Fig 1: In vivo analysis of a-tubulin modifications. (A) Cartoon showing a-tubulin modifications (left) and sequence alignment of the C-terminal tails of human, mouse, fly, and worm TUBA1A orthologs (right). Red, ten amino acids that were deleted in aTub84B?C. Underline, three residues (EEY) deleted in aTub84B?3. (B–D) Microtubules in developing fly embryos are modified, and da neuron axons are enriched in acetylated microtubules (D–D?). Embryos were stained for HRP (green), a neuronal membrane marker, as well as polyglutamylated (magenta, B), acetylated (red, C,D) and tyrosinated (blue, C,D) microtubules. Arrowheads, da neuron cluster; arrows, axons. (E,F) In larval da neurons, microtubules in axons and dendrites are acetylated at equivalent levels. The acetylated microtubule signals in proximal axonal and dendritic segments (E) were compared as a ratio (mean±s.d.), n=7 class IV ddaC neurons (F). Green, acetylated microtubules; magenta, HRP. Arrowheads, dendrite and ddaC cell body marker; arrow, axons. Images are from fixed tissue. Scale bars: 50 µm. (G) Cartoon of the aTub84BKO-attP allele that enables rapid knock-in of designer alleles to interrogate a-tubulin function in vivo. Top, wild-type aTub84B (blue); middle, the major coding exon of aTub84B was deleted (brackets) and replaced by an attP site (yellow); bottom, knock-in of a mutant aTub84B (orange). (H) GFP-tagged aTub84B is broadly expressed in developing larvae. In muscles and epithelial cells, a filamentous pattern indicates GFP::aTub84B is likely incorporated into microtubules. Arrowheads, dendrites; arrow, axon. Green, GFP; magenta, CD4::Tomato. Scale bars: 25 µm (left), 10 µm (muscle and epithelial cell images, right). Images from live third-instar larvae. (I) Western blot analysis (left) and quantification (right) of a-tubulin levels (normalized to actin) in wild-type (WT) or mutant strains as indicated. All strains are homozygous. K40A 84DKO refers to the aTub84BK40A chromosome with aTub84D deleted (aTub84BK40A, aTub84DKO); K'in-WT is a knock-in for wild-type aTub84B. (J) Western blot of lysates from wild-type and aTub84B mutant fly heads probed for acetylated, tyrosinated and polyglutamylated a-tubulin, as indicated. KO, single aTub84D knockout. The two-lane blot (right) was probed for acetylated a-tubulin and includes lysate from double-mutant aTub84BK40A aTub84DKO fly heads. The aTub84B ?3 mutation eliminates both the anti-tyrosinated and anti-polyglutamylated a-tubulin signals.