Motor neuron synapse and axon defects in a C. elegans alpha-tubulin mutant

Abstract

Regulation of microtubule dynamics underlies many fundamental cellular mechanisms including cell division, cell motility, and transport. In neurons, microtubules play key roles in cell migration, axon outgrowth, control of axon and synapse growth, and the regulated transport of vesicles and structural components of synapses. Loss of synapse and axon integrity and disruption of axon transport characterize many neurodegenerative diseases. Recently, mutations that specifically alter the assembly or stability of microtubules have been found to directly cause neurodevelopmental defects or neurodegeneration in vertebrates. We report here the characterization of a missense mutation in the C-terminal domain of C. elegans alpha-tubulin, tba-1(ju89), that disrupts motor neuron synapse and axon development. Mutant ju89 animals exhibit reduction in the number and size of neuromuscular synapses, altered locomotion, and defects in axon extension. Although null mutations of tba-1 show a nearly wild-type pattern, similar axon outgrowth defects were observed in animals lacking the beta-tubulin TBB-2. Genetic analysis reveals that tba-1(ju89) affects synapse development independent of its role in axon outgrowth. tba-1(ju89) is an altered function allele that most likely perturbs interactions between TBA-1 and specific microtubule-associated proteins that control microtubule dynamics and transport of components needed for synapse and axon growth.

Figure 1. GABAergic Motor Neuron Synapses are Defective in tba-1(ju89) Mutants.

(A) Diagram of en passant neuromuscular synapses in C. elegans. Dots represent synapses; dorsal D neurons (DDs) form synapses onto dorsal body-wall muscles, and ventral D neurons (VDs) synapse with ventral muscles. Presynaptic termini of the inhibitory GABAergic type D motor neurons are visualized with the synaptic vesicle marker P_unc-25SNB-1::GFP. Each GFP puncta visible by fluorescence microscopy corresponds to the cumulative signal from all GFP-tagged vesicles at an individual synapse. (B) Morphology of the six DD motor neurons. Cell bodies are located in the ventral nerve cord. Each D neuron extends a ventral process, branches to form a commissure, and bifurcates at the dorsal nerve cord to form a dorsal process along the dorsal nerve cord. At the distal tip of each process, gap junctions are formed with adjacent DD neurons . Expression pattern of SNB-1::GFP in wild-type (C) and mutant (D) dorsal nerve cord. Irregular size puncta and reduced numbers of puncta are evident in ju89 mutants. An arrow designates the position of a DD commissure. (E) SNT-1 (synaptotagmin) expression in the dorsal nerve cord of wild-type and (F) ju89 mutant animals. UNC-10::GFP expression along the dorsal nerve cord of (G) wild-type and (H) ju89 worms. (I) Expression of the GABA-B receptor subunit, UNC-49::GFP, along dorsal muscles of wild-type animals and (J) ju89 mutant worms. Gaps in UNC-49::GFP expression demonstrate the lack of post-synaptic structures in these regions. Wild-type C. elegans (K) have a larger body size than tba-1(ju89) (L). C. elegans move in a wavelike pattern. ju89 mutants are uncoordinated, and the amplitude of the wave pattern is severely reduced compared to wild-type animals.

Figure 2. Ultrastructure of tba-1(ju89) Motor Neuron Synapses.

The ultrastructure of one adult wild-type and two ju89 adult mutant worm were examined by EM (see Methods). 1500 continuous sections of 50 nm each were collected from 1 wild-type adult animal and 400 sections from each of two ju89 adult mutant worms. Sections were collected in the anterior of each animal between the nerve ring and the vulva. Inhibitory GABAergic motor neurons form synapses directly onto dorsal and ventral muscles; excitatory cholinergic motor neurons form dyadic synapses with muscles and a GABAergic motor neuron dendrite. Representative GABAergic motor neuron synapses with body wall muscle arms in (A) wild-type and (C) mutant tba-1(ju89) animals, and cholinergic motor neuron synapses in (B) wild-type and (D) ju89 worms. Fewer synaptic vesicles are visible in both types of synapses in the mutants. (E) The number of synaptic vesicles per active zone (p<.05), (F) length of the active zone in nm (p<.05), and (G) number of synaptic vesicles/active zone length for the cholinergic motor neuron synapses in one animal are shown. Cholinergic synapse size is reduced in the ju89 mutant. Error bars: S.E.M.

Figure 3. Axon Outgrowth Defects in Tubulin Mutants.

(A) Diagrams of defects in axon extension and commissure outgrowth observed in newly hatched mutant worms (see Table 2). The morphology of the DD neurons in wild-type and mutant worms was visualized with the cytoplasmic axon markers P_unc-25GFP or P_flp-13GFP. Examples of axon defects in tubulin mutants: (B), (C) short regions (arrows) where axons fail to extend to their full length along the dorsal nerve cord of tba-1(ju89) worms; (D) a commissure branches prematurely and fails to reach the dorsal nerve cord in a tbb-1(gk207) mutant animal; (E) axon outgrowth or extension defects in all six DD neurons of a newly hatched L1 larvae. The DD6 commissure stalls prematurely (arrow), and the DD2 cell body is also displaced anterior. (F) abnormal commissure branch (arrow) in a tba-1(ju89) mutant. Scale bar 20 µM.

Figure 4. Synapse Loss in DD Axons of tba-1(ju89) Animals.

The DD neuron axon marker P_flp-13::GFP was coexpressed with a presynaptic marker P_unc-25 mCherry::RAB-3 in wild-type (A, C, E) and mutant (B, D, F) tba-1(ju89) animals (see Methods). Axon extension was similar in both the wild-type (A) and mutant (B) axons depicted. mCherry::RAB-3 puncta were reduced or missing in the tba-1(ju89) mutant axon (D, F) compared to the wild-type worm (C, E). The bracket in (F) delineates a region in which mCherry::RAB-3 puncta are missing or greatly reduced in size in the mutant. Scale bar 20 µM.

Figure 5. ju89 is a Novel Allele of C. elegans Alpha-Tubulin tba-1.

(A) location of ju89 on C. elegans chromosome I based on genetic map data, (B) Transformation rescue of ju89 by F24E4.8 (tba-1). The minimal rescuing activity for ju89 was narrowed to an overlapping region of cosmid F26E4 and fosmid HO1I17 containing the tba-1-drsh-1 operon. pCZ485, a 4.5 kb subclone containing upstream sequence and only the tba-1 coding region (F26E4.8) is sufficient to rescue the SNB-1:GFP and locomotion defects of ju89 mutants. (C) ju89 is a missense mutation that converts a conserved glycine to arginine in the H11–H12 loop of the TBA-1 C-terminus. C-terminal domain structure based on Lowe et al., 2001. Sequences used for the alignment are C. elegans TBA-1 (CAB03001), TBA-2 (CAB16856), human TUBA3/TUBA1A (NP006000), Mus musculus TUBA1A (AAH83344), and Danio rerio (NP919369). (D) Crystal structure of alpha-beta tubulin dimer generated by Polyview based on Nogales et al, 1998 and Lowe et al., 2001 (PDB#1JFF). Red highlights the residue altered in ju89 mutants (G414 in TBA-1 and G416 in TUBA1A) located at the beginning of helix 12. Blue highlights the R402 residue mutated in human lissencephaly (R400 in TBA-1) in the H11–H12 loop adjacent to H11.

Figure 6. Expression of Mutant tba-1(ju89) from Extrachromosomal Arrays in Wild-Type C. elegans Phenocopies tba-1 (ju89) Defects.

PCR products corresponding to the tba-1 4.5 kb rescuing region of tba-1 were amplified from wild-type (N2) or ju89 mutant animals and injected into wild-type juIs1 hermaphrodites to generate transgenic lines (see Methods). 14/27 lines expressing the mutant tba-1 gene, Extba-1(R414) exhibited uncoordinated movement defects and altered SNB-1::GFP patterns similar to ju89 homozygotes. All seven lines expressing wild-type tba-1 (Extba-1) were wild-type. Expression of the juIs1 SNB-1::GFP transgene in the dorsal nerve cord of wild-type N2 worms expressing (A) Extba-1; or (B) Extba-1(R414). The body size and locomotion of transgenic animals expressing wild-type tba-1 arrays (C) was the same as wild-type worms, whereas N2 worms expressing arrays of tba-1 amplified from the ju89 mutant (D) exhibited the smaller body size and uncoordinated movement characteristic of ju89 mutants. Scale bar 5 µM.