Drosophila melanogaster lipins are tissue-regulated and developmentally regulated and present specific subcellular distributions Valeria Valente 1,2, *, Rafaela Martins Maia 1, *, Murilo Carlos Bizam Vianna 1 and Maria Luisa P ac¸o ´ -Larson 1 1 Department of Cellular and Molecular Biology, Ribeira˜o Preto School of Medicine, University of Sa˜o Paulo, Brazil 2 Department of Surgery and Anatomy, Ribeira˜o Preto School of Medicine, University of Sa˜o Paulo, Brazil Introduction The lipins constitute a highly conserved family of proteins encountered in diverse organisms such as single-cell eukaryotes, plants, invertebrates and mammals [1,2]. The Saccharomyces cerevisae lipin Pah1p and all three lipins of mammals (lipin 1, lipin 2 and lipin 3) have been biochemically characterized as Keywords Drosophila; isoforms; lipin; Pah1p; subcellular localization Correspondence M. L. Pac¸o ´ -Larson, Av. Bandeirantes, 3900, Ribeira˜o Preto, Sa˜o Paulo, Brazil Fax: +55 16 36331786 Tel: +55 16 36023347 E-mail: mlplarso@fmrp.usp.br *These authors contributed equally to this work (Received 12 February 2010, revised 6 August 2010, accepted 9 September 2010) doi:10.1111/j.1742-4658.2010.07883.x Lipins constitute a novel family of Mg 2+ -dependent phosphatidate phos- phatases that catalyze the dephosphorylation of phosphatidic acid to yield diacylglycerol, an important intermediate in lipid metabolism and cell signaling. Whereas a single lipin is detected in less complex organisms, in mammals there are distinct lipin isoforms and paralogs that are differen- tially expressed among tissues. Compatible with organism tissue complex- ity, we show that the single Drosophila Lpin1 ortholog (CG8709, here named DmLpin) expresses at least three isoforms (DmLpinA, DmLpinK and DmLpinJ) in a temporal and spatially regulated manner. The highest levels of lipin in the fat body, where DmLpinA and DmLpinK are expressed, correlate with the highest levels of triacylglycerol (TAG) mea- sured in this tissue. DmLpinK is the most abundant isoform in the central nervous system, where TAG levels are significantly lower than in the fat body. In the testis, where TAG levels are even lower, DmLpinJ is the pre- dominant isoform. Together, these data suggest that DmLpinA might be the isoform that is mainly involved in TAG production, and that DmLpinK and DmLpinJ could perform other cellular functions. In addi- tion, we demonstrate by immunofluorescence that lipins are most strongly labeled in the perinuclear region of the fat body and ventral ganglion cells. In visceral muscles of the larval midgut and adult testis, lipins present a sarcomeric distribution. In the ovary chamber, the lipin signal is concen- trated in the internal rim of the ring canal. These specific subcellular local- izations of the Drosophila lipins provide the basis for future investigations on putative novel cellular functions of this protein family. Abbreviations BDGP, Berkeley Drosophila Genome Project; CLPIN, conserved domain characteristic of the C-terminal region of lipin family; CNS, central nervous system; DAG, diacylglycerol; DAPI, 6-diamidino-2-phenylindole; DmLipin, Drosophila melanogaster lipin; ER, endoplasmic reticulum; NLPIN, conserved domain characteristic of the N-terminal region of lipin family; PA, phosphatidate; PAP, phosphatidate phosphatase; PPAR, peroxisome proliferator-activated receptor; TAG, triacylglycerol. FEBS Journal 277 (2010) 4775–4788 Journal compilation ª 2010 FEBS. No claim to original Brazilian government works 4775 Mg 2+ -dependent phosphatidate (PA) phosphatases (PAPs) [3,4]. PAP1 catalyzes the dephosphorylation of PA to diacylglycerol (DAG), which is used for the syn- thesis of phosphatidylcholine, phosphatidylethanol- amine and the storage lipid triacylglycerol (TAG). In addition to their biosynthetic roles, PA and DAG are also important in signal transduction cascades. Regula- tion of PA and DAG availability involves a second type of PAP activity, known as PAP2 or lipid phos- phate phosphohydrolase, which does not require Mg 2+ as a cofactor [5]. Lipins of different species have conserved amino acid stretches in the N-terminal and C-terminal domains, respectively [6]. The C-terminal domain contains two functionally critical motifs: the halo- acid dehalogenase motif DXDXT, characteristic of Mg 2+ -dependent phosphatases, and the transcription factor-binding motif LXXIL. These motifs confer dual molecular functions for the members of the lipin family: the enzymatic activity as phosphatases, and the coactivator properties by interaction with transcription factors. Mammalian lipin 1 and lipin 2 have been dem- onstrated to act in adipocyte differentiation and liver metabolism as transcriptional coactivators, indepen- dently of their PAP1 activity, by physical interactions with peroxisome proliferator-activated receptor (PPAR)a, PPAR coactivator-1a and PPARc 2 [7–9]. Also, in agreement with the roles of lipins in metabolic homeostasis, their activity is regulated by phosphoryla- tion in an insulin-dependent manner [9–11], and Lpin1 gene expression is induced by glucocorticoids [12]. In accordance with the molecular functions described above, it was reported that yeast Pah1p con- trols phospholipid biosynthesis at the nuclear ⁄ endo- plasmic reticulum (ER) membrane and coordinates nuclear growth with the cell cycle, in a manner regu- lated by phosphorylation [13,14]. The Caenorhabd- itis elegans lipin is also needed for the maintenance of normal nuclear ⁄ ER morphology and for lipid storage. Loss of function of the nematode lpin-1 leads to decreased lipid accumulation, disorganization of the peripheral ER and abnormal nuclear morphology, with the presence of paired nuclei, and defects in chromo- some segregation [15,16]. Moreover, Lpin1 loss of function is associated with severe metabolic abnormali- ties in the mouse strain with fatty liver dystrophy (fld) [6], which exhibits a dramatic reduction in fat pad mass, triglyceride-filled fatty liver, insulin resistance and a peripheral neuropathy caused by axonal demye- lination as a consequence of PA accumulation [6,17–23]. Recently, it was demonstrated that Majeed syndrome, an inflammatory disorder characterized by osteomyelitis, congenital dyserythropoietic anemia and cutaneous inflammation [24,25], is associated with mutations in the LPIN2 locus [9]. In mammals, besides the three lipin paralogs, alter- natively spliced isoforms (lipin 1-a and lipin 1-b) are generated from the Lpin1 locus [6,26]. Expression anal- ysis in a panel of mouse and human tissues revealed that these proteins present a distinct but, to some extent, overlapping expression pattern [4]. Lipin 1 mRNAs are enriched in adipose tissue and skeletal muscle, with lower expression in the intestine, brain and liver. Lipin 2 mRNA was detected at higher levels in the brain, liver, placenta and adipose tissue, and at lower levels in the intestine. Lipin 3 mRNA expression is predominant in the stomach, pancreas and intestinal tract, but was also detected in the heart, liver, kidney, adipose tissue and placenta [4]. Additionally, lipin 1 isoforms are differentially expressed during adipocyte differentiation and have divergent subcellular localiza- tions, consistent with their individual roles in this tissue. Lipin 1-a, which is primarily involved in the differentiation process, is mainly detected in the nuclei of preadipocytes, whereas lipin 1-b, which is related to lipogenesis and adipocyte growth, is predominantly localized to the cytoplasm of these cells [7,26]. Besides lipin 1, the subcellular localization of lipin 2 was deter- mined by analysis of tagged protein in HeLa cells [27]. The only lipin ortholog evaluated with respect to the subcellular distribution of the endogenous protein was from C. elegans. Immunolocalization experiments showed that the nematode lipin 1 was distributed throughout the cytoplasm and the nucleoplasm of embryo cells, but was enriched in the ER and in the nuclear envelope [16], in accordance with a possible function in the regulation of nuclear–ER membrane dynamics [16]. The fundamental roles of lipins, reflected in their involvement in several cellular and physiological pro- cesses, justify further investigation of these proteins in different models. This is the first report regarding the characterization of the Drosophila melanogaster lipin gene (DmLpin). We show that, despite the fact that the Drosophila genome contains a single lipin ortholog (CG8709), it expresses at least three isoforms (DmLpinA, DmLpinK and DmLpinJ), which exhibit distinct expression patterns during development and among different tissues. The observation of differen- tial correlation between the levels of lipin isoforms and TAG amounts in the tissues suggests that lipin isoforms are involved in different cellular functions. Also, the specific subcellular distribution for Drosoph- ila lipins observed among tissues indicates putative novel regulatory functions for the members of this protein family. Drosophila lipins molecular and cellular analysis V. Valente et al. 4776 FEBS Journal 277 (2010) 4775–4788 Journal compilation ª 2010 FEBS. No claim to original Brazilian government works Results DmLpin encodes different isoforms generated by alternative splicing and the transcription initiation start site To isolate full-length cDNAs corresponding to CG8709, we screened embryonic and larval ⁄ pupal cDNA libraries using a previously generated ORES- TES [28] that mapped in the CG8709 genomic region not covered by expressed sequence tags at that time. We obtained two cDNAs, of 4384 bp (from the embry- onic library) and 3517 bp (from larvae ⁄ pupae), with an exon–intron organization that differed from that of the full-length cDNA available for this locus. The sequence of the shorter cDNA was extended at the 5¢-end by RT-PCR with a pair of primers anchored at the 5 ¢ -extremity of the isolated cDNA and in several upstream positions, resulting in a 462 bp PCR prod- uct, which was cloned and sequenced. The sequence thus obtained was assembled with the 3517 bp cDNA, producing a consensus sequence of 3979 bp. These cDNAs correspond to the predicted CG8709-RK and CG8709-RJ isoforms, which were defined in the last FlyBase annotation performed on the basis of genome- wide sequence data (release FB2010_03). These two newly identified mRNAs and a third cDNA of 3992 bp (AY089509), which has been completely sequenced by the Berkeley Drosophila Genome Project (BDGP), represent three isoforms from the CG8709 locus, herein named DmLpin. To be consistent with the current FlyBase nomenclature of the predicted iso- forms, we have named the isoforms characterized in this study accordingly. The isoform represented by the cDNA AY089509 was named DmLpinA, and the isoforms represented by the novel cDNAs completely sequenced in this study were named DmLpinK (GU327733) and DmLpinJ (GU327734). These mRNAs are generated by alternative splicing and the use of different transcription initiation sites (Fig. 1). Although most of the exons of DmLpin mRNAs are shared by the three cDNAs, they have particular struc- tures at the 5¢-ends and 3¢-ends (Fig. 1). The first exon of DmLpinA is absent in the other two isoforms. Simi- larly, the first exon of DmLpinK is not present in the DmLpinA and DmLpinJ mRNAs. Additionally, exon 1K is shared with mRNAs of the adjacent gene (Kermit), whose 5¢-end is located more than 10 kb upstream of DmLpin exon 1A (Fig. 1). The first exon of DmLpinJ includes part of the second exon, the sec- ond intron and the third exon of DmLpinA ⁄ K. DmLpinA and DmLpinJ ⁄ K contain alternative last ex- ons (Fig. 1). These data reveal the occurrence of alter- native splicing, and support the existence of alternative transcription start sites for DmLpin. DmLpinA, DmLpinK and DmLpinJ encode distinct polypeptides of 1089, 1035 and 962 amino acids, whose molecular masses were estimated to be 120.9, 114.5 and 106.6 kDa, respectively. The amino acid sequences for the N-terminal and C-terminal regions in which these isoforms differ are shown in Fig. 2. As the first exons of DmLpinA and DmLpinK are not coding sequences, the deduced proteins (DmLipinA and DmLipinK) are identical from the first methionine to the amino acid, at position 971. The C-terminal regions of DmLipinA and DmLipinK, which are encoded by alternative final exons, have distinct sequences (Fig. 2). DmLipinJ does not contain the first 73 amino acids of DmLipinA ⁄ DmLipinK, and is iden- tical to DmLipinK in the rest of the molecule (Fig. 2). The conserved domain characteristic of the N-terminal region (NLPIN, [6]) is fully present in DmLipinA and 4 025 000 4 030 000 4 035 000 4 040 000 DmLpin K Kermit DmLpinA DmLpinJ pK-FpK-RpJ-FpJ-RpA-FpA-R Chr 2R Fig. 1. Structure of the DmLpin locus; schematic representation of the exon–intron organization. The nucleotide sequences of DmLpinA (AY089509), DmLpinK (GU327733) and DmLpinJ (GU327734) cDNAs, and predicted mRNAs of the Kermit gene, were aligned with the gen- ome sequence by use of the BLAT tool (http://genome.ucsc.edu/cgi-bin/hgBlat?command=start). The bar at the top represents the genome region of chromosome 2, where the cDNA sequences are mapped. The exons are indicated by bars and the introns by lines. Noncoding sequences are shown as narrower portions of the exon bars. The arrowheads in the intron lines indicate the transcription direction. The primers used for PCR amplifications are indicated by the arrowheads as follows: from right to left, pK-F and pK-R for DmLpinK, pJ-F and pJ-R for DmLpinJ, and pA-F and pA-R for DmLpinA. V. Valente et al. Drosophila lipins molecular and cellular analysis FEBS Journal 277 (2010) 4775–4788 Journal compilation ª 2010 FEBS. No claim to original Brazilian government works 4777 DmLipinK. In DmLipinJ, only the last 32 amino acids of NLPIN are present; however, the Gly84, which is important for PAP1 activity in the mouse (17), is included (Fig. 3A). The conserved domain characteris- tic of the C-terminal region (CLPIN, [6]) of the lipin family is present in all three Drosophila isoforms (Fig. 2). This domain includes the motif DXDXGT, which is responsible for PAP1 catalytic activity, and the LXXIL motif required for the transcriptional coactiva- tion function of mouse lipin 1 [8], which are conserved among lipins from yeast to human (Fig. 3A). In addi- tion, putative nuclear localization signals are observed in the three DmLipin proteins (Fig. 2). Phylogenetic analysis based on CLPIN amino acid sequences of diverse organisms revealed that Drosophila lipins are more closely related to mammalian lipins than to yeast Pah1p or the worm lipin (Fig. 3B). DmLpin expression is regulated during Drosophila development The hybridization of a [ 32 P]DmLpinK cDNA with north- ern blots of polyA + RNA extracted from embryos, larvae, pupa and adults revealed mRNAs o f approximately 4 kb at all stages of development (Fig. 4A). The length of these mRNAs agrees with the length of DmLpin cDNAs described here, demonstrating that they represent full- length transcripts. Using cDNA probes specific for the isoforms DmLpinK and DmLpinJ, we observed that whereas DmLpinK mRNA was present at all develop- mental stages analyzed, DmLpinJ was detected in pupae and in higher amounts in adult males, but not in embryos or adult females (Fig. 4A¢). To extend the investigation of the expression pattern of each DmLpin isoform during development, we Fig. 2. Multiple alignment of lipin protein isoforms of D. melanogaster. The deduced amino acid sequences of the longest ORFs for the mRNAs DmLpinA, DmLpinK and DmLpinJ were aligned with the CLUSTALW2 tool (http://www.ebi.ac.uk/Tools/clustalw2/ index.html). Gray boxes delimit regions corresponding to NLPIN and CLPIN [6], respectively. The amino acid stretches in bold and underlined indicate putative nuclear localization signals. The boxes indicate the conserved catalytic motif characteristic of PAP1 enzymes, DXDXT, and the conserved coactivator motif, LXXIL. Drosophila lipins molecular and cellular analysis V. Valente et al. 4778 FEBS Journal 277 (2010) 4775–4788 Journal compilation ª 2010 FEBS. No claim to original Brazilian government works performed semiquantitative RT-PCR experiments with primers directed to isoform-specific exons (Fig. 1). Whereas DmLpinA and DmLpinK were expressed at similar levels at all developmental stages evaluated, the DmLpinJ mRNA was found only in pupae and adult males (Fig. 4D). The investigation of DmLpin expres- sion was extended to the protein level by the use of an affinity-purified antibody (a-NLip; Fig. S1) that recog- nizes a region common to all isoforms of lipin. In wes- tern blots of protein extracts of embryos, larvae (first, second and third instar), pupae and adults (male and female), a-NLip detected major bands in the range of 130–140 kDa in all stages analyzed (Fig. 4B). Quantifi- cation of the bands of three immunoblots indicated that the total amount of lipin varied during develop- ment. However, in contrast to expectations based on the mRNAs analysis, the amounts of these proteins were significantly higher in larvae and pupae (P < 0.001) than in embryos and male and female adults (Fig. 4C). DmLpin expression and triglyceride levels in Drosophila tissues In order to gain some insights into the physiological roles of the distinct Drosophila lipin isoforms, we evaluated the expression of DmLpin and the triglycer- ide levels in different tissues. Northern blot experi- ments with RNA of third instar larvae have shown that DmLpin is expressed in all tissues analyzed: cen- tral nervous system (CNS), intestinal tract ⁄ Malpighian tubules, fat body and carcass (Fig. 5A). With the use of exon-specific probes, DmLpinA and DmLpinK were mainly detected in the larval intestinal tract ⁄ Malpi- ghian tubules and in the CNS, respectively, whereas DmLpinJ was not detected in these tissues (data not shown). Faint bands for DmLpinA were also observed in the larval fat body and carcass, suggesting the pres- ence of lower levels of this mRNA in these tissues (Fig. 5A). Moreover, with multiplex RT-PCR, DmLp- inA was detected in both the ovary and the testis from adult flies, whereas DmLpinK was present in ovary and DmLpinJ in the testis (Fig. 5B). Semiquantitative RT-PCR analysis agreed with these results, and showed that, despite being predominantly expressed in the intestinal tract ⁄ Malpighian tubules, DmLpinA was expressed in all tissues analyzed. Also, DmLpinK mRNA, which was highly abundant in the larval CNS, was also detected at lower levels in the intestinal tract ⁄ Malpighian tubules, fat body, carcass and adult ovary. DmLpinJ was exclusively expressed in the testis (Fig. 5C). Fig. 3. Comparison of the DmLipin isoforms with lipins of diverse organisms. (A) Multiple alignment of 60 amino acids of NLPIN and CLPIN of DmLipin isoforms with the corre- sponding regions of the lipins of humans (HmLipin), mouse (MmLipin) and worm (CeLipin), and yeast Pah1p (ScLipin). Amino acid sequences were aligned with the CLUSTALW2 tool (http://www.ebi.ac.uk/Tools/ clustalw2/index.html). Boxes indicate the conserved Gly84 and Ser106 of mammalian lipin-1, the catalytic motif characteristic of PAP1 enzymes, DXDXT, and the conserved coactivator motif, LXXIL. (*), amino acids identical in all proteins aligned; (:) and (.) conserved and semiconserved substitutions, respectively. (B) The phylogram tree of the indicated lipin proteins was generated by CLUSTALW2 on the basis of multiple alignment of the entire CLPINs. V. Valente et al. Drosophila lipins molecular and cellular analysis FEBS Journal 277 (2010) 4775–4788 Journal compilation ª 2010 FEBS. No claim to original Brazilian government works 4779 Western blots carrying protein extracts of dissected tissues, probed with a-NLip, showed the presence of lipin proteins in all tissues evaluated of larvae (CNS, fat body, intestinal tract ⁄ Malpighian tubules and car- cass) and adults (ovary and testis) (Fig. 5D). Densi- tometry analysis of three immunoblots (Fig. 5E) revealed that the highest levels of lipin were present in the larval fat body: twice the amount detected in the CNS, and four to five times as much as observed in the other tissues analyzed. Measurement of TAG levels showed that the fat body of third instar larvae contains 20-fold more TAG than the other tissues analyzed. In addition, we observed that the TAG levels were  2.5-fold higher in the ovary than in the larval CNS, intestinal tract ⁄ Malpighian tubules, carcass and adult testis (Fig. 5F). Lipins have distinct subcellular distributions in different tissues To extend the characterization of Drosophila lipins, we evaluated the subcellular localization of these proteins in the fat body, CNS and intestinal tract of third instar larvae, and adult testis and ovary. Whole mounted dis- sected tissues were immunostained with a-NLip and analyzed by confocal microscopy. In the fat body, the label was diffusely distributed throughout the cyto- plasm and nucleoplasm, and was strongly concentrated in the perinuclear region of adipocytes (Fig. 6). In the CNS, the lipin signal was strongly detected in the cyto- plasm of cells of the brain hemispheres (not shown) and ventral ganglion (Fig. 6). Lipins were also present in the peripheral glia, but not in the axons of the seg- mental nerves (Fig. 6, arrows). In whole mount preparations of gut and testis, a-NLip demonstrated the presence of lipins in the vis- ceral musculature that circumvents both organs (Figs 7 and 8), but not in the gut epithelia or in the germ line cells of the testis (data not shown). In the intestinal tract, the lipin label was restricted to the midgut region, where the longitudinal musculature showed intense and striated labeling (Fig. 7). Similarly, in the testis musculature, lipin showed a striated pattern of labeling, and it was not detected in the muscles of the seminal vesicles, ejaculatory duct or accessory glands (Fig. 8). The striated labeling of lipin in the muscles of A D BC A′ Fig. 4. Expression pattern of DmLpin during development. (A) Northern blots containing polyA + RNA from whole animals in different devel- opmental stages after hybridization with the following probes: the full-length cDNA of DmLpinK (DmLpin), a fragment of DmLpinK first exon (DmLpinK) and a 120 bp fragment of DmLpinJ exon 1 that is specific for this isoform (DmLpinJ). The stages analyzed were embryo (E), larvae at first plus second stages (L1 ⁄ 2), larvae at the third stage (L3), prepupae and pupae (P), and adults (A). The membrane was stripped and reprobed with the internal control rp49. (B) Western blots containing protein of whole embryos (E), larvae at first plus second (L1 ⁄ 2) and third (L3) stages, pupae (P), male adults (M) and female adults (F) were probed with a-NLip and a-tubulin (Sigma). (C) Densitometry analyses of the bands corresponding to lipin and a-tubulin proteins were performed with IMAGEJ (http://rsbweb.nih.gov/ij/). The values are means (±standard deviations) of the pixel intensities corresponding to the normalized amounts of DmLipin expressed in arbitrary units (a.u.); n =3. (D) Amplification products of semiquantitative PCR reactions were separated eletrophoretically in agarose gels, and stained with ethidium bromide. RT-PCR reactions were performed with total RNA from animals at the indicated stages and primers directed to each DmLpin isoform (Fig. 1) and to the rp49 transcript. Amplification products were collected at PCR cycles 23, 26, 29 and 32. Drosophila lipins molecular and cellular analysis V. Valente et al. 4780 FEBS Journal 277 (2010) 4775–4788 Journal compilation ª 2010 FEBS. No claim to original Brazilian government works both the midgut and testis was interposed with actin signals, revealing that Drosophila lipins are concen- trated in the A band of these visceral muscle fibers (Figs 7 and 8, inserts). a-NLip showed diffuse labeling within the cytoplasm of the follicular cells, nurse cells and oocyte, through- out oogenesis (Fig. 8). Interestingly, lipins were enriched in the ring canals that connect nurse cells with each other and with the developing oocyte (Fig. 9), and colocalized with actin filaments in the inner rim of these canals (Fig. 9, lower panel). In addi- tion, relatively more intense labeling was observed in the cytoplasm of migrating polar ⁄ border cells (Fig. 9, inserts). Lipin was absent from the somatic muscle sheath that surrounds each ovariole (data not shown). Discussion We have characterized the molecular structure and the expression pattern of the lipin 1 ortholog of D. mela- nogaster (CG8709), named DmLpin. Our data demon- strated that this gene encodes three protein isoforms, referred to as DmLipinA, DmLipinK and DmLipinJ. Comparative analysis of the amino acid sequences of the conserved C-terminal domain shows that Drosoph- ila lipins are more closely related to the mammalian lipins than to C. elegans and S. cerevisae orthologs (Fig. 3B). Also, all D. melanogaster lipins have the DXDXT and the LXXIL motifs that are critical for the lipin PAP1 activity [29] and transcriptional coacti- vator function [8], respectively. Moreover, Gly84 and Ser106, which are required for lipin 1 activity in the A B C D E F Fig. 5. Expression pattern of DmLpin and levels of TAG in different tissues. (A) Northern blots containing 10 lg of total RNA from the CNS, intestinal tract plus Malpighian tubules (IT), fat body (FB) and carcass (CA) from L3 larvae were hybridized with the following probes: the full- length cDNA of DmLpinK (DmLpin), a fragment of the DmLpinK first exon (DmLpinK) and a fragment of the DmLpinA last exon (DmLpinA). (B) Multiplex RT-PCR for the DmLpin isoforms. RT-PCR reactions were performed with total RNA from ovary (Ov) and testis (Te) of adults and primers (Fig. 1) directed to each DmLpin isoform (DmLpinA, DmLpinK and DmLpinJ ). (C) Semiquantitative RT-PCR for the DmLpin iso- forms in different tissues. RT-PCR reactions were performed with total RNA of the indicated tissues and primers directed to each DmLpin isoform (Fig. 1) and to the rp49 transcript. Amplification products were collected at PCR cycles 26, 29, 32 and 35. (D) Western blots with protein extracts of carcass, fat body, intestinal tract plus Malpighian tubules and CNS from L3 larvae, and ovary and testis dissected from adults, were probed with a-NLip, stripped and reprobed with antibody against a-tubulin. The asterisk indicates an inconsistently detected signal. (E) Densitometry analysis of the bands corresponding to lipin and a-tubulin proteins was performed with IMAGEJ (http://rsbweb.nih.gov/ij/). The values are means (± standard deviations) of the pixel intensities corresponding to the normalized amounts of DmLipin expressed in arbitrary units (a.u.); n = 3. (F) Triglyceride content of carcass, fat body, intestinal tract plus Malpighian tubules and CNS from third instar larvae, and ovary and testis from adults. Errors bars represent the standard deviations for three independent assays. V. Valente et al. Drosophila lipins molecular and cellular analysis FEBS Journal 277 (2010) 4775–4788 Journal compilation ª 2010 FEBS. No claim to original Brazilian government works 4781 mouse [6,11], and Ser734, which is essential for the PAP1 activity of human lipin 2 [9], are conserved in DmLipin isoforms. The exon–intron organization of lipin mRNAs (Fig. 1) revealed that DmLpin is a complex locus. Dif- ferent regulatory and splicing mechanisms, such as alternative promoter ⁄ first exon, alternative terminal exon and intron retention [30], generate the Drosophila lipin isoforms. The three mRNAs have mutually exclu- sive first exons located at distances up to approxi- mately 14 kb (Fig. 1). This gene structure indicates the existence of alternative promoters and possible differ- ent transcriptional regulation for DmLpin expression, similar to what is found for mouse and human lipin 1 [26]. Consistent with this, although DmLpin is widely expressed, the levels of its transcripts vary during development and among tissues (Figs 4 and 5). DmLpinJ mRNA was detected from stage L3 throughout adult- hood, specifically in the testis. DmLpinK mRNA was most abundant in the larval CNS, and DmLpinA mRNA was present at higher levels in the intestinal tract ⁄ Malpighian tubules of larvae (Fig. 5 ). Regulated patterns of lipin tissue expression have also been reported for the mammalian paralogs [4]. Therefore, the existence of various forms of lipins expressed in a regu- lated manner seems to be related to the tissue complexity of the organism. Fat bodyVentral ganglion Fig. 6. Subcellular localization of DmLipins in the fat body and in the ventral ganglion of Drosophila larvae. Tissues were dissected from third instar larvae, immunolabeled with a-NLip and a-rabbit IgG conjugated to Alexa-594 (red), and stained with DAPI for chro- matin (blue) visualization. Arrows point to the abdominal nerves. The images are stacks of five optical sections (0.5 lm) from the central plane of the cells. Scale bars: 10 lm. Gut Fig. 7. Subcellular localization of DmLipins in the intestinal tract of third instar larvae. Tissues were dissected and immunostained by the use of a-NLpi and a-rabbit IgG conjugated to Alexa-488 (green). F-actin and nuclei were labeled with phalloidin conjugated to Alexa-594 (red) and DAPI (blue), respectively. MG, midgut; HD, hindgut. The insert shows higher magnification of muscle cells: the sarcomeric pattern of lipin labeling can be clearly observed. The images are stacks of 10 optical sections (0.5 lm) in the plane of the muscle layers. Scale bars: 10 lm. Drosophila lipins molecular and cellular analysis V. Valente et al. 4782 FEBS Journal 277 (2010) 4775–4788 Journal compilation ª 2010 FEBS. No claim to original Brazilian government works Although the highest levels of the lipin proteins were observed in the larval fat body (Fig. 5E), both DmLp- inA and DmLpinK mRNAs were present in lower amounts in this tissue than in the intestinal tract ⁄ Mal- pighian tubules (DmLpinA) and CNS (DmLpinK) (Fig. 5A,C). Divergences between the levels of lipin proteins and mRNAs were also observed during devel- opment. Lipin proteins reached their maximum levels at the larval ⁄ pupal stages (Fig. 4C), whereas lipin mRNAs were detected in similar amounts at all stages (Fig. 4D). These data indicate that DmLpin expression is regulated at the protein level in addition to the mRNA transcription. Interestingly, regulation at the translational level has been recently described for mouse lipin 2. It was demonstrated that in the fld mouse strain, which is deficient for lipin 1, the rate of lipin 2 protein synthesis was increased in hepatocytes, although the lipin 2 mRNA amounts in wild-type and fld animals were similar [31]. The Drosophila lipins migrate in the range of 130– 140 kDa in SDS ⁄ PAGE (Fig. 4B and 5D), which is different from what is expected for the polypeptides on the basis of the mRNA sequences, which gave estimated masses of 120.9 kDa (DmLipinA), 114.5 kDa (DmLipinK) and 106.6 kDa (DmLipinJ). A shift in SDS ⁄ PAGE migration seems to be characteristic of this protein family, as lipin 1 of rat migrates at  140 kDa [10], and endogenous mouse [4] and C. elegans [16] lipins migrate well above 97 kDa, although their deduced molecular masses are between 51 and 103 kDa. The small differences observed in the migration distances of Drosophila lipin among samples (Figs 4B and 5D) may be attributable to the predominance of a particular isoform (DmLipinA, DmLipinK or DmLipinJ). Indeed, in the testis, where the smaller isoform D is specifically expressed, a lipin that migrates faster than those detected in the other tissues was clearly observed (Fig. S2). Also, variations in the phosphorylation status might influence the migration of the lipins. Lipins contain several poten- tial phosphorylation sites, and some of them have been proven to be phosphorylated in vivo. In fact, phosphorylation regulates the protein activity and subcellular localization of Pah1p and mammalian lipin 1 [11,14]. The observation of the highest levels of lipin pro- teins and TAG in the fat body of third instar larvae (Fig. 5E,F) is compatible with the activity of lipin in the production of storage lipids [26]. As DmLipinA and DmLipinK are expressed in this tissue, both of these proteins could be involved with TAG biosynthe- sis. However, in the CNS, where the TAG levels are significantly lower than in the fat body and ovary, DmLpinK mRNA is the most abundant. Also, in the testis, where TAG levels are even lower, DmLpinJ is the predominant isoform. Together, these data suggest Testis Fig. 8. Subcellular localization of DmLipins in the testis of adults. Tissues were dissected and immunostained by the use of a-NLip and a-rabbit IgG conjugated to Alexa-488 (green). F-actin and nuclei were labeled with phalloidin conjugated to Alexa-594 (red) and DAPI (blue), respectively. ED, ejaculatory duct; AG, accessory gland; SV, seminal vesicle; T, testis. The insert shows higher magnifications of muscle cells: the sarcomeric pattern of lipin labeling can be clearly observed. The images are stacks of 10 optical sections (0.5 lm) in the plane of the muscle layers. Scale bars: 20 lm. V. Valente et al. Drosophila lipins molecular and cellular analysis FEBS Journal 277 (2010) 4775–4788 Journal compilation ª 2010 FEBS. No claim to original Brazilian government works 4783 that DmLipinA might be the isoform mainly involved with TAG production. On the other hand, DmLipinK and DmLipinJ, which is an N-terminal-truncated version of DmLipinK, could perform other cellular functions. In the fat body and ventral ganglion cells of feeding L3 larvae, lipins were concentrated in the perinuclear region, consistent with an enrichment in the ER and nuclear envelope, as shown by immunolocalization of C. elegans LPIN-1 [11,16,27]. Downregulation of C. elegans LPIN-1 disrupts ER organization and causes the appearance of membrane sheets, in addition to defects in nuclear structure and chromosome segre- gation [15,16]. Genetic and biochemical studies per- formed mainly in yeast have linked Pah1p function to nuclear membrane biogenesis [2]. Yeast mutants with deletion of PAH1 display irregularly shaped nuclei with long stacks of membranes that contain nuclear pores and appear to be in contact with the nuclear envelope [13]. Similar defects are observed in Ned1 lipin mutants of the fission yeast, which also show impairment of chromosome maintenance [32]. In the intestinal tract of larvae, lipin was readily detected in the midgut longitudinal muscle fibers. In Fig. 9. Subcellular localization of DmLipins in egg chambers of adults. Ovaries were dissected and immunostained by the use of a-NLip and anti-rabbit IgG conjugated to Alexa-594 (red, upper panel) or to Alexa-488 (green, bottom panel). F-actin and chromatin were labeled with phalloidin conjugated to Alexa-594 (red) and DAPI (blue), respec- tively. Note the colocalization of lipin and the actin filaments in the ring channels, which make connections between the cytoplasm of the nurse cells and the oocyte. Insert: migrating polar ⁄ border cells. The images in the upper panels are stacks of 10 optical sections (2 lm). In the lower panel, the images are stacks of three optical sections (0.1 lm) in the equatorial plane of a ring canal. Scale bar: 10 lm. Drosophila lipins molecular and cellular analysis V. Valente et al. 4784 FEBS Journal 277 (2010) 4775–4788 Journal compilation ª 2010 FEBS. No claim to original Brazilian government works [...]... cytoskeleton, and did not exhibit membrane ruffles after insulin treatment [20] Although our data do not provide a direct functional link between lipin and tissue development or actin filament dynamics, they should stimulate further investigations in this regard Drosophila lipins molecular and cellular analysis Experimental procedures Drosophila lines and culture Canton S and yw flies were grown on standard... considered Lipins could be required for the differentiation or maintenance of the tissue by controlling PA or DAG availability, the key elements in lipid mediated-signaling In fact, wunen and wunen-2, Drosophila homologs of mammalian PAP2, are known to regulate the migration and survival of primordial germ cells during embryogenesis [36] Active lipins are mainly localized to membranes [11,13,14,27,37], and. .. gut, and the muscles of the accessory glands, seminal vesicle and ejaculatory duct (Figs 7 and 8), and the muscle cells of the ovary (Fig 9), lipins were undetected The reason for lipin expression in specific visceral muscles is unclear Correlation of lipin levels with mammalian muscle activity has been recently reported [33] However, differences in the activity of the analyzed visceral muscles are improbable... Genetic Analyzer (Applied Biosytems-HITACHI, Foster City, CA, USA) Sequence analysis and clustering were performed with the staden software package, and the amino acid sequence alignments were performed with clustalw RNA extraction, northern blot and RT-PCR Drosophila total RNA was extracted with Trizol reagent (Invitrogen, Aukland, NZ), according to the manufacturer’s instructions, with an extra phenol ⁄... photoshop 6.0 for contrast and brightness only Acknowledgements We are grateful to G M Rubin, who provided the BDGP cDNA libraries We thank C A.V Saraiva for her dedicated technical assistance, B O de Souza and S Regina Andrade for keeping the laboratory materials, and T Paula Aquino Defina for DNA sequencing assistance This work was supported by grants from FAPESP proc# 2007 ⁄ 50173-8 and CNPq (proc# 479444/2008-0)... Infection decreases fatty acid oxidation and nuclear hormone receptors in the diaphragm J Lipid Res 50, 2055–2063 34 Bodenstein D (1994) The postembryonic development of Drosophila In Biology of Drosophila (Demerec M FEBS Journal 277 (2010) 4775–4788 Journal compilation ª 2010 FEBS No claim to original Brazilian government works 4787 Drosophila lipins molecular and cellular analysis 35 36 37 38 39 40... for CNS, 10 for intestinal tract, and five for carcass) and adult flies (five for ovary and 60 for testis) were dissected in cold NaCl ⁄ Pi Tissues were then homogenized in 100 lL of 0.1% Tween-20 plus protease inhibitors, heated at 70 °C for 5 min to inactivate endogenous enzymes, and centrifuged The homogenates were spun at 2700 g on a tabletop centrifuge for 1 min, and the supernatant was then additionally... mainly localized to membranes [11,13,14,27,37], and in both midgut longitudinal muscles and muscles of the testis, lipins were concentrated in the A band (Figs 7 and 8), a sarcomeric region enriched in ER membranes In the ovary chamber cells, lipin was clearly detected in the ring canals, starting in the germarium and continuing throughout later oogenesis Lipin was remarkably enriched in the internal... proc# 2007 ⁄ 50173-8 and CNPq (proc# 479444/2008-0) V Valente and R M Maia were supported by fellowships from FAPESP, and M Vianna received a fellowship from CAPES M L Paco-Larson ¸ ´ is a CNPq fellow References 1 Reue K (2009) The lipin family: mutations and metabolism Curr Opin Lipidol 20, 165–170 2 Siniossoglou S (2009) Lipins, lipids and nuclear envelope structure Traffic 10, 1181–1187 3 Han GS,... mutant mouse with a developmental abnormality in triglyceride metabolism and associated tissue -specific defects in lipoprotein lipase and hepatic lipase activities J Biol Chem 264, 7994–8003 18 Langner CA, Birkenmeier EH, Roth KA, Bronson RT & Gordon JI (1991) Characterization of the peripheral neuropathy in neonatal and adult mice that are homozygous for the fatty liver dystrophy (fld) mutation J Biol Chem . Drosophila melanogaster lipins are tissue -regulated and developmentally regulated and present specific subcellular distributions Valeria. Drosophila lipins are more closely related to mammalian lipins than to yeast Pah1p or the worm lipin (Fig. 3B). DmLpin expression is regulated during Drosophila