of CycD/Cdk4, regulates growth and is required for inhibition of the hypoxia-inducible transcription factor 1 (Hif-1). Both functions depend on mRpL12 dosage, suggesting that CycD/Cdk4, mRpL12 and Hph function together in a common pathway that controls cell growth via affecting mitochondrial activity.

Figure 1

CycD/Cdk4 requires mRpL12 to drive growth in the Drosophila eye. (A) SEM images at × 500 magnification. Genotypes: GMR-Gal4/+; +/+ (left), GMR-Gal4 UAS-CycD UAS-Cdk4/+;+/+ (second from left), GMR-Gal4/+; Df(3L)Scf-R6/+ (third from left) and GMR-Gal4 UAS-CycD UAS-Cdk4/+; Df(3L)Scf-R6/+ (right). Scale bar, 20 μm. (B) Break points of the deficiencies used. ‘+' indicates suppression of the overgrowth phenotype whereas ‘−' indicates no change as compared to a wild-type background. (C) SEM at × 120 magnification. CycD/Cdk4 is driven from the GMR-Gal4 driver. ‘+/+' indicates precise excisions of P{PZ}10534. Scale bar: 100 μm. All flies in A and C are females, reared at 22.5°C. (D) Genomic locus of MRPL12. The lethal P-element (P{PZ}10534) insertion into the 5′UTR of MRPL12 is indicated. The 3′UTR of CG5008 ends 294 bp upstream of MRPL12, and the 5′UTR of CG13313 starts 600 bp downstream of MRPL12.

Figure 2

mRpL12¹⁰⁵³⁴ suppresses CycD/Cdk4-driven growth in the eye, wing and fat body. (A) FACS of eye imaginal discs from third instar larvae. The forward scatter (FSC; x-axis), indicative of the cell size, is blotted against the number of cells (y-axis). The black line represents posterior cells expressing CycD/Cdk4 together with GFP, compared to posterior cells expressing GFP only in the same background (filled gray). Insets are anterior cells. (B) FACS of pupal eye imaginal discs 48 h after white prepupae formation. DNA content (x-axis) is blotted against cell number (y-axis). GFP-negative cells are non-eye disc cells. (C) Drawing of third instar eye and antenna imaginal discs showing the morphogenetic furrow (arrow) moving from posterior to anterior. GMR-Gal4 leads to the expression of UAS transgenes in the posterior compartment. (D) Random clones in wing imaginal discs expressing GFP or CycD/Cdk4 plus GFP in mRpL12¹⁰⁵³⁴ or wild-type backgrounds were induced and the median clone areas were measured as described in Materials and methods. (E) Fat body clones expressing CycD/Cdk4 plus GFP were induced during embryogenesis in the fat body in wild-type (top) or an mRpL12¹⁰⁵³⁴/+ background (bottom). Larvae were fed until 72 h AED and starved for 5 days in 20% sucrose in 1 × PBS. Fat bodies were stained with DAPI, mounted and imaged as described in Materials and methods. CycD/Cdk4 leads to a 3.2-fold increase in DNA content in a wild-type background (n=33, P<0.01 compared to GFP alone). In the mRpL12¹⁰⁵³⁴/+ background, the increase in DNA is 1.6-fold (n=18, P>0.5). Scale bar, 20 μm.

Figure 3

Homozygous mRpL12¹⁰⁵³⁴ cells have a cell-autonomous growth defect. (A) Images of larvae at 70 h AED. (B) Homozygous mRpL12¹⁰⁵³⁴ or control clones were induced at 66 h and dissected at 114 h AED, and wing discs were stained with DAPI and imaged. The area without GFP (−/−) and two copies of GFP (+/+) was measured in Photoshop. Genotypes: hs-Flp¹²²; FRT80B mRpL12¹⁰⁵³⁴ or FRT80B/FRT80B Ub-GFP^13A. (C) Wing discs from (B) were analyzed by FACS and GFP-negative cells (black line) were separated from GFP-positive cells (one or two copies; filled gray histogram. Shown are the forward scatters and DNA contents blotted against cell numbers (left). DAPI and GFP staining of a representative FRT80B mRpL12¹⁰⁵³⁴/FRT80B Ub-GFP^13A twinspot (right). (D) Homozygous mRpL12¹⁰⁵³⁴ cells were induced in the fat body during embryogenesis by ionizing radiation, and third instar larvae were dissected and their fat body mounted. Homozygous mutant cells are marked by the absence of GFP (white arrows). Scale bar, 10 μm.

Figure 4

CycD/Cdk4 regulates mitochondrial function. (A–E) Fat body from third instar larvae were stained using MitoTracker red (left; see Materials and methods). (A) Ectopic expression of UAS-mRpL12-GFP in the fat body using the hs-Flp Act>CD2>Gal4 system. (B) Cells homozygous mutant for mRpL12¹⁰⁵³⁴ were induced by ionizing radiation. Mutant cells lack GFP (arrows). (C–F) Ectopic expression of CycD/Cdk4 or dMyc using the hs-Flp Act>CD2>Gal4 UAS-GFP system in a wild-type (C, E and F) or mRpL12¹⁰⁵³⁴/+ background (D). Expression was induced during embryogenesis without any heat shock. CycD/Cdk4- or dMyc-expressing cells are GFP positive. (E) Trachea were stained with TMRM. Scale bar, 20 μm.

Figure 5

CycD/Cdk4 stimulates mitochondria in fat body cells. (A) COX (top) and SDH (bottom) activity in fat body cells. CycD/Cdk4-expressing cells were detected by coexpression of GFP in random clones, and are marked by arrows. (B) COX staining in second instar larvae, 77 h AED for the midgut and the proventriculus (left) and 96 h AED for the fat body (right). Images of heterozygous (mrpl12¹⁰⁵³⁴ or Df(3L)Scf-R6/+) or homozygous mutant (mrpl12¹⁰⁵³⁴/Df(3L)Scf-R6) larvae were exposed and treated identically in Photoshop. (C) Random clones expressing CycD/Cdk4 (top) or dMyc (bottom) were induced as described in Figure 2E, and marked by coexpression of GFP. At 24 h AED, larvae were transferred to normal food, or food supplemented with rotenone (5 μg/ml), antimycin A (5 μg/ml), or normal food and larvae were incubated at 7% O₂. All were dissected at 116 h AED, fixed and stained with DAPI, and fat bodies were mounted. Numbers indicate the percentages of GFP-positive cells that are increased in ploidity, compared to neighboring cells. Quantifications were carried out in blind (n⩾150). Scale bar, 20 μm.

Figure 6

Normal mRpL12 levels are required for Hph function. (A) Hph was overexpressed in random clones in wing imaginal discs and clone area was measured as in Figure 2D. (B) Cells homozygous mutant for mRpL12 were induced by a heat shock 48 h AED. Larvae were either left at normoxia (left) or incubated at 2.5% oxygen for the last 7 h before dissection (right). Wing discs from third instar larvae were stained for lacZ or DNA, or imaged for GFP. Arrows point to a homozygous mutant clone.

Figure 7

Model for the CycD/Cdk4–mRpL12–Hph pathway. mRpL12 and Hph are required for CycD/Cdk4-stimulated growth, but not proliferation, suggesting that they function downstream of CycD/Cdk4. CycD/Cdk4 has a dual function: post-transcriptional regulation of Hph protein levels (Frei and Edgar, 2004), and induction of mitochondrial activity, which is dependent on mRpL12. Furthermore, Hph activity depends on mRpL12, hence may be regulated in response to mitochondrial activity. Hph hydroxylation activity is required for stimulation of growth and inhibition of the transcription factor Hif-1α/Sima, which is essential for the cellular response to hypoxia.