Experimental taphonomy of giant sulphur bacteria: implications for the interpretation of the embryo-like Ediacaran Doushantuo fossils.

The Ediacaran Doushantuo biota has yielded fossils interpreted as eukaryotic organisms, either animal embryos or eukaryotes basal or distantly related to Metazoa. However, the fossils have been interpreted alternatively as giant sulphur bacteria similar to the extant Thiomargarita. To test this hypothesis, living and decayed Thiomargarita were compared with Doushantuo fossils and experimental taphonomic pathways were compared with modern embryos. In the fossils, as in eukaryotic cells, subcellular structures are distributed throughout cell volume; in Thiomargarita, a central vacuole encompasses approximately 98 per cent cell volume. Key features of the fossils, including putative lipid vesicles and nuclei, complex envelope ornament, and ornate outer vesicles are incompatible with living and decay morphologies observed in Thiomargarita. Microbial taphonomy of Thiomargarita also differed from that of embryos. Embryo tissues can be consumed and replaced by bacteria, forming a replica composed of a three-dimensional biofilm, a stable fabric for potential fossilization. Vacuolated Thiomargarita cells collapse easily and do not provide an internal substrate for bacteria. The findings do not support the hypothesis that giant sulphur bacteria are an appropriate interpretative model for the embryo-like Doushantuo fossils. However, sulphur bacteria may have mediated fossil mineralization and may provide a potential bacterial analogue for other macroscopic Precambrian remains.

Genetics of microtubule systems.

In most eucaryotes the tubulin genes comprise small multigene families with approximately equal numbers of genes for alpha- and beta-tubulin, the structural proteins of microtubules. The recent isolation of tubulin mutations in several species is proving to be a powerful tool for examining the structure and function of specific sets of microtubules. In Drosophila melanogaster, genetic analysis of a testis-specific beta-tubulin gene has shown that a single tubulin gene product may fulfill a number of different microtubule functions. In addition to tubulin mutations, mutations in other genes whose products are involved in the regulation or structure of specific microtubule arrays have also been isolated. The combination of analysis of both classes of mutations is beginning to allow a molecular description of the construction and function of three-dimensional cellular structures. In addition, such studies may also shed light on the evolutionary pressures that gave rise to and serve to maintain small families of genes encoding very similar proteins.

The effects of adenosine triphosphate and related compounds on some hydrodynamic properties of glycerinated cilia.

Cilia were isolated from Tetrahymena pyriformis by a glycerol method. The addition of low concentrations of ATP, but not of other nucleoside triphosphates, caused an increase of up to twofold in the amount of cilia pelleted in a low-speed centrifugation assay and decreased the density of the pellets compared to control pellets. Pellet size and density depend on pH, both in the absence and in the presence of low concentrations of ATP. High concentrations (5 mM and above) of ATP and of other nucleoside triphosphates tend to "dissolve" the cilia. Heating the cilia for 11 min at 40 degrees C abolishes the increase in pellet size and the decrease in pellet density caused by low ATP, but slightly increases the ATPase activity of the cilia. Heat treatment, however, does not prevent the dissolving effect of high ATP. There are, thus, two independent effects of ATP on the hydrodynamic properties of cilia suspensions.

Two Drosophila beta tubulin isoforms are not functionally equivalent.

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.

Genetic analysis of microtubule structure: a beta-tubulin mutation causes the formation of aberrant microtubules in vivo and in vitro.

A recessive male sterile mutation (B2t8) that encodes a stable variant of the testis-specific beta 2-tubulin of Drosophila causes the assembly of aberrant microtubules both in vivo and in vitro. The B2t8 mutation appears to cause defects in the formation of interprotofilament bonds. In testes from homozygous mutant males, the most commonly observed aberrant structures were sheets of protofilaments curved to form an S in cross section rather than a normal, closed microtubule. These characteristic S-shaped structures appear in the meiotic spindle, in place of axonemes in differentiating spermatids, and in cytoplasmic microtubules, including those that lie next to the nucleus during nuclear elongation. Homozygous mutant males exhibit defects in chromosome movement and cytokinesis during meiosis, flagellar elongation, and nuclear shaping, indicating that the ability to form normal closed microtubules is required for each of these events. The presence of the aberrant microtubules in three architecturally different microtubule arrays demonstrates conclusively the multifunctional nature of the beta 2-tubulin gene product. Although the mutant beta 2-tubulin subunit causes assembly of aberrant microtubules in vitro and in homozygous males, in the presence of wild-type beta 2-tubulin in heterozygous males, the variant subunit coassembles with the wild-type subunit into functional sperm.

Mutations that encode partially functional beta 2 tubulin subunits have different effects on structurally different microtubule arrays.

The testis-specific beta 2 tubulin of Drosophila is required for assembly and function of at least three architecturally different microtubule arrays (Kemphues et al., 1982). Two recessive male-sterile mutations in the B2t locus that encode partially functional, stable, variant forms of beta 2 tubulin cause defects in only certain microtubule-based processes during spermatogenesis. These mutations could thus identify aspects of beta tubulin primary structure critical for function only in specific microtubule arrays. In males carrying the B2t6 mutation, meiotic chromosome segregation and nuclear shaping are normal and flagellar axonemes are formed, but there is a subtle defect in axoneme structure; the outer doublet microtubules fill in with a central core normally seen only in the central pair and accessory microtubules. In homozygous B2t7 males, chromosome movement is usually normal during meiosis but cytokinesis often fails, cytoplasmic microtubules are assembled and nuclear shaping appears to be normal, but the flagellar axoneme lacks structural integrity. In contrast, the B2t8 allele affects a general property of tubulin, the ability to form normal side-to-side association of protofilaments (Fuller et al., 1987), and causes defects in meiosis, axoneme assembly and nuclear shaping. Certain combinations of these beta 2 tubulin mutations show interallelic complementation; in B2t6/B2t8 males functional sperm are produced and both variant subunits are incorporated into mature sperm, in the absence of wild-type beta 2 tubulin. Comparison of the phenotypes of the three partially functional beta 2 tubulin alleles reveals some aspects of tubulin primary structure more important for function in specific subsets of microtubule arrays, and other aspects required for the construction of microtubules in general.

Microtubule architecture specified by a beta-tubulin isoform.

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.

Evidence that the head of kinesin is sufficient for force generation and motility in vitro.

Kinesin is a mechanochemical protein that converts the chemical energy in adenosine triphosphate into mechanical force for movement of cellular components along microtubules. The regions of the kinesin molecule responsible for generating movement were determined by studying the heavy chain of Drosophila kinesin, and its truncated forms, expressed in Escherichia coli. The results demonstrate that (i) kinesin heavy chain alone, without the light chains and other eukaryotic factors, is able to induce microtubule movement in vitro, and (ii) a fragment likely to contain only the kinesin head is also capable of inducing microtubule motility. Thus, the amino-terminal 450 amino acids of kinesin contain all the basic elements needed to convert chemical energy into mechanical force.

Molecular phylogeny of the animal kingdom.

A rapid sequencing method for ribosomal RNA was applied to the resolution of evolutionary relationships among Metazoa. Representatives of 22 classes in 10 animal phyla were used to infer phylogenetic relationships, based on evolutionary distances determined from pairwise comparisons of the 18S ribosomal RNA sequences. The classical Eumetazoa are divided into two groups. Cnidarians arose from a protist ancestry different from the second group, the Bilateria. Within the Bilateria, an early split gave rise to Platyhelminthes (flatworms) and the coelomate lineage. Coelomates are thus monophyletic, and they radiated rapidly into four groups: chordates, echinoderms, arthropods, and eucoelomate protostomes.

Axoneme-specific beta-tubulin specialization: a conserved C-terminal motif specifies the central pair.

Axonemes are ancient organelles that mediate motility of cilia and flagella in animals, plants, and protists. The long evolutionary conservation of axoneme architecture, a cylinder of nine doublet microtubules surrounding a central pair of singlet microtubules, suggests all motile axonemes may share common assembly mechanisms. Consistent with this, alpha- and beta-tubulins utilized in motile axonemes fall among the most conserved tubulin sequences [1, 2], and the beta-tubulins contain a sequence motif at the same position in the carboxyl terminus [3]. Axoneme doublet microtubules are initiated from the corresponding triplet microtubules of the basal body [4], but the large macromolecular "central apparatus" that includes the central pair microtubules and associated structures [5] is a specialization unique to motile axonemes. In Drosophila spermatogenesis, basal bodies and axonemes utilize the same alpha-tubulin but different beta-tubulins [6--13]. beta 1 is utilized for the centriole/basal body, and beta 2 is utilized for the motile sperm tail axoneme. beta 2 contains the motile axoneme-specific sequence motif, but beta 1 does not [3]. Here, we show that the "axoneme motif" specifies the central pair. beta 1 can provide partial function for axoneme assembly but cannot make the central microtubules [14]. Introducing the axoneme motif into the beta 1 carboxyl terminus, a two amino acid change, conferred upon beta 1 the ability to assemble 9 + 2 axonemes. This finding explains the conservation of the axoneme-specific sequence motif through 1.5 billion years of evolution.

Conserved axoneme symmetry altered by a component beta-tubulin.

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.

Three Drosophila beta-tubulin sequences: a developmentally regulated isoform (beta 3), the testis-specific isoform (beta 2), and an assembly-defective mutation of the testis-specific isoform (B2t8) reveal both an ancient divergence in metazoan isotypes and structural constraints for beta-tubulin function.

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.

Structurally similar Drosophila alpha-tubulins are functionally distinct in vivo.

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.

Tubulin sorting during dimerization in vivo.

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.

The beta 3-tubulin gene of Drosophila melanogaster is essential for viability and fertility.

We have previously shown that the beta 3-tubulin gene of Drosophila melanogaster encodes a divergent isoform expressed in a complex developmental pattern. The beta 3 gene is transiently expressed in the embryo and again in the pupa at high levels in the developing musculature, and at lower levels in several different pupal tissues of ectodermal origin. Adult expression is confined to specific somatic cells in the gonads. In some of the cell types in which it is expressed, beta 3 is the sole or predominant beta-tubulin, while in others the beta 3 protein is a minor component of the beta-tubulin pool. The sites and timing of beta 3 expression demonstrated that beta 3-tubulin is utilized primarily in cytoplasmic microtubule arrays involved in changes in cell shape and tissue organization, and suggested to us that this isoform may be functionally specialized. To determine whether the expression of the beta 3 gene is essential for normal development, and to examine the specific functions of this divergent isoform, we have generated mutations within the gene. We determined that the small deficiency Df(2R)Px2, which deletes the 60C5,6-60D9,10 region of chromosome 2, removes all of the beta 3 coding sequences, and that the distal breakpoint of the deficiency is approximately 2 kb upstream from the start of transcription of the beta 3 gene. We have generated a total of 31 ethyl methanesulfonate- or diepoxybutane-induced recessive lethal or visible mutations which map within the deficiency. These mutations define 12 new lethal complementation groups, which together with two previously identified visible mutations, altogether identify 14 genes in this interval of the second chromosome. A lethal complementation group comprising mutations in the beta 3-tubulin gene (beta Tub60D) was identified by rescue of their lethality by a wild-type copy of the gene introduced into the genome via P element-mediated germ line transformation. Analysis of the homozygous and transheterozygous phenotypes of the five beta 3 mutations recovered (alleles designated B3t1-B3t5) demonstrates that beta 3-tubulin is essential for viability and fertility.

Genetic analysis of B2t, the structural gene for a testis-specific beta-tubulin subunit in Drosophila melanogaster.

Genetic analysis of the B2t locus has resulted in the recovery of four recessive mutations in the B2t structural gene and a deficiency that deletes the locus. Two of the mutations were recovered as suppressors of B2tD, a dominant male sterile mutation at the locus, and two were induced on wild-type chromosomes. All four mutant genes encode beta 2-tubulin subunits that are synthesized at normal rates but do not accumulate. All mutants are completely male sterile as homozygotes.

Structural analysis of mutations in the Drosophila beta 2-tubulin isoform reveals regions in the beta-tubulin molecular required for general and for tissue-specific microtubule functions.

We have determined the lesions in a number of mutant alleles of beta Tub85D, the gene that encodes the testis-specific beta 2-tubulin isoform in Drosophila melanogaster. Mutations responsible for different classes of functional phenotypes are distributed throughout the beta 2-tubulin molecule. There is a telling correlation between the degree of phylogenetic conservation of the altered residues and the number of different microtubule categories disrupted by the lesions. The majority of lesions occur at positions that are evolutionarily highly conserved in all beta-tubulins; these lesions disrupt general functions common to multiple classes of microtubules. However, a single allele B2t6 contains an amino acid substitution within an internal cluster of variable amino acids that has been identified as an isotype-defining domain in vertebrate beta-tubulins. Correspondingly, B2t6 disrupts only a subset of microtubule functions, resulting in misspecification of the morphology of the doublet microtubules of the sperm tail axoneme. We previously demonstrated that beta 3, a developmentally regulated Drosophila beta-tubulin isoform, confers the same restricted morphological phenotype in a dominant way when it is coexpressed in the testis with wild-type beta 2-tubulin. We show here by complementation analysis that beta 3 and the B2t6 product disrupt a common aspect of microtubule assembly. We therefore conclude that the amino acid sequence of the beta 2-tubulin internal variable region is required for generation of correct axoneme morphology but not for general microtubule functions. As we have previously reported, the beta 2-tubulin carboxy terminal isotype-defining domain is required for suprastructural organization of the axoneme. We demonstrate here that the beta 2 variant lacking the carboxy terminus and the B2t6 variant complement each other for mild-to-moderate meiotic defects but do not complement for proper axonemal morphology. Our results are consistent with the hypothesis drawn from comparisons of vertebrate beta-tubulins that the two isotype-defining domains interact in a three-dimensional structure in wild-type beta-tubulins. We propose that the integrity of this structure in the Drosophila testis beta 2-tubulin isoform is required for proper axoneme assembly but not necessarily for general microtubule functions. On the basis of our observations we present a model for regulation of axoneme microtubule morphology as a function of tubulin assembly kinetics.

Embryonic expression of the divergent Drosophila beta3-tubulin isoform is required for larval behavior.

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.

Microtubule protein pools in early development.

Microtubule protein pools have been demonstrated to exist in unfertilized eggs and the early embryonic stages of several organisms. The microtubule pool of the sea urchin embryo is constant in size (about 0.4% of the total embryo protein) throughout early development. Protein withdrawn from this pool for organelle assembly is replaced by new synthesis. Eggs and embryos of Drosophila similarly contain a pool of microtubule proteins (larger than or equal to 0.4% of the total embryo protein, congruent to 3% of the soluble protein), which is constant in size throughout early development. The Drosophila egg microtubule proteins are easily purified by self-assembly in vitro of microtubules, and are similar to microtubule proteins from other organisms in molecular weight and other properties. Synthesis of microtubule proteins in sea urchin embryos is supported by oogenetic mRNA. This appears also to be the case in molluscan (Ilyanassa) embryos. It is not known whether Drosophila embryos synthesize microtubule proteins during the early stages of development.