RESUMEN
Regular chromosome segregation during the first meiotic division requires prior pairing of homologous chromosomes into bivalents. During canonical meiosis, linkage between homologous chromosomes is maintained until late metaphase I by chiasmata resulting from meiotic recombination in combination with distal sister chromatid cohesion. Separase-mediated elimination of cohesin from chromosome arms at the end of metaphase I permits terminalization of chiasmata and homolog segregation to opposite spindle poles during anaphase I. Interestingly, separase is also required for bivalent splitting during meiosis I in Drosophila males, where homologs are conjoined by an alternative mechanism independent of meiotic recombination and cohesin. Here we report the identification of a novel alternative homolog conjunction protein encoded by the previously uncharacterized gene univalents only (uno). The univalents that are present in uno null mutants at the start of meiosis I, instead of normal bivalents, are segregated randomly. In wild type, UNO protein is detected in dots associated with bivalent chromosomes and most abundantly at the localized pairing site of the sex chromosomes. UNO is cleaved by separase. Expression of a mutant UNO version with a non-functional separase cleavage site restores homolog conjunction in a uno null background. However, separation of bivalents during meiosis I is completely abrogated by this non-cleavable UNO version. Therefore, we propose that homolog separation during Drosophila male meiosis I is triggered by separase-mediated cleavage of UNO.
Asunto(s)
Proteínas de Drosophila/genética , Meiosis/genética , Separasa/genética , Factores de Transcripción/genética , Animales , Proteínas de Ciclo Celular/genética , División del Núcleo Celular/genética , Centrómero/genética , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/genética , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica/genética , Masculino , Metafase/genética , Cromosomas Sexuales/genética , CohesinasRESUMEN
CRISPR-Cas9-based genome editing has transformed the life sciences, enabling virtually unlimited genetic manipulation of genomes: The RNA-guided Cas9 endonuclease cuts DNA at a specific target sequence and the resulting double-strand breaks are mended by one of the intrinsic cellular repair pathways. Imprecise double-strand repair will introduce random mutations such as indels or point mutations, whereas precise editing will restore or specifically edit the locus as mandated by an endogenous or exogenously provided template. Recent studies indicate that CRISPR-induced DNA cuts may also result in the exchange of genetic information between homologous chromosome arms. However, conclusive data of such recombination events in higher eukaryotes are lacking. Here, we show that in Drosophila, the detected Cas9-mediated editing events frequently resulted in germline-transmitted exchange of chromosome arms-often without indels. These findings demonstrate the feasibility of using the system for generating recombinants and also highlight an unforeseen risk of using CRISPR-Cas9 for therapeutic intervention.
Asunto(s)
Cromosomas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Roturas del ADN de Doble Cadena , Recombinación Homóloga , Animales , Secuencia de Bases , Sistemas CRISPR-Cas , Drosophila/genética , Edición Génica , Expresión Génica , Marcación de Gen , Genes Reporteros , Conformación de Ácido Nucleico , Fenotipo , ARN Guía de Kinetoplastida/genéticaRESUMEN
Muscles organise pseudo-crystalline arrays of actin, myosin and titin filaments to build force-producing sarcomeres. To study sarcomerogenesis, we have generated a transcriptomics resource of developing Drosophila flight muscles and identified 40 distinct expression profile clusters. Strikingly, most sarcomeric components group in two clusters, which are strongly induced after all myofibrils have been assembled, indicating a transcriptional transition during myofibrillogenesis. Following myofibril assembly, many short sarcomeres are added to each myofibril. Subsequently, all sarcomeres mature, reaching 1.5 µm diameter and 3.2 µm length and acquiring stretch-sensitivity. The efficient induction of the transcriptional transition during myofibrillogenesis, including the transcriptional boost of sarcomeric components, requires in part the transcriptional regulator Spalt major. As a consequence of Spalt knock-down, sarcomere maturation is defective and fibers fail to gain stretch-sensitivity. Together, this defines an ordered sarcomere morphogenesis process under precise transcriptional control - a concept that may also apply to vertebrate muscle or heart development.
Asunto(s)
Drosophila melanogaster/genética , Vuelo Animal/fisiología , Morfogénesis , Músculos/fisiología , Sarcómeros/metabolismo , Transcriptoma/genética , Animales , Regulación del Desarrollo de la Expresión Génica , Desarrollo de Músculos/genética , Análisis de Componente Principal , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
The ease of generating genetically modified animals and cell lines has been markedly increased by the recent development of the versatile CRISPR/Cas9 tool. However, while the isolation of isogenic cell populations is usually straightforward for mammalian cell lines, the generation of clonal Drosophila cell lines has remained a longstanding challenge, hampered by the difficulty of getting Drosophila cells to grow at low densities. Here, we describe a highly efficient workflow to generate clonal Cas9-engineered Drosophila cell lines using a combination of cell pools, limiting dilution in conditioned medium and PCR with allele-specific primers, enabling the efficient selection of a clonal cell line with a suitable mutation profile. We validate the protocol by documenting the isolation, selection and verification of eight independently Cas9-edited armadillo mutant Drosophila cell lines. Our method provides a powerful and simple workflow that improves the utility of Drosophila cells for genetic studies with CRISPR/Cas9.
Asunto(s)
Sistemas CRISPR-Cas , Cartilla de ADN/química , Drosophila/genética , Ingeniería Genética/métodos , Genoma de los Insectos , Análisis de la Célula Individual/métodos , Alelos , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , Mutación , Homología de SecuenciaRESUMEN
Using label-free ToF-SIMS imaging mass spectrometry, we generated a map of small molecules differentially expressed in the Drosophila wing imaginal disc. The distributions of these moieties were in line with gene expression patterns observed during wing imaginal disc development. Combining ToF-SIMS imaging and coherent anti-Stokes Raman spectroscopy (CARS) microspectroscopy allowed us to locally identify acylglycerols as the main constituents of the pattern differentiating the future body wall tissue from the wing blade tissue. The findings presented herein clearly demonstrate that lipid localization patterns are strongly correlated with a developmental gene expression. From this correlation, we hypothesize that lipids play a so far unrecognized role in organ development.
Asunto(s)
Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/genética , Perfilación de la Expresión Génica , Glicéridos/análisis , Discos Imaginales/crecimiento & desarrollo , Espectrometría de Masa de Ion Secundario , Alas de Animales/crecimiento & desarrollo , Animales , Drosophila melanogaster/anatomía & histología , Glicéridos/genética , Discos Imaginales/anatomía & histología , Espectrometría Raman , Factores de Tiempo , Alas de Animales/anatomía & histologíaRESUMEN
The hallmark of canonical Wnt signaling is the transcriptional induction of Wnt target genes by the beta-catenin/TCF complex. Several studies have proposed alternative interaction partners for beta-catenin or TCF, but the relevance of potential bifurcations in the distal Wnt pathway remains unclear. Here we study on a genome-wide scale the requirement for Armadillo (Arm, Drosophila beta-catenin) and Pangolin (Pan, Drosophila TCF) in the Wnt/Wingless(Wg)-induced transcriptional response of Drosophila Kc cells. Using somatic genetics, we demonstrate that both Arm and Pan are absolutely required for mediating activation and repression of target genes. Furthermore, by means of STARR-sequencing we identified Wnt/Wg-responsive enhancer elements and found that all responsive enhancers depend on Pan. Together, our results confirm the dogma of canonical Wnt/Wg signaling and argue against the existence of distal pathway branches in this system.
Asunto(s)
Proteínas del Dominio Armadillo/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Vía de Señalización Wnt , Proteína Wnt1/metabolismo , Animales , Proteínas del Dominio Armadillo/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Genoma de los Insectos , Proteínas Represoras/genética , Análisis de Secuencia de ARN , Transducción de Señal , Factores de Transcripción/genética , Proteína Wnt1/genéticaRESUMEN
In animals, microRNAs frequently form families with related sequences. The functional relevance of miRNA families and the relative contribution of family members to target repression have remained, however, largely unexplored. Here, we used the Caenorhabditis elegans miR-58 miRNA family, composed primarily of the four highly abundant members miR-58.1, miR-80, miR-81, and miR-82, as a model to investigate the redundancy of miRNA family members and their impact on target expression in an in vivo setting. We found that miR-58 family members repress largely overlapping sets of targets in a predominantly additive fashion. Progressive deletions of miR-58 family members lead to cumulative up-regulation of target protein and RNA levels. Phenotypic defects could only be observed in the family quadruple mutant, which also showed the strongest change in target protein levels. Interestingly, although the seed sequences of miR-80 and miR-58.1 differ in a single nucleotide, predicted canonical miR-80 targets were efficiently up-regulated in the mir-58.1 single mutant, indicating functional redundancy of distinct members of this miRNA family. At the aggregate level, target binding leads mainly to mRNA degradation, although we also observed some degree of translational inhibition, particularly in the single miR-58 family mutants. These results provide a framework for understanding how miRNA family members interact to regulate target mRNAs.
Asunto(s)
Caenorhabditis elegans/genética , MicroARNs/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , Regulación hacia Arriba , Animales , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Represión Epigenética , MicroARNs/metabolismo , Proteómica , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN , TranscriptomaRESUMEN
In bottom-up mass spectrometry-based proteomics analyses, variability at any step of the process, particularly during sample proteolysis, directly affects the sensitivity, accuracy, and precision of peptide detection and quantification. Currently, no generic internal standards are available to control the quality of sample processing steps. This makes it difficult to assess the comparability of MS proteomic data obtained under different experimental conditions. Here, we describe the design, synthesis, and validation of a universal protein standard, called DIGESTIF, that can be added to any biological sample. The DIGESTIF standard consists of a soluble recombinant protein scaffold to which a set of 11 artificial peptides (iRT peptides) with good ionization properties has been incorporated. In the protein scaffold, the amino acids flanking iRT peptide cleavage sites were selected either to favor or hinder protease cleavage. After sample processing, the retention time and relative intensity pattern of the released iRT peptides can be used to assess the quality of sample workup, the extent of digestion, and the performance of the LC-MS system. Thus, DIGESTIF can be used to standardize a broad spectrum of applications, ranging from simple replicate measurements to large-scale biomarker screening in biomedical applications.
Asunto(s)
Proteínas/química , Proteómica , Secuencia de Aminoácidos , Animales , Biomarcadores/química , Cromatografía Liquida , Humanos , Cinética , Masculino , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Proteolisis , Control de CalidadRESUMEN
The in silico prediction of the best-observable "proteotypic" peptides in mass spectrometry-based workflows is a challenging problem. Being able to accurately predict such peptides would enable the informed selection of proteotypic peptides for targeted quantification of previously observed and non-observed proteins for any organism, with a significant impact for clinical proteomics and systems biology studies. Current prediction algorithms rely on physicochemical parameters in combination with positive and negative training sets to identify those peptide properties that most profoundly affect their general detectability. Here we present PeptideRank, an approach that uses learning to rank algorithm for peptide detectability prediction from shotgun proteomics data, and that eliminates the need to select a negative dataset for the training step. A large number of different peptide properties are used to train ranking models in order to predict a ranking of the best-observable peptides within a protein. Empirical evaluation with rank accuracy metrics showed that PeptideRank complements existing prediction algorithms. Our results indicate that the best performance is achieved when it is trained on organism-specific shotgun proteomics data, and that PeptideRank is most accurate for short to medium-sized and abundant proteins, without any loss in prediction accuracy for the important class of membrane proteins. BIOLOGICAL SIGNIFICANCE: Targeted proteomics approaches have been gaining a lot of momentum and hold immense potential for systems biology studies and clinical proteomics. However, since only very few complete proteomes have been reported to date, for a considerable fraction of a proteome there is no experimental proteomics evidence that would allow to guide the selection of the best-suited proteotypic peptides (PTPs), i.e. peptides that are specific to a given proteoform and that are repeatedly observed in a mass spectrometer. We describe a novel, rank-based approach for the prediction of the best-suited PTPs for targeted proteomics applications. By building on methods developed in the field of information retrieval (e.g. web search engines like Google's PageRank), we circumvent the delicate step of selecting positive and negative training sets and at the same time also more closely reflect the experimentalist´s need for selecting e.g. the 5 most promising peptides for targeting a protein of interest. This approach allows to predict PTPs for not yet observed proteins or for organisms without prior experimental proteomics data such as many non-model organisms.
Asunto(s)
Algoritmos , Proteínas Bacterianas/genética , Bartonella henselae/genética , Bases de Datos de Proteínas , Proteínas de Drosophila/genética , Leptospira interrogans/genética , Péptidos/genética , Saccharomyces cerevisiae/genética , Análisis de Secuencia de Proteína/métodos , Animales , Proteínas Bacterianas/metabolismo , Bartonella henselae/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Leptospira interrogans/metabolismo , Péptidos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiaeRESUMEN
Imaginal discs, especially the wing imaginal disc, are powerful model systems to study organ development. The traditional methods to analyze wing imaginal discs depend on the laborious and time-consuming dissection of larvae. "Omics"-based approaches, such as RNA-seq, ChIP-seq, proteomics and lipidomics, offer new opportunities for the systems-level investigation of organ development. However, it is impractical to manually isolate the required starting material. This is even more problematic when experiments strive for enhanced temporal and spatial resolution. The mass isolation workflow discussed in this review, solves this problem. The semi-automated sorting of 1000 wing imaginal discs in less than 3h forms the basis of a workflow that can be connected to biochemical analyses of organ patterning and growth. In addition to the mass isolation workflow we briefly describe key "omics" technologies and their applications. The combination of mass isolation and "omics"-approaches ensures that the wing imaginal disc will continue to be a key model organ for studying developmental processes, both on the genetic, but increasingly also on the biochemical level.
Asunto(s)
Biología Evolutiva/métodos , Proteínas de Drosophila/biosíntesis , Discos Imaginales/metabolismo , Alas de Animales/metabolismo , Animales , Movimiento Celular/genética , Drosophila , Regulación del Desarrollo de la Expresión Génica , Discos Imaginales/crecimiento & desarrollo , Larva , Proteómica/métodos , Alas de Animales/crecimiento & desarrolloRESUMEN
Time-of-flight secondary ion mass spectrometry imaging is a rapidly evolving technology. Its main application is the study of the distribution of small molecules on biological tissues. The sequential image acquisition process remains susceptible to measurement distortions that can render imaging data less analytically useful. Most of these artifacts show a repetitive nature from tile to tile. Here we statistically describe these distortions and derive two different algorithms to correct them. Both a generalized linear model approach and the linear discriminant analysis approach are able to increase image quality for negative and positive ion mode data sets. Additionally, performing simulation studies with repetitive and nonrepetitive tiling error we show that both algorithms are only removing repetitive distortions. It is further shown that the spectral component of the data set is not altered by the use of these correction methods. Both algorithms presented in this work greatly increase the image quality and improve the analytical usefulness of distorted images dramatically.
Asunto(s)
Algoritmos , Espectrometría de Masas/métodos , Análisis DiscriminanteRESUMEN
The co-operation of specialized organ systems in complex multicellular organisms depends on effective chemical communication. Thus, body fluids (like blood, lymph or intraspinal fluid) contain myriads of signaling mediators apart from metabolites. Moreover, these fluids are also of crucial importance for immune and wound responses. Compositional analyses of human body fluids are therefore of paramount diagnostic importance. Further improving their comprehensiveness should increase our understanding of inter-organ communication. In arthropods, which have trachea for gas exchange and an open circulatory system, the single dominating interstitial fluid is the hemolymph. Accordingly, a detailed analysis of hemolymph composition should provide an especially comprehensive picture of chemical communication and defense in animals. Therefore we used an extensive protein fractionation workflow in combination with a discovery-driven proteomic approach to map out the detectable protein composition of hemolymph isolated from Drosophila larvae. Combined mass spectrometric analysis revealed more than 700 proteins extending far beyond the previously known Drosophila hemolymph proteome. Moreover, by comparing hemolymph isolated from either fed or starved larvae, we provide initial provisional insights concerning compositional changes in response to nutritional state. Storage proteins in particular were observed to be strongly reduced by starvation. Our hemolymph proteome catalog provides a rich basis for data mining, as exemplified by our identification of potential novel cytokines, as well as for future quantitative analyses by targeted proteomics.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Hemolinfa/metabolismo , Animales , Proteínas Sanguíneas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Privación de Alimentos/fisiología , Larva/crecimiento & desarrollo , Larva/metabolismo , Anotación de Secuencia Molecular , Proteoma/genética , Proteoma/metabolismoRESUMEN
The community working on model organisms is growing steadily and the number of model organisms for which proteome data are being generated is continuously increasing. To standardize efforts and to make optimal use of proteomics data acquired from model organisms, a new Human Proteome Organisation (HUPO) initiative on model organism proteomes (iMOP) was approved at the HUPO Ninth Annual World Congress in Sydney, 2010. iMOP will seek to stimulate scientific exchange and disseminate HUPO best practices. The needs of model organism researchers for central databases will be better represented, catalyzing the integration of proteomics and organism-specific databases. Full details of iMOP activities, members, tools and resources can be found at our website http://www.imop.uzh.ch/ and new members are invited to join us.
Asunto(s)
Arabidopsis/química , Modelos Animales , Proteoma , Animales , Animales de Laboratorio , Bases de Datos de Proteínas , HumanosRESUMEN
Proteomes, the ensembles of all proteins expressed by cells or tissues, are typically analysed by mass spectrometry. Recent technical and computational advances have greatly increased the fraction of a proteome that can be identified and quantified in a single study. Current mass spectrometry-based proteomic strategies have the potential to reproducibly, accurately, quantitatively and comprehensively measure any protein or whole proteomes from cells and tissues at different states. Achieving these goals will require complete proteome maps and analytical strategies that use these maps as prior information and will greatly enhance the impact of proteomics on biological and clinical research.
Asunto(s)
Espectrometría de Masas/métodos , Proteínas/análisis , Proteoma/análisis , Proteómica/métodos , Animales , Humanos , Reproducibilidad de los ResultadosAsunto(s)
Bacterias/metabolismo , Eucariontes/metabolismo , Modelos Biológicos , Proteoma/análisis , Proteómica/métodos , Bacterias/genética , Proteínas Bacterianas/análisis , Electroforesis en Gel Bidimensional/métodos , Eucariontes/genética , Proteínas Fúngicas/análisis , Espectrometría de Masas/métodos , Modelos Animales , Procesamiento Proteico-PostraduccionalRESUMEN
Wingless (Wg) signaling regulates expression of its target genes via Pangolin and Armadillo, and their interacting cofactors. In the absence of Wg, Pangolin mediates transcriptional repression. In the presence of Wg, Pangolin, Armadillo, and a cohort of coactivators mediate transcriptional activation. Here we uncover Coop (corepressor of Pan) as a Pangolin-interacting protein. Coop and Pangolin form a complex on DNA containing a Pangolin/TCF-binding motif. Overexpression of Coop specifically represses Wg target genes, while loss of Coop function causes derepression. Finally, we show that Coop antagonizes the binding of Armadillo to Pangolin, providing a mechanism for Coop-mediated repression of Wg target gene transcription.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Regulación de la Expresión Génica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Proteína Wnt1/metabolismo , Animales , Proteínas del Dominio Armadillo/metabolismo , Proteínas Co-Represoras/metabolismo , Drosophila melanogaster/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: The TSC-22 domain family (TSC22DF) consists of putative transcription factors harboring a DNA-binding TSC-box and an adjacent leucine zipper at their carboxyl termini. Both short and long TSC22DF isoforms are conserved from flies to humans. Whereas the short isoforms include the tumor suppressor TSC-22 (Transforming growth factor-beta1 stimulated clone-22), the long isoforms are largely uncharacterized. In Drosophila, the long isoform Bunched A (BunA) acts as a growth promoter, but how BunA controls growth has remained obscure. RESULTS: In order to test for functional conservation among TSC22DF members, we expressed the human TSC22DF proteins in the fly and found that all long isoforms can replace BunA function. Furthermore, we combined a proteomics-based approach with a genetic screen to identify proteins that interact with BunA. Madm (Mlf1 adapter molecule) physically associates with BunA via a conserved motif that is only contained in long TSC22DF proteins. Moreover, Drosophila Madm acts as a growth-promoting gene that displays growth phenotypes strikingly similar to bunA phenotypes. When overexpressed, Madm and BunA synergize to increase organ growth. CONCLUSIONS: The growth-promoting potential of long TSC22DF proteins is evolutionarily conserved. Furthermore, we provide biochemical and genetic evidence for a growth-regulating complex involving the long TSC22DF protein BunA and the adapter molecule Madm.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila/fisiología , Drosophila/crecimiento & desarrollo , Leucina Zippers/fisiología , Proteínas Represoras/química , Proteínas Supresoras de Tumor/fisiología , Animales , Secuencia Conservada , Dípteros/genética , Drosophila/genética , Crecimiento/genética , Humanos , Receptores Citoplasmáticos y Nucleares , Alineación de Secuencia , Transducción de Señal/fisiología , Factores de Transcripción/química , Factores de Transcripción/fisiología , Factor de Crecimiento Transformador beta1/fisiología , Proteínas de Transporte VesicularRESUMEN
Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (X)PX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (X)PX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Acetilación , Alanina/genética , Alanina/metabolismo , Animales , Animales Modificados Genéticamente , Sitios de Unión/genética , Western Blotting , Carboxiliasas/genética , Carboxiliasas/metabolismo , Línea Celular , Bases de Datos de Proteínas , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Células HeLa , Humanos , Inmunoprecipitación , Espectrometría de Masas , Mutación , Biosíntesis de Proteínas , Serina/genética , Serina/metabolismo , Treonina/genética , Treonina/metabolismo , Transgenes/genéticaRESUMEN
Autophagy is a lysosomal-mediated degradation process that promotes cell survival during nutrient-limiting conditions. However, excessive autophagy results in cell death. In Drosophila, autophagy is regulated nutritionally, hormonally and developmentally in several tissues, including the fat body, a nutrient-storage organ. Here we use a proteomics approach to identify components of starvation-induced autophagic responses in the Drosophila fat body. Using cICAT labeling and mass spectrometry, differences in protein expression levels of normal compared to starved fat bodies were determined. Candidates were analyzed genetically for their involvement in autophagy in fat bodies deficient for the respective genes. One of these genes, Desat1, encodes a lipid desaturase. Desat1 mutant cells fail to induce autophagy upon starvation. The desat1 protein localizes to autophagic structures after nutrient depletion and is required for fly development. Lipid analyses revealed that Desat1 regulates the composition of lipids in Drosophila. We propose that Desat1 exerts its role in autophagy by controlling lipid biosynthesis and/or signaling necessary for autophagic responses.