RESUMO
A key challenge of functional genomics today is to generate well-annotated data sets that can be interpreted across different platforms and technologies. Large-scale functional genomics data often fail to connect to standard experimental approaches of gene characterization in individual laboratories. Furthermore, a lack of universal annotation standards for phenotypic data sets makes it difficult to compare different screening approaches. Here we address this problem in a screen designed to identify all genes required for the first two rounds of cell division in the Caenorhabditis elegans embryo. We used RNA-mediated interference to target 98% of all genes predicted in the C. elegans genome in combination with differential interference contrast time-lapse microscopy. Through systematic annotation of the resulting movies, we developed a phenotypic profiling system, which shows high correlation with cellular processes and biochemical pathways, thus enabling us to predict new functions for previously uncharacterized genes.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Desenvolvimento Embrionário/genética , Genoma , Interferência de RNA , Animais , Caenorhabditis elegans/fisiologia , Biologia Computacional , Genes de Helmintos/genética , Genômica , Fenótipo , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Regulation of ligand-gated ion channel (LGIC) function and trafficking by cytoskeleton proteins has been the topic of recent research. Here, we report that the light chain (LC1) of microtubule-associated protein 1B (MAP1B) specifically interacted with the 5-HT(3A) receptor, a predominant serotonin-gated ion channel in the brain. LC1 and 5-HT(3A) receptors were colocalized in central neurons and in HEK 293 cells expressing 5-HT(3A) receptors. LC1 reduced the steady-state density of 5-HT(3A) receptors at the membrane surface of HEK 293 cells and significantly accelerated receptor desensitization time constants from 3.8 +/- 0.3 s to 0.8 +/- 0.1 s. However, LC1 did not significantly alter agonist binding affinity and single-channel conductance of 5-HT(3A) receptors. On the other hand, application of specific LC1 antisense oligonucleotides and nocodazole, a microtubule disruptor, significantly prolonged the desensitization time of the recombinant and native neuronal 5-HT(3) receptors by 3- to 6-fold. This kinetic change induced by nocodazole was completely rescued by addition of LC1 but not GABA(A) receptor-associated protein (GABARAP), suggesting that LC1 can specifically interact with 5-HT(3A) receptors. These observations suggest that the LC1-5-HT(3A) receptor interaction contributes to a mechanism that regulates receptor desensitization kinetics. Such dynamic regulation may play a role in reshaping the efficacy of 5-HT(3) receptor-mediated synaptic transmission.
Assuntos
Rim/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores 5-HT3 de Serotonina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Linhagem Celular , Membrana Celular/metabolismo , Células Cultivadas , Feminino , Hipocampo/citologia , Humanos , Rim/citologia , Rim/embriologia , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Nocodazol/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores 5-HT3 de Serotonina/efeitos dos fármacos , Moduladores de Tubulina/farmacologiaRESUMO
Recent reports suggest the high frequency of nickel induced allergic contact dermatitis and the absence in some patients of a clinical improvement following topical avoidance of sensitising allergen. In this study we evaluated the role of food ingested nickel in the maintenance and exacerbations of the disease. A high frequency of cutaneous exacerbations was obtained after oral challenge with a low nickel containing diet. These results suggest a pathogenetic role of nickel present in foods and indicate that an appropriate diet may be useful in the prophylaxis and in the therapy of nickel dermatitis in sensitized patients.
Assuntos
Dermatite de Contato/etiologia , Hipersensibilidade Alimentar/etiologia , Níquel/efeitos adversos , Dermatite de Contato/prevenção & controle , Dieta , Feminino , Humanos , Masculino , Níquel/administração & dosagemRESUMO
Genome sequencing projects generate a wealth of information; however, the ultimate goal of such projects is to accelerate the identification of the biological function of genes. This creates a need for comprehensive studies to fill the gap between sequence and function. Here we report the results of a functional genomic screen to identify genes required for cell division in Caenorhabditis elegans. We inhibited the expression of approximately 96% of the approximately 2,300 predicted open reading frames on chromosome III using RNA-mediated interference (RNAi). By using an in vivo time-lapse differential interference contrast microscopy assay, we identified 133 genes (approximately 6%) necessary for distinct cellular processes in early embryos. Our results indicate that these genes represent most of the genes on chromosome III that are required for proper cell division in C. elegans embryos. The complete data set, including sample time-lapse recordings, has been deposited in an open access database. We found that approximately 47% of the genes associated with a differential interference contrast phenotype have clear orthologues in other eukaryotes, indicating that this screen provides putative gene functions for other species as well.