betaTubulin60D (beta3 tubulin) part 1/2

BIOLOGICAL OVERVIEW

The four ß-tubulin genes of Drosophila each encode a distinct isoform. Beta tubulins are found dimerized with alpha tubulins for which there are also four isoforms in Drosophila. In all cases an alpha tubulin - beta tubulin heterodimer is the functional form of tubulin in the cell. This overview will discuss two of them, ß1 and ß3, but predominantly, the latter.

In early Drosophila development, the microtubule cytoskeleton is involved in prepositioning of developmental cues in the oocyte as well as in the mitotic divisions and cytoplasmic reorganizations in the embryo (Theurkauf, 1993 and 1994). All of these early developmental functions are supported by microtubules assembled from the ß1-tubulin isoform, which is both maternally supplied and zygotically expressed (Raff, 1982, Bialojan, 1984, Gasch, 1988 and Buttgereit, 1991).

The ß3-tubulin gene encodes a divergent isoform expressed in a complex developmental pattern. The ß3 gene is transiently expressed in the embryo and later, in the pupa, at higher 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, ß3 is the sole or predominant beta-tubulin, while in others the ß3 protein is a minor component of the beta-tubulin pool. The sites and timing of ß3 expression demonstrate that ß3-tubulin is utilized primarily in cytoplasmic microtubule arrays involved in changes in cell shape and tissue organization (Kimble, 1989).

ß3 is the predominant ß-tubulin isoform found in developing muscle cells. In late stage embryos, zygotic expression of the ß1 gene supports accumulation of ß1 in the developing nervous system and in the segmental muscle attachment cells (the apodemes) but ß1 is not zygotically expressed in other tissues. Thus myogenic cells descend from cells wherein ß1 is maternally loaded, but ß1 in muscles cannot be detected above the generalized background caused by the presence of ß1 throughout the embryo.

In spite of the presence of ß3 in muscle cells, examination of the microtubules in ß3 mutants reveals no disruption in the microtubule arrays that precede myofibril formation. These microtubule arrays are associated with growing myofibriles in embryonic body wall and gut muscle. Microtubule assembly precedes sarcomere formation, and microtubules are later intermingled among growing sarcomere elements. Muscle-associated microtubules are transient and are disassembled after completion of myogenesis. The presence of microtubules in ß3 mutants suggests that these microtubules must have another source. Microtubule assembly in the myogenic cells begins in stage 12. It is thought that these microtubules utilize the ß1 tubulin from the maternal pool.

Loss of ß3 function has no discernible effects, neither in the ultrastructure nor the function of either embryonic or larval muscle. Although ß3 is dispensable for larval muscle, it is essential for development of at least a subset of adult muscles, since two viable combinations of ß3 alleles are flightless. Nevertheless, organization and structure of the sarcomeres in the flightless mutants can not be distinguished from wild type. What then does ß3 do, if it is dispensable for development of the tissues in which it is expressed (Dettman, 1996)?

Clue to an answer come from the mutant phenotype. The most severe lethals produce first instar larvae that never grow and fail to molt into the second instar even though they live for several days as first instar larvae. A test was performed on newly hatched larvae to see if they could perform the muscle movements necessary for scraping food during feeding. As with all other behavior tests, no specific defect was seen in the ability to perform feeding movements or in the amount of time spent feeding. There was a manifestation of starvation, known to be the cause of larval arrest in other species of insects. Starving wild-type larvae manifest a similar phenotype to ß3 mutation. The partial phenocopy of mutation to the effects of starvation suggested that mutant larvae live under starvation conditions. Interest turned from analysis of the mesoderm to analysis of the affects of mutation on the gut (Dettman, 1996).

In the developing gut, microtubules are assembled into at least two distinct arrays: those that accompany morphogenesis of the gut musculature and those that mediate constriction of the midgut. Both expression patterns are confined to the mesoderm. ß3 localization in the visceral mesoderm during development in the gut is consistent with a role in both of these processes. Examination of the overall pattern of folding of the midgut in mutant larvae reveals that it is often smaller than normal and irregular in shape. Nevertheless, there was no relationship in mutant flies between the size of the gut and the ability to grow. However, an examination of food uptake and movement through the gut and the capability for nutrient absorption revealed that ß3 mutants were unable to absorb food. This difference did not reflect any difference in visceral musculature, as the rate at which the visceral muscle moved food through the gut was unaffected in ß3 mutants (Dettman, 1996).

Since ß3 is expressed in visceral mesodermal cells associated with constriction and folding of the midgut, and since the gut is often abnormally shaped in ß3 mutants, it is likely that ß3 is required in gut constriction furrow microtubules. Gut constriction microtubules are assembled later than the early myogenic microtubules; their function may be optimal in the presence of ß3 because by this time maternal ß1-tubulin has undergone some depletion.

A brief aside before resuming the starvation phenotype question: Cell migration and the establishment of the midgut requires cues from the visceral mesoderm. (Reuter, 1993). To form the midgut, endodermal cells migrate from anterior and posterior midgut primordia to form a single luminal epithelial cell layer that is surrounded by the mesodermally derived muscle cell layer. Both muscular and endodermal cells undergo changes in shape and layering. Thus it is thought that ß3 tubulin somehow is required to satisfy tubulin requirements where the microtubule cytoskeleton is involved in mediating cell-cell interactions (Dettman, 1996). Therefore the defect in gut constriction may have a complex origin preceding the event itself, a defect in mesodermal - endodermal interaction regulating cell migration.

The mesoderm-endoderm interaction is not the only place that ß3 exerts such an effect. In the testis, ß is expressed in the somatic cells that enclose each cyst of developing germ cells (Kimble, 1989). Loss of ß3 function in somatic cells disrupts crucial developmental events in the germ cells (Dettman, 1996).

Returning to the starvation phenotype: could it be due to a behavioral defect? Mutant larvae appear not to have a wandering behavior exhibited by wild-type larvae, suggesting that mutant larvae cannot sense the presence of food. ß3 accumulates in the peripheral nervous system in the support cells of the chordotonal organs, the mechanosensory organs in the body wall, and also in sets of sensory organs in the anterior-most portion of the embryo. In addition, lethal phenotypes reveal a requirement for ß3 in ecdysis. At death, mutants often posses multipe mouth parts, suggesting difficulty in shedding the cuticle after a molt, and small spots of tanned cuticle, showing incomplete entry into the pupal stage. ß3 is expressed in the ring gland, the release site of Ecdysone hormone, required for induction of larval molts and pupariation, and ß3 is an ecdysone-responsive gene. One class of ß3 mutation more closely resembles the effect of excess rather than insufficient levels of Ecdysone (Dettman, 1996).

It is clear that ß3, in spite of its seeming importance in gut morphogenesis has just as complex a function in other aspects of development. This is due to the multiple functions of the cytoskeleton. Microtubule involvement in mitosis is only the tip of the iceberg. Tubulins are involved in intracompartmental and axonic transport, interact with the actin based cytoskeleton, and have essential roles in cell polarity. Apparently, tubulin is also involved in mediating the efficacy of cell interaction. Another lesson here is that phenotype is a complex phenomenon, not always revealing of function.

GENE STRUCTURE

Exons - 4

PROTEIN STRUCTURE

Amino Acids - 454

Structural Domains and Evolutionary Homologs

Three of the four ß-tubulins of Drosophila exhibit an 87% homology in overall amino acid sequence. The first and second introns of ß3-tubulin occur in positions identical to those of the first two introns in the vertebrate beta-tubulin genes. The fly ß3-tubulin contains an additional six amino acids not present in any other beta-tubulin. The position of the third intron differs from that of the vertebrate beta-tubulin genes, but is identical in position to the testis-specific ß2 isoform. The testis-specific isoform and the ubiquitously expressed ß1 isoform are highly homologous both to each other and to the vertebrate major neural form. The ß3 isoform is more divergent, and reveals an ancient divergence in beta-tubulin sequences which must have occurred at or before the split between lines leading to insects and vertebrates. For ß tubulins there appear to be two major regions that differ in their secondary structures, that is in the folding of the polypeptide chain. A predicted alpha-helical region is present in the carboxyl third of the molecule, and a predicted beta-pleated sheet region falls primarily in the amino-terminal two-thirds of the molecule (Rudolph, 1987).

The ß1-, ß2- and ß3-tubulin-specific sequences in the chromosomes of Drosophila auraria are found in the same polytene band in region 32C of the 2L polytene chromosome. In contrast, the three beta tubulin genes in D. melanogaster are not closely linked (Scouras, 1994).

Dictyostelium is unusual in that its genome contains single alpha- and beta-tubulin genes, rather than the multi-gene family common in most eukaryotic organisms. The complete alpha-tubulin cDNA contains 1558 nucleotides with an open reading frame that encodes a protein of 457 amino acids. The complete beta-tubulin cDNA contains 1572 nucleotides and encodes a protein of 456 amino acids. Analysis of the deduced protein sequences indicates that while there is a significant degree of sequence similarity between the Dictyostelium tubulins and other known tubulins, the Dictyostelium alpha-tubulin displays the greatest sequence divergence yet described. Single alpha- and beta-tubulin transcripts are detected by northern blot analysis during all stages of Dictyostelium development. The highest message levels accumulate late in germinating spores and vegetative amoeba. Despite changes in alpha- and beta-tubulin mRNA levels, protein levels remain constant throughout development. Antisera recognize one alpha- and two beta-tubulin spots on western blots of 2-D gels and, by indirect immunofluorescence, both recognize the interphase and mitotic microtubule arrays in vegetative amoeba (Trivinos-Lagos, 1993).

Polyglutamylation is an important posttranslational modification of tubulin. It occurs in nerve cells where it is the main factor responsible for tubulin heterogeneity. Glutamylated alpha- and beta-tubulin both markedly accumulate during this process, with a time course remarkably similar to that observed in vivo during brain development. However, the characteristics of the glutamylation in the two subunits are not exactly the same. Glutamylated alpha-tubulin is already abundant in very young neurons. At this stage it displays a wide range of glutamylation (1 to 6 glutamyl units present in the lateral polyglutamyl chain), which remains unchanged during the entire period of the culture. Glutamylated beta-tubulin is present at very low levels in young neurons and its accumulation during differentiation is accompanied by a progressive increase in its degree of glutamylation from 2 to 6 glutamyl units. Posttranslational incorporation of [3H]glutamate into alpha- and beta-tubulin decreases during differentiation, as well as the rate of the reverse deglutamylation reaction, suggesting that accumulation of glutamylated tubulin is accompanied by a decrease in the turnover of glutamyl units onto tubulin. Neuronal differentiation is also accompanied by an increase of other posttranslationally modified forms of tubulin, including acetylated and non-tyrosinatable alpha-tubulin, which can occur in combination with polyglutamylation, contributing to the increase in complexity of tubulin in mature neurons (Audebert, 1994).

Promoter Structure

Continued: see betaTubulin60D part 2/2|

date revised:  2 nov 96

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