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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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In vitro DHC-1 MTBD sedimentation assay and model of dynein MTBD-tubulin dimer interaction.(A) SDS-PAGE gel image stained with Coomassie blue for microtubule sedimentation using purified wild-type MTBD or MTBD(D3323K) of DHC-1. Lanes from left to right represent serial 2-fold dilution of MTBD loading, with concentrations indicated. LC, loading control. (B) Quantification (mean ± S.E.M.) of MTBD co-sedimented with microtubules. Data were averaged from three independent experiments. (C) The structure of the dynein microtubule-binding domain (MTBD) complexed with the tubulin dimer. Data from Protein Data Bank (PDB#3J1T) were processed by UCSF Chimera software. The interaction surface between the H12 of the α-tubulin and the dynein MTBD was highlighted in the bottom panel. Residues of the α-tubulin were labled in black, and those from the dynein MTBD were labeled in blue. An intramolecular salt bridge was suggested to form between E3378 and R3382 of the dynein MTBD when dynein is at some distance from the tubulin surface. Upon approaching the tubulin dimer, negative charges of the H12 of tubulin dimers were hypothesized to break the MTBD intramolecular salt bridge and foster the interaction between the MTBD and the tubulin dimer [12].
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pgen-1004715-g008: In vitro DHC-1 MTBD sedimentation assay and model of dynein MTBD-tubulin dimer interaction.(A) SDS-PAGE gel image stained with Coomassie blue for microtubule sedimentation using purified wild-type MTBD or MTBD(D3323K) of DHC-1. Lanes from left to right represent serial 2-fold dilution of MTBD loading, with concentrations indicated. LC, loading control. (B) Quantification (mean ± S.E.M.) of MTBD co-sedimented with microtubules. Data were averaged from three independent experiments. (C) The structure of the dynein microtubule-binding domain (MTBD) complexed with the tubulin dimer. Data from Protein Data Bank (PDB#3J1T) were processed by UCSF Chimera software. The interaction surface between the H12 of the α-tubulin and the dynein MTBD was highlighted in the bottom panel. Residues of the α-tubulin were labled in black, and those from the dynein MTBD were labeled in blue. An intramolecular salt bridge was suggested to form between E3378 and R3382 of the dynein MTBD when dynein is at some distance from the tubulin surface. Upon approaching the tubulin dimer, negative charges of the H12 of tubulin dimers were hypothesized to break the MTBD intramolecular salt bridge and foster the interaction between the MTBD and the tubulin dimer [12].

Mentions: Previous structural studies suggest that dynein binds the H12 helix of α-tubulin [10], [12]. Redwine et al. proposed that E3378 and R3382 of the dynein microtubule-binding domain (MTBD) form intramolecular salt bridge, and upon approaching the tubulin dimer, negative charges of the α-tubulin H12 disrupt this salt bridge by attracting R3382, which carries positive charges (Figure 8C). An E3378K mutation of MTBD disrupted this intramolecular salt bridge and increased both the affinity and the run length of dynein on the microtubules [12]. We speculate that the E3378K mutation facilitates the electrostatic interaction between MTBD and the negative charges of the α-tubulin H12 domain. To test this, we expressed and purified a fragment of C. elegans DHC-1 MTBD, and showed that this MTBD precipitated with microtubules synthesized from purified bovine tubulin in the in vitro sedimentation experiment (Figure 8A and S9). Moreover, D3323K mutation, which is equivalent to E3378K mutation of the yeast dynein MTBD, enhanced MTBD-microtubule interaction across a range of tested concentrations (Figure 8A, 8B and S9). This result supports our hypothesis that exaggerated microtubule affinity for dynein critically depends on the electrostatic interaction between the EEGE-containing H12 helix of α-tubulin and the dynein MTBD (Figure 8C).


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)

In vitro DHC-1 MTBD sedimentation assay and model of dynein MTBD-tubulin dimer interaction.(A) SDS-PAGE gel image stained with Coomassie blue for microtubule sedimentation using purified wild-type MTBD or MTBD(D3323K) of DHC-1. Lanes from left to right represent serial 2-fold dilution of MTBD loading, with concentrations indicated. LC, loading control. (B) Quantification (mean ± S.E.M.) of MTBD co-sedimented with microtubules. Data were averaged from three independent experiments. (C) The structure of the dynein microtubule-binding domain (MTBD) complexed with the tubulin dimer. Data from Protein Data Bank (PDB#3J1T) were processed by UCSF Chimera software. The interaction surface between the H12 of the α-tubulin and the dynein MTBD was highlighted in the bottom panel. Residues of the α-tubulin were labled in black, and those from the dynein MTBD were labeled in blue. An intramolecular salt bridge was suggested to form between E3378 and R3382 of the dynein MTBD when dynein is at some distance from the tubulin surface. Upon approaching the tubulin dimer, negative charges of the H12 of tubulin dimers were hypothesized to break the MTBD intramolecular salt bridge and foster the interaction between the MTBD and the tubulin dimer [12].
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230729&req=5

pgen-1004715-g008: In vitro DHC-1 MTBD sedimentation assay and model of dynein MTBD-tubulin dimer interaction.(A) SDS-PAGE gel image stained with Coomassie blue for microtubule sedimentation using purified wild-type MTBD or MTBD(D3323K) of DHC-1. Lanes from left to right represent serial 2-fold dilution of MTBD loading, with concentrations indicated. LC, loading control. (B) Quantification (mean ± S.E.M.) of MTBD co-sedimented with microtubules. Data were averaged from three independent experiments. (C) The structure of the dynein microtubule-binding domain (MTBD) complexed with the tubulin dimer. Data from Protein Data Bank (PDB#3J1T) were processed by UCSF Chimera software. The interaction surface between the H12 of the α-tubulin and the dynein MTBD was highlighted in the bottom panel. Residues of the α-tubulin were labled in black, and those from the dynein MTBD were labeled in blue. An intramolecular salt bridge was suggested to form between E3378 and R3382 of the dynein MTBD when dynein is at some distance from the tubulin surface. Upon approaching the tubulin dimer, negative charges of the H12 of tubulin dimers were hypothesized to break the MTBD intramolecular salt bridge and foster the interaction between the MTBD and the tubulin dimer [12].
Mentions: Previous structural studies suggest that dynein binds the H12 helix of α-tubulin [10], [12]. Redwine et al. proposed that E3378 and R3382 of the dynein microtubule-binding domain (MTBD) form intramolecular salt bridge, and upon approaching the tubulin dimer, negative charges of the α-tubulin H12 disrupt this salt bridge by attracting R3382, which carries positive charges (Figure 8C). An E3378K mutation of MTBD disrupted this intramolecular salt bridge and increased both the affinity and the run length of dynein on the microtubules [12]. We speculate that the E3378K mutation facilitates the electrostatic interaction between MTBD and the negative charges of the α-tubulin H12 domain. To test this, we expressed and purified a fragment of C. elegans DHC-1 MTBD, and showed that this MTBD precipitated with microtubules synthesized from purified bovine tubulin in the in vitro sedimentation experiment (Figure 8A and S9). Moreover, D3323K mutation, which is equivalent to E3378K mutation of the yeast dynein MTBD, enhanced MTBD-microtubule interaction across a range of tested concentrations (Figure 8A, 8B and S9). This result supports our hypothesis that exaggerated microtubule affinity for dynein critically depends on the electrostatic interaction between the EEGE-containing H12 helix of α-tubulin and the dynein MTBD (Figure 8C).

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