Axoneme-specific β-tubulin specialization a conserved C-terminal motif specifies the central pair

AbstractAxonemes 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, α- and β-tubulins utilized in motile axonemes fall among the most conserved tubulin sequences [1, 2], and the β-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 α-tubulin but different β-tubulins [6–13]. β1 is utilized for the centriole/basal body, and β2 is utilized for the motile sperm tail axoneme. β2 contains the motile axoneme-specific sequence motif, but β1 does not [3]. Here, we show that the “axoneme motif” specifies the central pair. β1 can provide partial function for axoneme assembly but cannot make the central microtubules [14]. Introducing the axoneme motif into the β1 carboxyl terminus, a two amino acid change, conferred upon β1 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

Genetic Analysis of theDrosophilaβ3-Tubulin Gene Demonstrates That the Microtubule Cytoskeleton in the Cells of the Visceral Mesoderm Is Required for Morphogenesis of the Midgut Endoderm

AbstractWe have investigated the cellular basis for lethality of mutant alleles of theDrosophila melanogasterβ3-tubulin gene,βTub60D.Lethal β3 mutations can be grouped into two classes: the most severe mutations (Class I alleles) cause death during the first larval instar, while weaker alleles (Class II) cause death in later larval stages or in early pupal development. Since β3 is not expressed during larval development, lethality of the Class I mutations must reflect essential functions of β3 in embryogenesis. β3-tubulin is zygotically expressed during midembryogenesis in the developing mesoderm, and the major site of β3 accumulation is in the developing muscles during myogenesis. We show that the embryonic pattern of β3 expression, including accumulation in the developing musculature, is conserved in otherDrosophilaspecies. However, we found that loss of β3 function does not cause discernible defects in either the ultrastructure or function of the larval muscle. Thus β3-tubulin is dispensable in its highest site of accumulation. Rather, the essential site of function of β3 in embryos is in cells of the visceral mesoderm. Lethality of Class I alleles is caused by defects in midgut morphogenesis and failure of gut function. Although the folding pattern is irregular and the gut is smaller than normal, a complete folded gut forms in mutant larvae, and the visceral muscle functions normally to move food through the gut. However, mutant larvae cannot absorb nutrients across the gut wall. Thus loss of β3 function in the mesoderm results in defects in the underlying endodermally derived layer of the gut. Our data provide an assay for cellular interactions between mesoderm and endodermal tissues and reveal a role for the microtubule cytoskeleton of the visceral mesodermal cells in differentiation of the endodermal cell layer of the larval gut