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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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Negative charges of the EEGE cluster in the MEC-12 H12 helix instruct synaptic vesicle targeting.(A) Effects of G416 substitution on SV targeting in the PLM neuron. Expression of respective MEC-12 variants in the mec-12(e1607) was indicated by the cell-autonomous marker Pdpy-30::NLS::dsRed. SVs were labeled by jsIs821(Pmec-7::GFP::RAB-3). Arrows, mistargeted SVs. Asterisks, PLM soma. Scale bar  = 5 µm. (B) Quantification of SV mistargeting in animals expressing MEC-12 variants. Array loss, transgenic animals that lost the MEC-12-expressing array from the PLM. No array, non-transgenic animals. Scale bars  = 5 µm.
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pgen-1004715-g007: Negative charges of the EEGE cluster in the MEC-12 H12 helix instruct synaptic vesicle targeting.(A) Effects of G416 substitution on SV targeting in the PLM neuron. Expression of respective MEC-12 variants in the mec-12(e1607) was indicated by the cell-autonomous marker Pdpy-30::NLS::dsRed. SVs were labeled by jsIs821(Pmec-7::GFP::RAB-3). Arrows, mistargeted SVs. Asterisks, PLM soma. Scale bar  = 5 µm. (B) Quantification of SV mistargeting in animals expressing MEC-12 variants. Array loss, transgenic animals that lost the MEC-12-expressing array from the PLM. No array, non-transgenic animals. Scale bars  = 5 µm.

Mentions: The aforementioned data indicate that G416 of MEC-12 plays a critical role in determining the relative affinity of microtubules for dynein. To further decipher the mechanisms that instruct microtubule-dynein affinity, we systemically replaced G416 with acidic (aspartic acid/D) or basic (lysine/K, arginine/R) residues, as well as alanine (A) and glutamine (Q), the latter being similar to glutamic acid in side chain length but did not carry charges (Figure 7A, 7B). These MEC-12 species were expressed in the touch neurons of the mec-12(e1607) mutant. SV mistargeting was seen only with the expression MEC-12(G416D), but not other G416 substitutions (Figure 7A, 7B). These results suggest that SV mistargeting in the gm379 mutant was caused by the increased negative charges at the EEGE cluster of MEC-12.


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)

Negative charges of the EEGE cluster in the MEC-12 H12 helix instruct synaptic vesicle targeting.(A) Effects of G416 substitution on SV targeting in the PLM neuron. Expression of respective MEC-12 variants in the mec-12(e1607) was indicated by the cell-autonomous marker Pdpy-30::NLS::dsRed. SVs were labeled by jsIs821(Pmec-7::GFP::RAB-3). Arrows, mistargeted SVs. Asterisks, PLM soma. Scale bar  = 5 µm. (B) Quantification of SV mistargeting in animals expressing MEC-12 variants. Array loss, transgenic animals that lost the MEC-12-expressing array from the PLM. No array, non-transgenic animals. Scale bars  = 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004715-g007: Negative charges of the EEGE cluster in the MEC-12 H12 helix instruct synaptic vesicle targeting.(A) Effects of G416 substitution on SV targeting in the PLM neuron. Expression of respective MEC-12 variants in the mec-12(e1607) was indicated by the cell-autonomous marker Pdpy-30::NLS::dsRed. SVs were labeled by jsIs821(Pmec-7::GFP::RAB-3). Arrows, mistargeted SVs. Asterisks, PLM soma. Scale bar  = 5 µm. (B) Quantification of SV mistargeting in animals expressing MEC-12 variants. Array loss, transgenic animals that lost the MEC-12-expressing array from the PLM. No array, non-transgenic animals. Scale bars  = 5 µm.
Mentions: The aforementioned data indicate that G416 of MEC-12 plays a critical role in determining the relative affinity of microtubules for dynein. To further decipher the mechanisms that instruct microtubule-dynein affinity, we systemically replaced G416 with acidic (aspartic acid/D) or basic (lysine/K, arginine/R) residues, as well as alanine (A) and glutamine (Q), the latter being similar to glutamic acid in side chain length but did not carry charges (Figure 7A, 7B). These MEC-12 species were expressed in the touch neurons of the mec-12(e1607) mutant. SV mistargeting was seen only with the expression MEC-12(G416D), but not other G416 substitutions (Figure 7A, 7B). These results suggest that SV mistargeting in the gm379 mutant was caused by the increased negative charges at the EEGE cluster of MEC-12.

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