Limits...
Genetic analysis of a novel tubulin mutation that redirects synaptic vesicle targeting and causes neurite degeneration in C. elegans.

Hsu JM, Chen CH, Chen YC, McDonald KL, Gurling M, Lee A, Garriga G, Pan CL - PLoS Genet. (2014)

Bottom Line: This missense mutation replaced an absolutely conserved glycine in the H12 helix with glutamic acid, resulting in increased negative charges at the C-terminus of α-tubulin.By contrast, neurite swelling and neurodegeneration were independent of dynein and could be ameliorated by genetic paralysis of the animal.This suggests that mutant microtubules render the neurons susceptible to recurrent mechanical stress induced by muscle activity, which is consistent with the observation that microtubule network was disorganized under electron microscopy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Neuronal cargos are differentially targeted to either axons or dendrites, and this polarized cargo targeting critically depends on the interaction between microtubules and molecular motors. From a forward mutagenesis screen, we identified a gain-of-function mutation in the C. elegans α-tubulin gene mec-12 that triggered synaptic vesicle mistargeting, neurite swelling and neurodegeneration in the touch receptor neurons. This missense mutation replaced an absolutely conserved glycine in the H12 helix with glutamic acid, resulting in increased negative charges at the C-terminus of α-tubulin. Synaptic vesicle mistargeting in the mutant neurons was suppressed by reducing dynein function, suggesting that aberrantly high dynein activity mistargeted synaptic vesicles. We demonstrated that dynein showed preference towards binding mutant microtubules over wild-type in microtubule sedimentation assay. By contrast, neurite swelling and neurodegeneration were independent of dynein and could be ameliorated by genetic paralysis of the animal. This suggests that mutant microtubules render the neurons susceptible to recurrent mechanical stress induced by muscle activity, which is consistent with the observation that microtubule network was disorganized under electron microscopy. Our work provides insights into how microtubule-dynein interaction instructs synaptic vesicle targeting and the importance of microtubule in the maintenance of neuronal structures against constant mechanical stress.

Show MeSH

Related in: MedlinePlus

Genetic analysis of gm379 and other mec-12 alleles.(A) The gene structure and mutant alleles of mec-12, and comparison of α-tubulins in yeast, C. elegans, mice and human. Characters and numbers are nucleotides and their cDNA positions (upper panel) or amino acids (lower panel). (B) Predicted protein structure of MEC-12 generated by Polyview software. The G416E mutation in gm379 is located in the H12 helix. (C, D) RNAi against mec-12 completely suppressed axon defects and largely abolished SV mistargeting in the gm379 mutant, phenocopying the mec-12(e1607) and mec-12(tm5083)  mutants. Neurons and SVs were labeled by jsIs973 and jsIs821, respectively. Arrows indicate axon swellings in ALM and mistargeted SVs in PLM. Asterisks indicate neuronal soma. Anterior is to the left. Scale bars  = 5 µm. (E) Genetic analysis of various mec-12 alleles. -, no phenotypes; +, moderate defects; ++, prominent defects.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230729&req=5

pgen-1004715-g004: Genetic analysis of gm379 and other mec-12 alleles.(A) The gene structure and mutant alleles of mec-12, and comparison of α-tubulins in yeast, C. elegans, mice and human. Characters and numbers are nucleotides and their cDNA positions (upper panel) or amino acids (lower panel). (B) Predicted protein structure of MEC-12 generated by Polyview software. The G416E mutation in gm379 is located in the H12 helix. (C, D) RNAi against mec-12 completely suppressed axon defects and largely abolished SV mistargeting in the gm379 mutant, phenocopying the mec-12(e1607) and mec-12(tm5083) mutants. Neurons and SVs were labeled by jsIs973 and jsIs821, respectively. Arrows indicate axon swellings in ALM and mistargeted SVs in PLM. Asterisks indicate neuronal soma. Anterior is to the left. Scale bars  = 5 µm. (E) Genetic analysis of various mec-12 alleles. -, no phenotypes; +, moderate defects; ++, prominent defects.

Mentions: We cloned gm379 by single nucleotide polymorphism (SNP) mapping, complementation test and DNA sequencing, and found that it contained a missense mutation of the touch neuron-specific α-tubulin mec-12 that alters an absolutely conserved C-terminal glycine residue to glutamate (G416E) [19], [17] (Figure 4A and 4B). Interestingly, gm379 animals were touch-insensitive (30% touch-sensitive, compared to wild type, 89%; and mec-12(e1607) , 12%, n>35). We found another intron mutation in gm379 that was distant to exon-intron junctions (nucleotide 828 of unspliced transcript, G to A mutation). Two mutants of mec-12, e1607 and tm5083, also had SV transport defects, but not SV mistargeting in the PLM or axon swelling in the touch neurons (Figure 4C, S3). RNAi against mec-12 in the gm379 mutant almost completely abolished axon swelling or SV mistargeting, indicating that these two phenotypes were neomorphic (Figure 4C, 4D). Expression of the MEC-12(G416E) mutant tubulin in the mec-12 mutants recapitulated the SV mistargeting phenotypes of the mec-12(gm379) mutant (67% of transgenic animals showed SV mistargeting, n = 21 v.s. 6.5% of array-loss siblings showing SV mistargeting, n = 77), confirming that SV mistargeting and neurite swelling were indeed caused by the mec-12(gm379) rather than other unidentified mutations in the background.


Genetic analysis of a novel tubulin mutation that redirects synaptic vesicle targeting and causes neurite degeneration in C. elegans.

Hsu JM, Chen CH, Chen YC, McDonald KL, Gurling M, Lee A, Garriga G, Pan CL - PLoS Genet. (2014)

Genetic analysis of gm379 and other mec-12 alleles.(A) The gene structure and mutant alleles of mec-12, and comparison of α-tubulins in yeast, C. elegans, mice and human. Characters and numbers are nucleotides and their cDNA positions (upper panel) or amino acids (lower panel). (B) Predicted protein structure of MEC-12 generated by Polyview software. The G416E mutation in gm379 is located in the H12 helix. (C, D) RNAi against mec-12 completely suppressed axon defects and largely abolished SV mistargeting in the gm379 mutant, phenocopying the mec-12(e1607) and mec-12(tm5083)  mutants. Neurons and SVs were labeled by jsIs973 and jsIs821, respectively. Arrows indicate axon swellings in ALM and mistargeted SVs in PLM. Asterisks indicate neuronal soma. Anterior is to the left. Scale bars  = 5 µm. (E) Genetic analysis of various mec-12 alleles. -, no phenotypes; +, moderate defects; ++, prominent defects.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230729&req=5

pgen-1004715-g004: Genetic analysis of gm379 and other mec-12 alleles.(A) The gene structure and mutant alleles of mec-12, and comparison of α-tubulins in yeast, C. elegans, mice and human. Characters and numbers are nucleotides and their cDNA positions (upper panel) or amino acids (lower panel). (B) Predicted protein structure of MEC-12 generated by Polyview software. The G416E mutation in gm379 is located in the H12 helix. (C, D) RNAi against mec-12 completely suppressed axon defects and largely abolished SV mistargeting in the gm379 mutant, phenocopying the mec-12(e1607) and mec-12(tm5083) mutants. Neurons and SVs were labeled by jsIs973 and jsIs821, respectively. Arrows indicate axon swellings in ALM and mistargeted SVs in PLM. Asterisks indicate neuronal soma. Anterior is to the left. Scale bars  = 5 µm. (E) Genetic analysis of various mec-12 alleles. -, no phenotypes; +, moderate defects; ++, prominent defects.
Mentions: We cloned gm379 by single nucleotide polymorphism (SNP) mapping, complementation test and DNA sequencing, and found that it contained a missense mutation of the touch neuron-specific α-tubulin mec-12 that alters an absolutely conserved C-terminal glycine residue to glutamate (G416E) [19], [17] (Figure 4A and 4B). Interestingly, gm379 animals were touch-insensitive (30% touch-sensitive, compared to wild type, 89%; and mec-12(e1607) , 12%, n>35). We found another intron mutation in gm379 that was distant to exon-intron junctions (nucleotide 828 of unspliced transcript, G to A mutation). Two mutants of mec-12, e1607 and tm5083, also had SV transport defects, but not SV mistargeting in the PLM or axon swelling in the touch neurons (Figure 4C, S3). RNAi against mec-12 in the gm379 mutant almost completely abolished axon swelling or SV mistargeting, indicating that these two phenotypes were neomorphic (Figure 4C, 4D). Expression of the MEC-12(G416E) mutant tubulin in the mec-12 mutants recapitulated the SV mistargeting phenotypes of the mec-12(gm379) mutant (67% of transgenic animals showed SV mistargeting, n = 21 v.s. 6.5% of array-loss siblings showing SV mistargeting, n = 77), confirming that SV mistargeting and neurite swelling were indeed caused by the mec-12(gm379) rather than other unidentified mutations in the background.

Bottom Line: This missense mutation replaced an absolutely conserved glycine in the H12 helix with glutamic acid, resulting in increased negative charges at the C-terminus of α-tubulin.By contrast, neurite swelling and neurodegeneration were independent of dynein and could be ameliorated by genetic paralysis of the animal.This suggests that mutant microtubules render the neurons susceptible to recurrent mechanical stress induced by muscle activity, which is consistent with the observation that microtubule network was disorganized under electron microscopy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Neuronal cargos are differentially targeted to either axons or dendrites, and this polarized cargo targeting critically depends on the interaction between microtubules and molecular motors. From a forward mutagenesis screen, we identified a gain-of-function mutation in the C. elegans α-tubulin gene mec-12 that triggered synaptic vesicle mistargeting, neurite swelling and neurodegeneration in the touch receptor neurons. This missense mutation replaced an absolutely conserved glycine in the H12 helix with glutamic acid, resulting in increased negative charges at the C-terminus of α-tubulin. Synaptic vesicle mistargeting in the mutant neurons was suppressed by reducing dynein function, suggesting that aberrantly high dynein activity mistargeted synaptic vesicles. We demonstrated that dynein showed preference towards binding mutant microtubules over wild-type in microtubule sedimentation assay. By contrast, neurite swelling and neurodegeneration were independent of dynein and could be ameliorated by genetic paralysis of the animal. This suggests that mutant microtubules render the neurons susceptible to recurrent mechanical stress induced by muscle activity, which is consistent with the observation that microtubule network was disorganized under electron microscopy. Our work provides insights into how microtubule-dynein interaction instructs synaptic vesicle targeting and the importance of microtubule in the maintenance of neuronal structures against constant mechanical stress.

Show MeSH
Related in: MedlinePlus