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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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Related in: MedlinePlus

SV mistargeting in the mec-12(gm379) mutant depends on dynein activity.(A) SV distribution in the PLM posterior process (upper panels) and the anterior PLM process (lower panels) visualized by jsIs821(Pmec-7::GFP::RAB-3). Anterior is to the left. Normal and mistargeted SVs were labeled by arrowheads and arrows, respectively. Asterisks, PLM soma. (B-C) Quantification of SV mistargeting in the mec-12(gm379); twnEx42[Pmec-7::dhc-1(sense/antisense)] (B) or in the unc-104; mec-12(gm379); twnEx42 (C) animals. yes, animals with the array expressed in the PLM; loss, transgenic animals with the array lost from the PLM; no, non-transgenic animals. (D) Quantification (mean ± S.E.M.) of SV density upon loss of DHC-1 functions in the mec-12(gm379) mutant. (E) Microtubule sedimentation assay. Microtubules were sedimented from animals overexpressing wild-type MEC-12 or MEC-12(G416E), and probed with antibodies against UNC-104 or DHC-1. Immunoreactivity for 6-11B-1 confirmed the presence of MEC-12 in the samples. Actin was present in the supernatant but not in the sediment, suggesting that the sedimentation procedure was highly successful. n = 3 for each experiment. Quantification (mean ± S.E.M.) and normalization of band intensity were described in Materials and Methods. (F) Epifluorescence images showing the recruitment of GFP::DHC-1 (arrowheads) to the PLM posterior process of the mec-12(gm379) but not the wild-type animals. Boxed regions were highlighted on the right. Axons were marked by jsIs973(Pmec-7::mRFP). Asterisks, PLM soma. Scale bars  = 5 µm. ***, p<0.0001, Mann-Whitney U test.
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pgen-1004715-g006: SV mistargeting in the mec-12(gm379) mutant depends on dynein activity.(A) SV distribution in the PLM posterior process (upper panels) and the anterior PLM process (lower panels) visualized by jsIs821(Pmec-7::GFP::RAB-3). Anterior is to the left. Normal and mistargeted SVs were labeled by arrowheads and arrows, respectively. Asterisks, PLM soma. (B-C) Quantification of SV mistargeting in the mec-12(gm379); twnEx42[Pmec-7::dhc-1(sense/antisense)] (B) or in the unc-104; mec-12(gm379); twnEx42 (C) animals. yes, animals with the array expressed in the PLM; loss, transgenic animals with the array lost from the PLM; no, non-transgenic animals. (D) Quantification (mean ± S.E.M.) of SV density upon loss of DHC-1 functions in the mec-12(gm379) mutant. (E) Microtubule sedimentation assay. Microtubules were sedimented from animals overexpressing wild-type MEC-12 or MEC-12(G416E), and probed with antibodies against UNC-104 or DHC-1. Immunoreactivity for 6-11B-1 confirmed the presence of MEC-12 in the samples. Actin was present in the supernatant but not in the sediment, suggesting that the sedimentation procedure was highly successful. n = 3 for each experiment. Quantification (mean ± S.E.M.) and normalization of band intensity were described in Materials and Methods. (F) Epifluorescence images showing the recruitment of GFP::DHC-1 (arrowheads) to the PLM posterior process of the mec-12(gm379) but not the wild-type animals. Boxed regions were highlighted on the right. Axons were marked by jsIs973(Pmec-7::mRFP). Asterisks, PLM soma. Scale bars  = 5 µm. ***, p<0.0001, Mann-Whitney U test.

Mentions: We wondered whether increased activity of the minus end motor dynein is responsible for SV targeting to the PLM posterior process in the mutant, based on the presence of minus end-out microtubules in the PLM posterior process and the unc-104 effects. dhc-1 encodes the heavy chain for cytoplasmic dynein in C. elegans[30]. If enhanced dynein activity is responsible for SV mistargeting in the mutant, elimination of dynein function should suppress it. We could observe SV mistargeted to the PLM posterior process as early as 2-3 fold embryos, before the animal hatched. With the available dhc-1 mutant alleles, it was not possible to lose DHC-1 functions at such early stages without compromising animals' viability. To eliminate DHC-1 functions as early as possible, and to circumvent lethality due to widespread DHC-1 loss, we specifically knocked down dhc-1 in the touch neurons, but not in other somatic tissues, by simultaneously expressing sense and antisense dhc-1 from the mec-7 promoter, which we named transgenic dhc-1 RNAi. Strikingly, transgenic dhc-1 RNAi significantly suppressed SV mistargeting of the mec-12(gm379) mutant, with about one third of the transgenic animals completely devoid of mistargeted SVs (Figure 6A, 6B). This result was confirmed by another independently generated dhc-1 RNAi array (Figure S8A). Transgenic dhc-1 RNAi also significantly reduced SV mistargeting in the unc-104; mec-12(gm379) mutant (Figure 6C). In the wild type, transgenic dhc-1 RNAi had little effects on the intensity of GFP::RAB-3 or SNB-1::GFP in the PLM soma or synapses (Figure S8B). These data indicate that SV mistargeting in the mec-12(gm379) mutant is mediated by the dynein motor. The neurite swelling phenotypes of the mutant, by contrast, were not changed by dhc-1 RNAi, suggesting that SV mistargeting and neurite defects are mechanistically distinct.


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)

SV mistargeting in the mec-12(gm379) mutant depends on dynein activity.(A) SV distribution in the PLM posterior process (upper panels) and the anterior PLM process (lower panels) visualized by jsIs821(Pmec-7::GFP::RAB-3). Anterior is to the left. Normal and mistargeted SVs were labeled by arrowheads and arrows, respectively. Asterisks, PLM soma. (B-C) Quantification of SV mistargeting in the mec-12(gm379); twnEx42[Pmec-7::dhc-1(sense/antisense)] (B) or in the unc-104; mec-12(gm379); twnEx42 (C) animals. yes, animals with the array expressed in the PLM; loss, transgenic animals with the array lost from the PLM; no, non-transgenic animals. (D) Quantification (mean ± S.E.M.) of SV density upon loss of DHC-1 functions in the mec-12(gm379) mutant. (E) Microtubule sedimentation assay. Microtubules were sedimented from animals overexpressing wild-type MEC-12 or MEC-12(G416E), and probed with antibodies against UNC-104 or DHC-1. Immunoreactivity for 6-11B-1 confirmed the presence of MEC-12 in the samples. Actin was present in the supernatant but not in the sediment, suggesting that the sedimentation procedure was highly successful. n = 3 for each experiment. Quantification (mean ± S.E.M.) and normalization of band intensity were described in Materials and Methods. (F) Epifluorescence images showing the recruitment of GFP::DHC-1 (arrowheads) to the PLM posterior process of the mec-12(gm379) but not the wild-type animals. Boxed regions were highlighted on the right. Axons were marked by jsIs973(Pmec-7::mRFP). Asterisks, PLM soma. Scale bars  = 5 µm. ***, p<0.0001, Mann-Whitney U test.
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Related In: Results  -  Collection

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

pgen-1004715-g006: SV mistargeting in the mec-12(gm379) mutant depends on dynein activity.(A) SV distribution in the PLM posterior process (upper panels) and the anterior PLM process (lower panels) visualized by jsIs821(Pmec-7::GFP::RAB-3). Anterior is to the left. Normal and mistargeted SVs were labeled by arrowheads and arrows, respectively. Asterisks, PLM soma. (B-C) Quantification of SV mistargeting in the mec-12(gm379); twnEx42[Pmec-7::dhc-1(sense/antisense)] (B) or in the unc-104; mec-12(gm379); twnEx42 (C) animals. yes, animals with the array expressed in the PLM; loss, transgenic animals with the array lost from the PLM; no, non-transgenic animals. (D) Quantification (mean ± S.E.M.) of SV density upon loss of DHC-1 functions in the mec-12(gm379) mutant. (E) Microtubule sedimentation assay. Microtubules were sedimented from animals overexpressing wild-type MEC-12 or MEC-12(G416E), and probed with antibodies against UNC-104 or DHC-1. Immunoreactivity for 6-11B-1 confirmed the presence of MEC-12 in the samples. Actin was present in the supernatant but not in the sediment, suggesting that the sedimentation procedure was highly successful. n = 3 for each experiment. Quantification (mean ± S.E.M.) and normalization of band intensity were described in Materials and Methods. (F) Epifluorescence images showing the recruitment of GFP::DHC-1 (arrowheads) to the PLM posterior process of the mec-12(gm379) but not the wild-type animals. Boxed regions were highlighted on the right. Axons were marked by jsIs973(Pmec-7::mRFP). Asterisks, PLM soma. Scale bars  = 5 µm. ***, p<0.0001, Mann-Whitney U test.
Mentions: We wondered whether increased activity of the minus end motor dynein is responsible for SV targeting to the PLM posterior process in the mutant, based on the presence of minus end-out microtubules in the PLM posterior process and the unc-104 effects. dhc-1 encodes the heavy chain for cytoplasmic dynein in C. elegans[30]. If enhanced dynein activity is responsible for SV mistargeting in the mutant, elimination of dynein function should suppress it. We could observe SV mistargeted to the PLM posterior process as early as 2-3 fold embryos, before the animal hatched. With the available dhc-1 mutant alleles, it was not possible to lose DHC-1 functions at such early stages without compromising animals' viability. To eliminate DHC-1 functions as early as possible, and to circumvent lethality due to widespread DHC-1 loss, we specifically knocked down dhc-1 in the touch neurons, but not in other somatic tissues, by simultaneously expressing sense and antisense dhc-1 from the mec-7 promoter, which we named transgenic dhc-1 RNAi. Strikingly, transgenic dhc-1 RNAi significantly suppressed SV mistargeting of the mec-12(gm379) mutant, with about one third of the transgenic animals completely devoid of mistargeted SVs (Figure 6A, 6B). This result was confirmed by another independently generated dhc-1 RNAi array (Figure S8A). Transgenic dhc-1 RNAi also significantly reduced SV mistargeting in the unc-104; mec-12(gm379) mutant (Figure 6C). In the wild type, transgenic dhc-1 RNAi had little effects on the intensity of GFP::RAB-3 or SNB-1::GFP in the PLM soma or synapses (Figure S8B). These data indicate that SV mistargeting in the mec-12(gm379) mutant is mediated by the dynein motor. The neurite swelling phenotypes of the mutant, by contrast, were not changed by dhc-1 RNAi, suggesting that SV mistargeting and neurite defects are mechanistically distinct.

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