Substitution of MEC-7/β-tubulin S172 led to neurite growth defects in C. elegans TRNs.

(A) TRN morphologies in wild-type animals, S172A, S172E, and S172P mutants. Specific neurites are labeled by arrows. Asterisks indicate the position of vulva. Dashed arrows point to the termini of neurites that are shortened in mutants. (B) The distance of ALM-AN terminus to the tip of the nose in various strains. The longer the distance, the shorter the ALM-AN. (C) The distance from the PLM-AN terminus to the vulva in various strains. If the anteriorly directed PLM-AN grew past the vulva, the distance is positive. If PLM-AN cannot reach the vulva, the distance is negative. (D) Quantification of ALM-PN length. The wild-type animals have no or very short ALM-PN. (E) Quantification of PLM-PN length. (F) Representative images of PLM-PN in various strains. Dashed arrows indicate the neurite termini of shortened PLM-PN. (G) Representative images of ALM-PN in mec-7(S172A) mutants. Arrowhead indicates the synaptic branch of PLM-AN in the wild-type animals. Dashed arrows point to the branching defects where the synaptic branch failed to extend to the ventral cord. (H) TRNs extracted from the embryos of S172 mutants and cultured in vitro; they were identified by their expression of mec-17p::TagRFP among the embryonic cells. TRNs from S172E mutants had no or very short neurites. (I) The length of the longest neurite of the in vitro cultured TRNs from the S172 mutants. Scale bar = 20 μm.

MEC-7/β-tubulin S172 phosphorylation regulates MT dynamics, cargo transport, mechanosensory function, and axonal regeneration.

(A) Representative kymographs of EBP-2::GFP dynamics in the PLM-AN of various strains. The wild-type animals and S172A mutants were subjected to a mild colchicine treatment to increase MT dynamics and imaged after one-hour recovery. (B) Quantification of the number of EBP-2 tracks. (C) Percentages of retrograde and anterograde movement for the EBP-2 comets. (D) Comparison of the EBP-2::GFP signal in the cell body of wild-type and mec-7(S172A) animals. Scale bar = 20 μm. (E) Distribution of mitochondria in the ALM-AN indicated by the jsIs609 [mec-7p::mitoGFP] signal. (F) The quantification of the number of mitoGFP puncta in the distal segment of ALM-AN (150-300 μm away from the cell body). (G) Localization of the synaptic vesicles (GFP::RAB-3) in the S172 mutants. Arrows indicate the normal localization of RAB-3 signal to the synapses made by synaptic branch of PLM-AN in the ventral nerve cord posterior to the vulval position (indicated by the asterisk). In S172A mutants, RAB-3 was mistargeted to the ALM-PN (dashed arrow) and accumulated at the PLM-AN (arrowhead) when the synaptic branch fails to extend. In S172E mutants, RBA-3 signal was trapped in the cell body as PLM-AN is severely shortened. (H) Anterior touch responses of various mec-7 mutants; mec-7(ok2151) is knockout deletion allele. (I) Representative axonal regrowth of PLM-AN following laser axotomy in various strains; arrows indicate the cut site. (J) Quantification of PLM regrowth length for the regrowth cases. (K) Examples of reconnections after laser axotomy; arrows indicate the cut site.

CDK-1 mediates MEC-7/β-tubulin S172 phosphorylation and regulates neurite growth.

(A) Images of TRN morphologies in cdk-1(he5)/+ heterozygotes and cdk-1(he5) homozygotes (escapers). Arrow points to the ectopic ALM-PN. Scale bar = 100 μm. (B) The length of ALM-PN in wild-type and cdk-1 mutants. ALM-PN length in the escapers of he-5 and ok1882 homozygous mutants were measured. (C) The ratio of ALM-PN length to ALM-AN length in various strains. (D) The ALM-PN length in animals carrying both mec-7(S172A) and cdk-1 mutations. (E) Western blot of the kinase reaction samples using anti-phospho-S172 antibodies. The kinase reaction was conducted in the MEM buffer with 475 nM CDK1/Cyclin B or 500 nM MBK-2m. The weak signal in the first lane of the left panel likely resulted from the nonspecific interaction of the anti-phospho-S172 antibodies with the unmodified MEC-12/MEC-7 heterodimer or the background phosphorylation level of the recombinant dimer. Anti-β-tubulin antibodies were used to ensure the equal loading of the tubulin dimers.

The effects of tubulin acetylation on neurite development.

(A) The PLM-PN length in mec-12 K40 mutants. (B) The length of ALM-PN in mec-12 K40 mutants. (C) 20% of PLM-AN failed to extend the synaptic branch in mec-12(K40Q) mutants. Arrow points to the normal synaptic branch in mec-12(K40R) mutants and the dashed arrow indicates the branching defects. (D) Quantification of the PLM regrowth length after laser axotomy in mec-12 K40 mutants. (E) Anterior touch responses of mec-17 and mec-12 mutants. The deletion allele ok2109 was used for mec-17(-) and the unk126 (G121W & G123W) allele was used for mec-17(dW). (F) Comparison of ALM-PN in mec-17(-) and mec-17(dW) mutants. (G) Quantification of ALM-PN lengths in mec-17 mutants. (H) The swelling and looping of the TRN neurite in mec-17 mutants (ALM-AN is shown). (I) The ALM and PLM morphologies in mec-7 K252 mutants. Dashed arrows point to the termini of the shortened ALM-AN or PLM-AN or PLM-PN. (J) Quantification of the shortening of the three neurites. For ALM-AN, the distance from the neurite ending to the nose tip was measured; the bigger the gap, the shorter the ALM-AN. For PLM-AN, the distance from the neurite terminus to the vulva was measured. Positive value means the neurite grew past the vulva and negative value means the neurite did not reach the vulva.

Tubulin polyglutamylation regulates neurite growth and regeneration.

(A) The growth ectopic ALM-PN (arrow) in mec-12 mutants lacking the polyglutamylation sites. (B) The quantification of ALM-PN length in various mec-12 strains. The changes made to the last twelve amino acids of MEC-12 in the various alleles are shown. (C) Kymographs showing EBP-2::GFP dynamics in wild-type and mec-12(4Es-A) animals after a mild colchicine treatment and a one-hour recovery. (D) Quantification of the number of EBP-2 comets in mec-12(4Es-A) mutants. (E) The percentage of retrograde and anterograde EBP-3 movements. (F) PLM regrowth following laser axotomy in mec-12(4Es-A) and ttll-5(tm3360) mutants and the double mutants; arrows indicate the cut site. (G) The quantification of PLM regrowth length in various strains. (H) ALM-PN lengths in spas-1(tm683), klp-7(unk179; Δ11bp), klp-7(unk171; Δ14bp), and klp-7; mec-12(4Es-A) double mutants. unk171 was generated by gene editing in both the wild-type and the mec-12(4Es-A) background, and #1 and #2 were two independent lines. See Figure 5-figure supplement 2 for the details of the klp-7 alleles.

Substitution of glutamates in MEC-12 and MEC-7 C-terminal tails eliminate tubulin polyglutamylation in the axons.

(A) Antibody staining of in vitro cultured TRNs (labeled by RFP) with IN105 anti-polyglutamylation antibodies that recognize a chain of four or more glutamates. (B) Antibody staining of in vitro cultured TRNs from various strains with GT335 anti-polyglutamylation antibodies that recognize the branchpoint and a side chain of two glutamyl units. CGZ1554 ttll-4(tm3310); ttll-5(tm3360); ttll-12(unk185); uIs115 and CGZ1475 ttll-4(tm3310); ttll-5(tm3360) ttll-9(tm3889) ttll-15 (tm3871); ttll-12(unk185); uIs115 were used in the experiment. Arrows point to the TRN cell bodies and arrowheads point to the staining signal in the axon. (C-D) Quantification of the fluorescent intensity (in arbitrary units or a.u.) for the polyE staining of TRN axons and cell bodies using GT335. Three and four asterisks indicate p < 0.001 and 0.0001, respectively, for statistical significance in comparison with the wild type. (D) RNA level of the ttll genes in ALM and PLM neurons according to the L4 stage single-cell transcriptomic data obtained from wormbase.org. (F) ALM-PN length in mec-7, mec-12, and ttll mutant strains. CGZ1554, CGZ1475, and CGZ1474 ttll-4(tm3310); ttll-11(tm4059); ttll-5(tm3360) ttll-9(tm3889) ttll-15(tm3871); ttll-12(unk185); zdIs5 were used.

The effects of tubulin detyrosination and Δ2 modification on neurite growth.

(A) The length of ALM-PN in strains deleting the terminal tyrosine or the last two residues. (B) The weak staining of wild-type TRNs with the anti-tyrosinated α-tubulin antibodies. (C) The staining of the wild-type animals and the various mec-12 mutants with anti-detyrosinated α-tubulin antibodies. Arrows point to the ALM cell bodies, ALM-AN, and PLM-AN. (D) Quantification of the fluorescent intensity of the anti-detyrosination staining signal in the axons. Arbitrary units (a.u.) were used. Four asterisks indicate p < 0.0001 in the comparison with the wild type.

mec-7 S172A and S172E mutations are semidominant.

(A) The length of ALM-PN in day-one adults (three days after bleaching) of wild-type animals, mec-7(S172A)/+ heterozygotes, and mec-7(S172A) homozygotes. (B) The distance from the PLM-AN terminus to the vulva in the wild-type animals, mec-7(S172E)/+ heterozygotes, and mec-7(S172E) homozygotes. If PLM-AN grew past the vulva, the distance is positive. If PLM-AN cannot reach the vulva, the distance is negative.

mec-7 S172 mutations altered β-tubulin S172 phosphorylation level.

(A) Anti-β-tubulin phospho-S172 antibody staining of in vitro cultured C. elegans embryonic cells from the indicated strains. Cells expressing the mec-17p::TagRFP were identified as the TRNs. Scale bar = 20 μm. (B) Fluorescent intensity in the axons were measured and quantified. Asterisks indicate statistical significance compared with the wild-type cells. Four asterisks indicate p < 0.0001.

Expression of the minibrain homologs, mbk-1, mbk-2, and hpk-1 in the TRNs.

(A) The expression of cmIs6 [mbk-1::GFP] in the TRNs labeled by mec-17p::TagRFP. Circles indicate the expression of GFP in TRNs, including ALM, PLM, and AVM neurons. (B) The expression of cmEx6 [mbk-2p::GFP] in the TRNs. (C) The expression of unkEx194 [hpk-1Bp::GFP] in TRNs. Scale bar = 20 μm.

Kinase assay for MBK-2 in BRB80 buffer. 500 nM recombinant MEC-12/MEC-7 heterodimer and 1 mM ATP were incubated with various concentration of MBK-2. Western blot of the kinase reaction samples was done using the anti-phospho-S172 antibodies.

mec-12 K40R and K40Q mutations and the combined loss of mec-17 and atat-2 affect α-tubulin K40 acetylation.

(A) Anti-acetyl-K40 staining of the wild-type, mec-12(K40R), and mec-12(K40Q) animals. Only the wild-type animals showed the staining in TRNs. Neither mec-12(K40R) nor mec-12(K40Q) mutants showed any staining. Arrows point to the staining signal in cell bodies or axons. Scale bar = 20 μm. (B-C) Anti-acetyl-K40 staining of acetyltransferase mutants. mec-17(dW), mec-17(-), and atat-2(-) single mutants showed clear staining signal, whereas the mec-17; atat-2 double mutants showed no signal at all. Arrows point to the staining signal in the neurites. Scale bar = 20 μm.

Sequence divergence of α-tubulin C-terminal tails in C. elegans.

Comparison of the amino acid sequences of the C-terminal region of the α-tubulin isotypes. The last twelve amino acids of MEC-12 are highly divergent among the isotypes (boxed region).

Mutations in klp-7 result in the growth of an ectopic ALM-PN.

(A) The gene structure of klp-7 (from Wormbase.org) and the nucleotides deleted in unk171 and unk179 alleles, which were generated by CRISPR/Cas9-mediated gene editing. (B) The ectopic ALM-PN in klp-7(unk171) mutants.

The lack of cargo transport and mechanosensory defects in mec-12(4Es-A) mutants.

(A) The normal localization of synaptic vesicles (GFP::RAB-3) to the synaptic branch (arrows) of PLM-AN in mec-12(4Es-A) mutants. Focal plane 1 focuses on the neurites, while focal plane 2 focuses on the ventral cord. (B) Distribution of mitochondria in the ALM-AN of mec-12(4Es-A) mutants. (C) Quantification of the number of mito::GFP puncta in the distal segment (150∼300 μm from the cell body) of ALM-AN. (D) Anterior touch response in the mec-12(4Es-A) mutants.

Sequence divergence of the C-terminal tail among β-tubulin isotypes and the molecular details of ttll-12 CRISPR allele. (A) Comparison of the amino acid sequences of the C-terminal region of the β-tubulin isotypes in C. elegans. The last twelve amino acids of MEC-7 are divergent among the isotypes (boxed region). (B) Five nucleotides (red with strikethrough) were deleted in ttll-12(unk185) allele.