PMC

Loss of dLipin results in ultrastructural changes affecting the cell nucleus, mitochondria, and autophagosome formation. (A and B) Transmission electron micrographs of fat body cells of control animals [dLipin^e00680/+ or Df(2R)Exel7095/+]. As reported previously (), fat body cells of late-third-instar larvae are characterized by the presence of autophagosomes (AP) that contain mitochondria (m) and other cytoplasmic remnants. LD, lipid droplet. (C and D) Fat body cell of a dLipin mutant [dLipin^e00680/Df(2R)Exel7095]. Most autophagosomes have a matrix that is less dense than the matrix of normal autophagosomes. (E and F) Mitochondria of mutant fat body cells exhibit changed morphology. Fat body mitochondria of control animals are elongated and densely packed with cristae (E, arrows). In contrast, mitochondria of dLipin mutants are rounded, contain only a few cristae, and often exhibit ruptured double membranes (F, arrowhead). (G) Misshapen cell nucleus (Nc) of a dLipin mutant fat body cell, featuring a long nuclear projection (arrowhead) and the intrusion of a fat droplet (LD, arrow). Bars, 0.5 μm (A, C, E, and F), 0.2 μm (B and D), and 1 μm (G).

dLipin is broadly expressed in Drosophila tissues and shows both cytoplasmic and nuclear localization. (A) dLipin is strongly expressed in both the cytoplasm and the nuclei of fat body cells (FB). The strong cytoplasmic staining usually masks the nuclear staining, which can be seen clearly in some regions (inset). In stark contrast to the fat body, the larval salivary glands (SG) show very little or no staining. Knockdown of dLipin expression by RNAi using the fat body-specific driver P{w+mC=Cg-GAL4.A}2 greatly reduced both cytoplasmic and nuclear staining, demonstrating the specificity of antibody binding. (B) Malpighian tubules (MT) show strong nuclear staining with the anti-dLipin antibody. (C) The midgut (MG) and ceca (Ca) show both nuclear and cytoplasmic staining, whereas the proventriculus (Pv) does not appear to express significant amounts of dLipin. (D) The ring gland (RG) shows very strong cytoplasmic staining and nuclear staining (inset). (E) In the ovary, strong nuclear dLipin staining is found in the nuclei of the nurse cells (Nc) and follicle cells (Fc) (arrows). Diffuse staining is also observed in the oocyte cytoplasm (Oo), suggesting that dLipin protein is provided maternally. Bars, 50 μm.

Lack of dLipin results in reduced lipid droplet size, reduced stores of neutral lipids, and increased ketogenesis. (A) The size of fat droplets is reduced in the fat bodies of dLipin mutants and after fat body-specific knockdown of dLipin expression by RNAi. Fat droplets were stained with Bodipy (green) or Nile red (red). The section marked with an arrowhead was subjected to image deconvolution in order to facilitate recognition of the very small lipid droplets in the dLipin mutant. Bars, 50 μm. (B) TAG levels are reduced in animals lacking dLipin. Total TAG and protein levels were measured in whole animals homozygous for dLipin^e00680 or transheterozygous for dLipin^e00680 and Df(2R)Exel7095 (dLipin/dLipin and dLipin/Df), and after fat body-specific knockdown of dLipin. Asterisks indicate a statistically significant decrease in the TAG/protein level from that for the w¹¹¹⁸ control. (C) Ketone body formation is increased in animals lacking dLipin. The ketone β-hydroxybutyrate was measured in animals with the genotype dLipin^e00680/Df(2R)Exel7095 and in control animals heterozygous for dLipin^e00680 or Df(2R)Exel7095 (Ctl). BW, body weight. Error bars in panels B and C indicate standard errors of the means. **, P < 0.01; ***, P < 0.0001.

dLipin promotes starvation resistance. (A) dLipin RNA is increased under starvation conditions. RNAs from normally fed and starved flies were quantitated by qRT-PCR. Asterisks indicate that the increase in the dLipin RNA level in starved flies over that in fed flies was statistically significant (***, P < 0.01). Quantitation of rp49 RNA was used for normalization. (B) RNAi-mediated knockdown of dLipin in the fat body leads to decreased starvation resistance. The survival of male and female flies of the indicated genotypes was monitored daily after food withdrawal. Flies were supplied with water only, except for flies fed with RU486 dissolved in 5% ethanol and control flies obtaining 5% ethanol alone (bottom graphs). The DJ761 driver is active in many tissues, including the fat body and the gut, whereas DJ649 is active in the gut and other tissues but not the fat body. The Lsp2 driver is active in the fat body only. Note that flies in the RU486 feeding experiment survived for an extended period, because they could use ethanol as an energy source (). Survival curves were compared using the log rank test. The P values shown either are for the least significant difference in pairwise comparisons between RNAi and control animals or apply to all comparisons.

Loss of dLipin causes defects in fat body development. (A and B) The size and shape of fat body cells is dramatically altered in dLipin-deficient larvae. (A) Bright-field microscopy indicates that, compared to wild-type cells (w¹¹¹⁸), fat body cells of third-instar larvae lacking dLipin are more variable in size, more rounded, and, in some regions, detached from one another. Large fat droplets that appear as bright inclusions, and are plentiful in the control fat body, are scarce in the fat body lacking dLipin. (B) Staining with the plasma membrane stain CellMask (red) highlights cell boundaries, confirming the cell shape changes, while staining with the DNA stain DAPI (blue) reveals that cell nuclei in some of the dLipin-deficient cells are fragmented (arrows). The arrowhead marks a cell in which the DNA is dispersed throughout the cell. Cells show a similar mutant phenotype in the presence of the apoptosis inhibitor p35 or after dLipin knockdown by RNAi. Bars, 50 μm. (C) The sizes of cells and nuclei are increased in the fat bodies, but not in the salivary glands, of dLipin mutants, indicating that the observed effects are specific for fat tissue. Asterisks indicate a significant difference from the mean for w¹¹¹⁸ flies (*, P < 0.05; **, P < 0.01; ***, P < 0.0001). Error bars indicate standard errors of the means.

Animals lacking dLipin are characterized by pharate adult lethality and underdevelopment of the larval fat body. (A) Genomic structure of the dLipin gene. The inverted triangle indicates the insertion site of the piggyBac transposon in the dLipin^e00680 allele. Black boxes represent exons. The deficiencies Df(2R)Exel7095 and Df(2R)NCX10 remove chromosomal regions 43E7 to 44C5 and 44B3 to 44C2, respectively, including the entire dLipin gene at 44B4 to 44B5. dLipin encodes two protein isoforms with different C termini that result from alternative splicing, as indicated. (B) Animals homozygous for dLipin^e00680, or transheterozygous for dLipin^e00680 and Df(2R)Exel7095 (dLipin/Df), contain reduced levels of dLipin mRNA and protein. RNA for Northern blot analysis was from whole prepupae and pupae, and protein for Western blot analysis was from wandering third-instar larvae. Control samples were obtained from w¹¹¹⁸ animals. Detection of rp49 mRNA and actin protein served as a control for loading and transfer. (C) Animals homozygous for dLipin^e00680 develop into pharate adult flies that do not show obvious developmental defects (animal in middle, dissected from pupal case). However, many of these flies die and become necrotic while still enclosed in the pupal case (animal on the right). Note that mutant and control pupae are similar in size, indicating that a lack of dLipin does not lead to an overall growth defect. (D) A lack of dLipin causes severe underdevelopment of the larval fat body. (Top) dLipin mutant larvae appear translucent due to a lack of fat tissue. (Center) Inclusion of the Dcg-GFP fat body marker () in the mutant background confirms that the amount of fat tissue is dramatically reduced. (Bottom) The same phenotype is obtained when dLipin expression is knocked down by RNAi using the ubiquitous tubulin driver. Bars, 1 mm.