RESUMEN
The microtubule-based motor kinesin-I is essential for the intracellular transport of membrane-bound organelles in the Drosophila nervous system and female germ line. A number of studies have demonstrated that kinesin-I binds to its intracellular cargos through protein-protein interactions between the kinesin tail domain and proteins on the cargo surface. To identify proteins that mediate or regulate kinesin-cargo interactions, we have performed yeast two-hybrid screens of a Drosophila embryonic cDNA library, using the tetratricopeptide repeats of the kinesin light chain and amino acids 675-975 of the kinesin heavy chain as baits. One of the proteins we have identified is YETI. Interestingly, YETI has the unique ability to bind specifically to both subunits of the kinesin tail domain. An epitope-tagged YETI fusion protein, when expressed in Drosophila S2 cultured cells, binds to kinesin-I in copurification assays, suggesting that YETI-kinesin-I interactions are context-independent. Immunostaining of cultured cells expressing YETI shows that YETI accumulates in the nucleus and cytosol. YETI is evolutionarily conserved, and its yeast homolog (AOR1) may have a role in regulating cytoskeletal dynamics or intracellular transport. Collectively, these results demonstrate that YETI interacts with both kinesin subunits of the kinesin tail domain, and is potentially involved in kinesin-dependent transport pathways.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Cinesinas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Línea Celular , Núcleo Celular/metabolismo , Citosol/metabolismo , Drosophila/citología , Drosophila/genética , Proteínas de Drosophila/genética , Cinesinas/genética , Microscopía Fluorescente , Datos de Secuencia Molecular , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Homología de Secuencia de Aminoácido , Transfección , Técnicas del Sistema de Dos Híbridos , Levaduras/genéticaRESUMEN
A broadly conserved membrane-associated protein required for the functional interaction of kinesin-I with axonal cargo was identified. Mutations in sunday driver (syd) and the axonal transport motor kinesin-I cause similar phenotypes in Drosophila, including aberrant accumulations of axonal cargoes. GFP-tagged mammalian SYD localizes to tubulovesicular structures that costain for kinesin-I and a marker of the secretory pathway. Coimmunoprecipitation analysis indicates that mouse SYD forms a complex with kinesin-I in vivo. Yeast two-hybrid analysis and in vitro interaction studies reveal that SYD directly binds kinesin-I via the tetratricopeptide repeat (TPR) domain of kinesin light chain (KLC) with K(d) congruent with 200 nM. We propose that SYD mediates the axonal transport of at least one class of vesicles by interacting directly with KLC.
Asunto(s)
Transporte Axonal , Proteínas Portadoras/metabolismo , Proteínas de Drosophila , Cinesinas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Secuencia de Aminoácidos , Animales , Conducta Animal , Transporte Biológico , Proteínas Portadoras/genética , Compartimento Celular , Clonación Molecular , Drosophila/genética , Proteínas de Insectos/metabolismo , Larva/genética , Proteínas de la Membrana/genética , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Unión Proteica , Subunidades de Proteína , Técnicas del Sistema de Dos Híbridos , Red trans-Golgi/químicaRESUMEN
In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150(Glued) (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150(Glued) were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.
Asunto(s)
Axones/metabolismo , Drosophila/genética , Dineínas/genética , Cinesinas/genética , Proteínas Asociadas a Microtúbulos/genética , Animales , Axones/ultraestructura , Citoplasma/química , Drosophila/embriología , Drosophila/metabolismo , Complejo Dinactina , Dineínas/metabolismo , Cinesinas/metabolismo , Microscopía Confocal , Microscopía Electrónica , Microscopía por Video , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Fenotipo , Pruebas de PrecipitinaRESUMEN
Kinesin is a heterotetramer composed of two 115-kD heavy chains and two 58-kD light chains. The microtubule motor activity of kinesin is performed by the heavy chains, but the functions of the light chains are poorly understood. Mutations were generated in the Drosophila gene Kinesin light chain (Klc), and the phenotypic consequences of loss of Klc function were analyzed at the behavioral and cellular levels. Loss of Klc function results in progressive lethargy, crawling defects, and paralysis followed by death at the end of the second larval instar. Klc mutant axons contain large aggregates of membranous organelles in segmental nerve axons. These aggregates, or organelle jams (Hurd, D.D., and W.M. Saxton. 1996. Genetics. 144: 1075-1085), contain synaptic vesicle precursors as well as organelles that may be transported by kinesin, kinesin-like protein 68D, and cytoplasmic dynein, thus providing evidence that the loss of Klc function blocks multiple pathways of axonal transport. The similarity of the Klc and Khc (. Cell 64:1093-1102; Hurd, D.D., and W.M. Saxton. 1996. Genetics 144: 1075-1085) mutant phenotypes indicates that KLC is essential for kinesin function, perhaps by tethering KHC to intracellular cargos or by activating the kinesin motor.
Asunto(s)
Transporte Axonal/fisiología , Drosophila/fisiología , Cinesinas/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Animales , Animales Modificados Genéticamente , Genes de Insecto/fisiología , Cinesinas/genética , Larva , Proteínas Asociadas a Microtúbulos/análisis , Proteínas Asociadas a Microtúbulos/genética , Movimiento , Mutación , Proteínas del Tejido Nervioso/análisis , Neuronas/química , Parálisis/genética , FenotipoAsunto(s)
Drosophila/genética , Cinesinas/química , Proteínas Asociadas a Microtúbulos/química , Secuencia de Aminoácidos , Animales , Secuencia de Consenso , Secuencia Conservada/genética , Bases de Datos Factuales , Cinesinas/genética , Proteínas Asociadas a Microtúbulos/genética , Datos de Secuencia Molecular , Proteínas de Complejo Poro Nuclear , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Alineación de SecuenciaRESUMEN
The receptor tyrosine phosphatases DPTP69D and DPTP99A are expressed on motor axons in Drosophila embryos. In mutant embryos lacking DPTP69D protein, motor neuron growth cones stop growing before reaching their muscle targets, or follow incorrect pathways that bypass these muscles. Mutant embryos lacking DPTP99A are indistinguishable from wild type. Motor axon defects in dptp69D dptp99A double mutant embryos, however, are much more severe than in embryos lacking only DPTP69D. Our results demonstrate that DPTP69D and DPTP99A are required for motor axon guidance and that they have partially redundant functions during development of the neuro-muscular system.
Asunto(s)
Axones/enzimología , Drosophila/embriología , Drosophila/enzimología , Embrión no Mamífero/enzimología , Neuronas Motoras/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Genes de Insecto/fisiología , Neuronas Motoras/ultraestructura , Mutación/fisiología , Vías Nerviosas , Fenotipo , Proteínas Tirosina Fosfatasas/genéticaRESUMEN
The Drosophila homeotic gene Sex combs reduced (Scr) controls the segmental identity of the labial and prothoracic segments in the embryo and adult. It encodes a sequence-specific transcription factor that controls, in concert with other gene products, differentiative pathways of tissues in which Scr is expressed. During embryogenesis, Scr accumulation is observed in a discrete spatiotemporal pattern that includes the labial and prothoracic ectoderm, the subesophageal ganglion of the ventral nerve cord and the visceral mesoderm of the anterior and posterior midgut. Previous analyses have demonstrated that breakpoint mutations located in a 75-kb interval, including the Scr transcription unit and 50 kb of upstream DNA, cause Scr misexpression during development, presumably because these mutations remove Scr cis-regulatory sequences from the proximity of the Scr promoter. To gain a better understanding of the regulatory interactions necessary for the control of Scr transcription during embryogenesis, we have begun a molecular analysis of the Scr regulatory interval. DNA fragments from this 75-kb region were subcloned into P-element vectors containing either an Scr-lacZ or hsp70-lacZ fusion gene, and patterns of reporter gene expression were assayed in transgenic embryos. Several fragments appear to contain Scr regulatory sequences, as they direct reporter gene expression in patterns similar to those normally observed for Scr, whereas other DNA fragments direct Scr reporter gene expression in developmentally interesting but non-Scr-like patterns during embryogenesis. Scr expression in some tissues appears to be controlled by multiple regulatory elements that are separated, in some cases, by more than 20 kb of intervening DNA. Interestingly, regulatory sequences that direct reporter gene expression in an Scr-like pattern in the anterior and posterior midgut are imbedded in the regulatory region of the segmentation gene fushi tarazu (ftz), which is normally located between 10 and 20 kb 5' of the Scr transcription start site. This analysis provides an entry point for the study of how Scr transcription is regulated at the molecular level.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica/genética , Genes Homeobox/genética , Hormonas de Insectos/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Elementos Transponibles de ADN/genética , Drosophila melanogaster/embriología , Ectodermo/metabolismo , Elementos de Facilitación Genéticos/genética , Factores de Transcripción Fushi Tarazu , Genes de Insecto/genética , Genes Reporteros/genética , Proteínas de Homeodominio/genética , Hormonas de Insectos/biosíntesis , Mesodermo/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Transcripción Genética/genéticaRESUMEN
The Drosophila homeotic gene Sex combs reduced (Scr) is necessary for the establishment and maintenance of the morphological identity of the labial and prothoracic segments. In the early embryo, its expression pattern is established through the activity of several gap and segmentation gene products, as well as other transcription factors. Once established, the Polycomb group (Pc-G) and trithorax group (trx-G) gene products maintain the spatial pattern of Scr expression for the remainder of development. We report the identification of DNA fragments in the Scr regulatory region that may be important for its regulation by Polycomb and trithorax group gene products. When DNA fragments containing these regulatory sequences are subcloned into P-element vectors containing a white minigene, transformants containing these constructs exhibit mosaic patterns of pigmentation in the adult eye, indicating that white minigene expression is repressed in a clonally heritable manner. The size of pigmented and nonpigmented clones in the adult eye suggests that the event determining whether a cell in the eye anlagen will express white occurs at least as early as the first larval instar. The amount of white minigene repression is reduced in some Polycomb group mutants, whereas repression is enhanced in flies mutant for a subset of trithorax group loci. The repressor activity of one fragment, normally located in Scr Intron 2, is increased when it is able to homologously pair, a property consistent with genetic data suggesting that Scr exhibits transvection. Another Scr regulatory fragment, normally located 40 kb upstream of the Scr promoter, silences ectopic expression of an Scr-lacZ fusion gene in the embryo and does so in a Polycomb-dependent manner. We propose that the regulatory sequences located within these DNA fragments may normally mediate the regulation of Scr by proteins encoded by members of the Polycomb and trithorax group loci.