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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.

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Genetic interaction between mec-12(gm379) and unc-104.(A-D) unc-104 mutations aggravated SV mistargeting in the mec-12(gm379) mutant. GFP is jsIs821(Pmec-7::GFP::RAB-3). (A) Epifluorescence images of the PLM cell body and the posterior process. Anterior is to the left. Asterisks, PLM cell body. Percentage of animals with SV mistargeting (B), the abundance of mistargeted SV (C) and the distribution of mistargeted SV (D), which was the most distal GFP::RAB-3 distribution as a percentage of the total length of the PLM posterior process. Quantifications are mean ± S.E.M. (E-H) UNC-104 overexpression rescued SV mistargeting in the mec-12(gm379) mutant. Epifluorescence images of the PLM with the same parameters as those in (A). With excess UNC-104, SVs accumulated in the PLM soma (F) or mistargeted to the PLM posterior process (G) were decreased, and they were able to enter the anterior PLM process again (H). Quantifications are mean ± S.E.M. Scale bars  = 5 µm. *, p<0.05; **, p<0.001; ***, p<0.0001, Mann-Whitney U test.
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pgen-1004715-g005: Genetic interaction between mec-12(gm379) and unc-104.(A-D) unc-104 mutations aggravated SV mistargeting in the mec-12(gm379) mutant. GFP is jsIs821(Pmec-7::GFP::RAB-3). (A) Epifluorescence images of the PLM cell body and the posterior process. Anterior is to the left. Asterisks, PLM cell body. Percentage of animals with SV mistargeting (B), the abundance of mistargeted SV (C) and the distribution of mistargeted SV (D), which was the most distal GFP::RAB-3 distribution as a percentage of the total length of the PLM posterior process. Quantifications are mean ± S.E.M. (E-H) UNC-104 overexpression rescued SV mistargeting in the mec-12(gm379) mutant. Epifluorescence images of the PLM with the same parameters as those in (A). With excess UNC-104, SVs accumulated in the PLM soma (F) or mistargeted to the PLM posterior process (G) were decreased, and they were able to enter the anterior PLM process again (H). Quantifications are mean ± S.E.M. Scale bars  = 5 µm. *, p<0.05; **, p<0.001; ***, p<0.0001, Mann-Whitney U test.

Mentions: It is possible that the interaction between KIF1A and microtubule was altered by the G416E mutation. We found that the strong loss-of-function unc-104(rh43)/KIF1A mutation caused completely penetrant SV transport defects and, surprisingly, low percentage of SV mistargeting in the PLM (Figure 5A and 5B). Moreover, this unc-104 mutation enhanced SV mistargeting of mec-12(gm379) rather than suppressing the phenotype, with more SVs mistargeted to the PLM posterior process and distributed more distally (Figure 5C, 5D). This result suggests that SV mistargeting in the mec-12(gm379) mutant is not caused by aberrant UNC-104 activity. Furthermore, overexpression of UNC-104 significantly rescued SV transport defects and mistargeting in the mec-12(gm379) mutant, with more SVs reaching synapses in the nerve ring or entering the anterior ALM and PLM processes (Figure 5E-5H, Figure S7A, S7B). While these data are consistent with the interpretation that UNC-104 activity was reduced in the mec-12(gm379) mutant, resulting in severe SV transport defects, they also indicate that SV mistargeting in the mutant requires the activity of an unknown molecule.


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 interaction between mec-12(gm379) and unc-104.(A-D) unc-104 mutations aggravated SV mistargeting in the mec-12(gm379) mutant. GFP is jsIs821(Pmec-7::GFP::RAB-3). (A) Epifluorescence images of the PLM cell body and the posterior process. Anterior is to the left. Asterisks, PLM cell body. Percentage of animals with SV mistargeting (B), the abundance of mistargeted SV (C) and the distribution of mistargeted SV (D), which was the most distal GFP::RAB-3 distribution as a percentage of the total length of the PLM posterior process. Quantifications are mean ± S.E.M. (E-H) UNC-104 overexpression rescued SV mistargeting in the mec-12(gm379) mutant. Epifluorescence images of the PLM with the same parameters as those in (A). With excess UNC-104, SVs accumulated in the PLM soma (F) or mistargeted to the PLM posterior process (G) were decreased, and they were able to enter the anterior PLM process again (H). Quantifications are mean ± S.E.M. Scale bars  = 5 µm. *, p<0.05; **, p<0.001; ***, p<0.0001, Mann-Whitney U test.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004715-g005: Genetic interaction between mec-12(gm379) and unc-104.(A-D) unc-104 mutations aggravated SV mistargeting in the mec-12(gm379) mutant. GFP is jsIs821(Pmec-7::GFP::RAB-3). (A) Epifluorescence images of the PLM cell body and the posterior process. Anterior is to the left. Asterisks, PLM cell body. Percentage of animals with SV mistargeting (B), the abundance of mistargeted SV (C) and the distribution of mistargeted SV (D), which was the most distal GFP::RAB-3 distribution as a percentage of the total length of the PLM posterior process. Quantifications are mean ± S.E.M. (E-H) UNC-104 overexpression rescued SV mistargeting in the mec-12(gm379) mutant. Epifluorescence images of the PLM with the same parameters as those in (A). With excess UNC-104, SVs accumulated in the PLM soma (F) or mistargeted to the PLM posterior process (G) were decreased, and they were able to enter the anterior PLM process again (H). Quantifications are mean ± S.E.M. Scale bars  = 5 µm. *, p<0.05; **, p<0.001; ***, p<0.0001, Mann-Whitney U test.
Mentions: It is possible that the interaction between KIF1A and microtubule was altered by the G416E mutation. We found that the strong loss-of-function unc-104(rh43)/KIF1A mutation caused completely penetrant SV transport defects and, surprisingly, low percentage of SV mistargeting in the PLM (Figure 5A and 5B). Moreover, this unc-104 mutation enhanced SV mistargeting of mec-12(gm379) rather than suppressing the phenotype, with more SVs mistargeted to the PLM posterior process and distributed more distally (Figure 5C, 5D). This result suggests that SV mistargeting in the mec-12(gm379) mutant is not caused by aberrant UNC-104 activity. Furthermore, overexpression of UNC-104 significantly rescued SV transport defects and mistargeting in the mec-12(gm379) mutant, with more SVs reaching synapses in the nerve ring or entering the anterior ALM and PLM processes (Figure 5E-5H, Figure S7A, S7B). While these data are consistent with the interpretation that UNC-104 activity was reduced in the mec-12(gm379) mutant, resulting in severe SV transport defects, they also indicate that SV mistargeting in the mutant requires the activity of an unknown molecule.

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