RESUMEN
We demonstrate sorting of beta-tubulins during dimerization in the Drosophila male germ line. Different beta-tubulin isoforms exhibit distinct affinities for alpha-tubulin during dimerization. Our data suggest that differences in dimerization properties are important in determining isoform-specific microtubule functions. The differential use of beta-tubulin during dimerization reveals structural parameters of the tubulin heterodimer not discernible in the resolved three-dimensional structure. We show that the variable beta-tubulin carboxyl terminus, a surface feature in the heterodimer and in microtubules, and which is disordered in the crystallographic structure, is of key importance in forming a stable alpha-beta heterodimer. If the availability of alpha-tubulin is limiting, alpha-beta dimers preferentially incorporate intact beta-tubulins rather than a beta-tubulin missing the carboxyl terminus (beta 2 Delta C). When alpha-tubulin is not limiting, beta 2 Delta C forms stable alpha-beta heterodimers. Once dimers are formed, no further sorting occurs during microtubule assembly: alpha-beta 2 Delta C dimers are incorporated into axonemes in proportion to their contribution to the total dimer pool. Co-incorporation of beta 2 Delta C and wild-type beta 2-tubulin results in nonmotile axonemes because of a disruption of the periodicity of nontubulin axonemal elements. Our data show that the beta-tubulin carboxyl terminus has two distinct roles: 1) forming the alpha-beta heterodimer, important for all microtubules and 2) providing contacts for nontubulin components required for specific microtubule structures, such as axonemes.
Asunto(s)
Tubulina (Proteína)/metabolismo , Animales , Dimerización , Drosophila melanogaster/metabolismo , Masculino , Microtúbulos/fisiología , Isoformas de Proteínas/metabolismo , Transporte de ProteínasRESUMEN
We have sought to define the developmental and cellular roles played by differential expression of distinct beta-tubulins. Drosophila beta3-tubulin (beta3) is a structurally divergent isoform transiently expressed during midembryogenesis. Severe beta3 mutations cause larval lethality resulting from failed gut function and consequent starvation. However, mutant larvae also display behavioral abnormalities consistent with defective sensory perception. We identified embryonic beta3 expression in several previously undefined sites, including different types of sensory organs. We conclude that abnormalities in foraging behavior and photoresponsiveness exhibited by prelethal mutant larvae reflect defective beta3 function in the embryo during development of chordotonal and other mechanosensory organs and of Bolwig's organ and nerve. We show that microtubule organization in the cap cells of chordotonal organs is altered in mutant larvae. Thus transient zygotic beta3 expression has permanent consequences for the architecture of the cap cell microtubule cytoskeleton in the larval sensilla, even when beta3 is no longer present. Our data provide a link between the microtubule cytoskeleton in embryogenesis and the behavioral phenotype manifested as defective proprioreception at the larval stage.
Asunto(s)
Drosophila/genética , Larva/fisiología , Tubulina (Proteína)/genética , Animales , Drosophila/embriología , Drosophila/crecimiento & desarrollo , Inmunohistoquímica , Microscopía Electrónica , Especificidad de la EspecieRESUMEN
Ninefold microtubule symmetry of the eukaryotic basal body and motile axoneme has been long established [1-3]. In Drosophila, these organelles contain distinct but similar beta-tubulin isoforms [4-10]: basal bodies contain only beta1-tubulin, and only beta2-tubulin is used for assembly of sperm axonemes. A single alpha-tubulin functions throughout spermatogenesis [11,12]. Thus, differences in organelle assembly reside in beta-tubulin. We tested the ability of beta1 to function in axonemes and found that beta1 alone could not generate axonemes. Small sequence differences between the two isoforms therefore mediate large differences in assembly capacity, even though these two related organelles have a common evolutionarily ancient architecture. In males with equal beta1 and beta2, beta1 was co-incorporated at equimolar ratio into functional sperm axonemes. When beta1 exceeded beta2, however, axonemes with 10 doublets were produced, an alteration unprecedented in natural phylogeny. Addition of the tenth doublet occurred by a novel mechanism, bypassing the basal body. It has been assumed that the instructions for axoneme morphogenesis reside primarily in the basal body, which normally serves as the axonemal template. Our data reveal that beta-tubulin requirements for basal bodies and axonemes are distinct, and that key information for axoneme architecture resides in the axonemal beta-tubulin.
Asunto(s)
Microtúbulos/metabolismo , Cola del Espermatozoide/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Drosophila melanogaster/citología , Electroforesis en Gel Bidimensional , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Proteínas de Insectos/ultraestructura , Masculino , Microtúbulos/diagnóstico por imagen , Microtúbulos/genética , Isoformas de Proteínas/metabolismo , Motilidad Espermática , Cola del Espermatozoide/química , Cola del Espermatozoide/ultraestructura , Espermátides/metabolismo , Espermátides/ultraestructura , Tubulina (Proteína)/análogos & derivados , Tubulina (Proteína)/genética , UltrasonografíaRESUMEN
Drosophila beta3-tubulin is an essential isoform expressed during differentiation of many cell types in embryos and pupae. We report here that during pupal development transient beta3 expression demarcates a unique subset of neurons in the developing adult visual system. beta3 is coassembled into microtubules with beta1, the sole beta-tubulin isoform in the permanent microtubule cytoskeleton of the adult eye and brain. Examination of beta3 mutant phenotypes showed that beta3 is required for axonal patterning and connectivity and for spatial positioning within the optic lobe. Comparison of the phenotypes of beta3 mutations with those that result from disruption of the Hedgehog signaling pathway shows that beta3 functions early in the establishment of the adult visual system. Our data support the hypothesis that beta3 confers specialized properties on the microtubules into which it is incorporated. Thus a transient specialization of the microtubule cytoskeleton during differentiation of a specific subset of the neurons has permanent consequences for later cell function.
Asunto(s)
Tipificación del Cuerpo , Drosophila melanogaster/embriología , Drosophila melanogaster/crecimiento & desarrollo , Embrión no Mamífero/fisiología , Ojo/embriología , Ojo/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Microtúbulos/fisiología , Tubulina (Proteína)/genética , Animales , Animales Modificados Genéticamente , Axones/fisiología , Citoesqueleto/fisiología , Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas de Insectos/genética , Larva , Mutagénesis , Lóbulo Óptico de Animales no Mamíferos/embriología , Lóbulo Óptico de Animales no Mamíferos/crecimiento & desarrollo , Regiones Promotoras Genéticas , Pupa , Retina/embriología , Retina/crecimiento & desarrollo , Ubiquitinas/genéticaRESUMEN
We used transgenic analysis in Drosophila to compare the ability of two structurally similar alpha-tubulin isoforms to support microtubule assembly in vivo. Our data revealed that even closely related alpha-tubulin isoforms have different functional capacities. Thus, in multicellular organisms, even small changes in tubulin structure may have important consequences for regulation of the microtubule cytoskeleton. In spermatogenesis, all microtubule functions in the postmitotic male germ cells are carried out by a single tubulin heterodimer composed of the major Drosophila alpha-84B tubulin isoform and the testis-specific beta 2-tubulin isoform. We tested the ability of the developmentally regulated alpha 85E-tubulin isoform to replace alpha 84B in spermatogenesis. Even though it is 98% similar in sequence, alpha 85E is not functionally equivalent to alpha 84B. alpha 85E can support some functional microtubules in the male germ cells, but alpha 85E causes dominant male sterility if it makes up more than one-half of the total alpha-tubulin pool in the spermatids. alpha 85E does not disrupt meiotic spindle or cytoplasmic microtubules but causes defects in morphogenesis of the two classes of singlet microtubules in the sperm tail axoneme, the central pair and the accessory microtubules. Axonemal defects caused by alpha 85E are precisely reciprocal to dominant defects in doublet microtubules we observed in a previous study of ectopic germ-line expression of the developmentally regulated beta 3-tubulin isoform. These data demonstrate that the doublet and singlet axoneme microtubules have different requirements for alpha- and beta-tubulin structure. In their normal sites of expression, alpha 85E and beta 3 are coexpressed during differentiation of several somatic cell types, suggesting that alpha 85E and beta 3 might form a specialized heterodimer. Our tests of different alpha-beta pairs in spermatogenesis did not support this model. We conclude that if alpha 85E and beta 3 have specialized properties required for their normal functions, they act independently to modulate the properties of microtubules into which they are incorporated.
Asunto(s)
Tubulina (Proteína)/química , Tubulina (Proteína)/fisiología , Animales , Células Cultivadas , Dimerización , Drosophila , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica/fisiología , Genes Dominantes , Células Germinativas/química , Células Germinativas/metabolismo , Células Germinativas/ultraestructura , Isomerismo , Masculino , Microtúbulos/genética , Microtúbulos/fisiología , Microtúbulos/ultraestructura , Oligonucleótidos Antisentido/farmacología , Procesamiento Proteico-Postraduccional , ARN Mensajero/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Espermatozoides/ultraestructura , Relación Estructura-Actividad , Tubulina (Proteína)/genéticaRESUMEN
In Drosophila melanogaster, a testis-specific beta-tubulin (beta2) is required for spermatogenesis. A sequence motif was identified in carboxyl termini of axonemal beta-tubulins in diverse taxa. As a test of whether orthologous beta-tubulins from different species are functionally equivalent, the moth Heliothis virescens beta2 homolog was expressed in Drosophila testes. When coexpressed with beta2, the moth isoform imposed the 16-protofilament structure characteristic of that found in the moth on the corresponding subset of Drosophila microtubules, which normally contain only 13-protofilament microtubules. Thus, the architecture of the microtubule cytoskeleton can be directed by a component beta-tubulin.
Asunto(s)
Microtúbulos/ultraestructura , Espermátides/ultraestructura , Tubulina (Proteína)/fisiología , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Drosophila melanogaster/genética , Humanos , Masculino , Microtúbulos/química , Datos de Secuencia Molecular , Mariposas Nocturnas/genética , Espermátides/química , Espermátides/fisiología , Espermatogénesis , Tubulina (Proteína)/química , Tubulina (Proteína)/genéticaRESUMEN
In this study we examined two aspects of beta-tubulin function in Drosophila spermatogenesis: 1) beta-tubulin structural requirements for assembly of different categories of microtubules and 2) regulatory requirements for production of the correct tubulin protein level. In normal Drosophila spermatogenesis, the testis-specific beta 2-tubulin isoform supports multiple microtubule functions. Our previous work showed that another Drosophila isoform, beta 3, cannot support spermatogenesis, whereas a carboxyl-truncated form of beta 2, beta 2 delta C, can at least to some extent provide all of beta 2's normal functions, save one: beta 2 delta C cannot support organization of axonemal microtubules into the supramolecular architecture of the axoneme. Here, to test whether beta 2 carboxyl sequences can rescue the functional failure of the beta 3 isoform in spermatogenesis, we constructed a gene encoding a chimeric protein, beta 3 beta 2C, in which beta 3 sequences in the carboxyl region are replaced with those of beta 2. Unlike either beta 3 or beta 2 delta C, beta 3 beta 2C can provide partial function for both assembly of axonemal microtubules and their organization into the supramolecular architecture of the axoneme. In particular, the beta 2 carboxyl sequences mediate morphogenesis of the axoneme doublet tubule complex, including accessory microtubule assembly and attachment of spokes and linkers. However, our data also reveal aspects of beta 2-specific function that require structural features other than the primary sequence of the isotype-defining variable regions, the C terminus and the internal variable region. Tests of fecundity in males that coexpress beta 2 and the chimeric beta 3 beta 2C protein showed that in Drosophila there are differential requirements for sperm motility in the male and in the female reproductive tract. Since some aspects of microtubule function in spermatogenesis are sensitive to the tubulin pool size, we examined the mechanisms for control of tubulin protein levels in the male germ cells. We found that both beta 2-tubulin mRNA accumulation and protein synthesis are dependent on gene dose, and that the level of expression is regulated by 3' noncoding sequences in the beta 2 gene. Our data show that the regulatory mechanisms that control tubulin pool levels in the Drosophila male germ line differ from those observed in cultured animal somatic cells. Finally, expression of transgenic constructs is consistent with early cessation of X chromosome expression in Drosophila spermatogenesis.
Asunto(s)
Drosophila/genética , Espermatogénesis , Tubulina (Proteína)/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Drosophila/fisiología , Masculino , Microtúbulos/metabolismo , Microtúbulos/fisiología , Mitosis , Datos de Secuencia Molecular , Secuencias Reguladoras de Ácidos Nucleicos , Testículo/metabolismo , Transcripción Genética , Tubulina (Proteína)/genéticaRESUMEN
We have tested the functional capacity of different beta tubulin isoforms in vivo by expressing beta 3-tubulin either in place of or in addition to beta 2-tubulin in the male germ line of Drosophila melanogaster. The testes-specific isoform, beta 2, is conserved relative to major metazoan beta tubulins, while the developmentally regulated isoform, beta 3, is considerably divergent in sequence. beta 3-tubulin is normally expressed in discrete subsets of cells at specific times during development, but is not expressed in the male germ line. beta 2-Tubulin is normally expressed only in the postmitotic germ cells of the testis, and is required for all microtubule-based functions in these cells. The normal functions of beta 2-tubulin include assembly of meiotic spindles, axonemes, and at least two classes of cytoplasmic microtubules, including those associated with the differentiating mitochondrial derivatives. A hybrid gene was constructed in which 5' sequences from the beta 2 gene were joined to protein coding and 3' sequences of the beta 3 gene. Drosophila transformed with the hybrid gene express beta 3-tubulin in the postmitotic male germ cells. When expressed in the absence of the normal testis isoform, beta 3-tubulin supports assembly of one class of functional cytoplasmic microtubules. In such males the microtubules associated with the membranes of the mitochondrial derivatives are assembled and normal mitochondrial derivative elongation occurs, but axoneme assembly and other microtubule-mediated processes, including meiosis and nuclear shaping, do not occur. These data show that beta 3 tubulin can support only a subset of the multiple functions normally performed by beta 2, and also suggest that the microtubules associated with the mitochondrial derivatives mediate their elongation. When beta 3 is coexpressed in the male germ line with beta 2, at any level, spindles and all classes of cytoplasmic microtubules are assembled and function normally. However, when beta 3-tubulin exceeds 20% of the total testis beta tubulin pool, it acts in a dominant way to disrupt normal axoneme assembly. In the axonemes assembled in such males, the doublet tubules acquire some of the morphological characteristics of the singlet microtubules of the central pair and accessory tubules. These data therefore unambiguously demonstrate that the Drosophila beta tubulin isoforms beta 2 and beta 3 are not equivalent in intrinsic functional capacity, and furthermore show that assembly of the doublet tubules of the axoneme imposes different constraints on beta tubulin function than does assembly of singlet microtubules.
Asunto(s)
Drosophila melanogaster/fisiología , Microtúbulos/metabolismo , Huso Acromático/metabolismo , Tubulina (Proteína)/fisiología , Animales , Fertilidad/genética , Regulación de la Expresión Génica , Masculino , Meiosis , Familia de Multigenes , Proteínas Recombinantes de Fusión/metabolismo , Espermátides/citología , Espermátides/metabolismo , Espermatogénesis/fisiología , Transfección , Tubulina (Proteína)/genéticaRESUMEN
The genomic DNA sequence and deduced amino acid sequence are presented for three Drosophila melanogaster beta-tubulins: a developmentally regulated isoform beta 3-tubulin, the wild-type testis-specific isoform beta 2-tubulin, and an ethyl methanesulfonate-induced assembly-defective mutation of the testis isoform, B2t8. The testis-specific beta 2-tubulin is highly homologous to the major vertebrate beta-tubulins, but beta 3-tubulin is considerably diverged. Comparison of the amino acid sequences of the two Drosophila isoforms to those of other beta-tubulins indicates that these two proteins are representative of an ancient sequence divergence event which at least preceded the split between lines leading to vertebrates and invertebrates. The intron/exon structures of the genes for beta 2- and beta 3-tubulin are not the same. The structure of the gene for the variant beta 3-tubulin isoform, but not that of the testis-specific beta 2-tubulin gene, is similar to that of vertebrate beta-tubulins. The mutation B2t8 in the gene for the testis-specific beta 2-tubulin defines a single amino acid residue required for normal assembly function of beta-tubulin. The sequence of the B2t8 gene is identical to that of the wild-type gene except for a single nucleotide change resulting in the substitution of lysine for glutamic acid at residue 288. This position falls at the junction between two major structural domains of the beta-tubulin molecule. Although this hinge region is relatively variable in sequence among different beta-tubulins, the residue corresponding to glu 288 of Drosophila beta 2-tubulin is highly conserved as an acidic amino acid not only in all other beta-tubulins but in alpha-tubulins as well.
Asunto(s)
Drosophila melanogaster/genética , Genes , Mutación , Tubulina (Proteína)/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Enzimas de Restricción del ADN , Exones , Homocigoto , Intrones , Masculino , Microtúbulos/ultraestructura , Testículo/metabolismo , Testículo/ultraestructuraRESUMEN
The DNAase I sensitivities of the somatic-type 5 S DNA and oocyte-type 5 S DNA have been compared in nuclei from adult somatic tissues of Xenopus laevis. Neither of these Type III genes is expressed in mature erythrocytes and only somatic-type 5 S DNA is expressed in liver. We find that somatic-type 5 S DNA is DNAase-I-sensitive in liver nuclei and less sensitive in erythrocyte nuclei, while oocyte-type 5 S DNA is insensitive in both tissues. The DNAase I sensitivity appears to be uniform across each active somatic-type 5 S DNA repeat. Two regions slightly hypersensitive to DNAase I are found only in liver somatic-type 5 S DNA. One of these regions is within the gene, overlapping with the binding site of the transcription factor (TF III A) required for 5 S RNA synthesis. Thus, the correlation between DNAase I sensitivity and gene activity previously seen for protein-coding genes also holds for these Type III genes. Our data lead us to suggest that the fully DNAase-I-sensitive chromatin conformation on 5 S DNA requires the presence both of transcription factors and RNA polymerase.
Asunto(s)
Cromatina/metabolismo , ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Oocitos/análisis , Animales , Secuencia de Bases , Sitios de Unión , Eritrocitos/análisis , Femenino , Hígado/análisis , Conformación de Ácido Nucleico , Especificidad por Sustrato , Factor de Transcripción TFIIIA , Factores de Transcripción/metabolismo , Transcripción Genética , Xenopus laevisRESUMEN
The frequency of cytosine methylation at specific sites in the somatic 5S DNA (X1s) and trace oocyte 5S DNA (X1t) of X. laevis has been determined using restriction enzymes that are inhibited by the presence of 5-methylcytosine (5mC) within their cleavage sequences. 5S DNA methylation patterns were determined in genomic DNA from mature red blood cells, which express neither type of 5S gene, and from liver, which expresses only X1s. All the sites examined in X1t are greater than 95% methylated in red cells and liver. In the X1s of red cells all the sites examined are methylated in greater than 95% of repeats, while in liver some sites are modified in only 90% of repeats. Repeats containing unmethylated sites are randomly distributed throughout the tandem arrays in both red cells and liver. The high levels of methylation for X1s are in marked contrast to the situation with other Xenopus genes which do have sites of significant undermethylation in tissues where they are active. Thus, undermethylation in active genetic regions may not be a general feature for all classes of eukaryotic genes.