Display Settings:

Items per page

Results: 8

1.
Figure 1

Figure 1. From: Hypoxia regulates glutamate receptor trafficking through an HIF-independent mechanism.

Oxygen levels and EGL-9 regulate GLR-1 trafficking. GLR-1∷GFP fluorescence in (A) wild-type animals under normoxia, (B) egl-9(sa307) mutants under normoxia, (C) wild-type animals under hypoxia, and (D) egl-9(sa307) mutants under hypoxia. GLR-1 is localized to elongated accumulations (arrows), quantified per length of ventral cord dendrites in (E, F). Red bars indicate normoxia, whereas blue bars indicate hypoxia. ANOVA followed by Dunnett's multiple comparison with wild type, normoxia (*P<0.01). N=15–35 animals per condition and/or genotype. Error bars indicate s.e.m. Bar, 5 μm.

Eun Chan Park, et al. EMBO J. 2012 March 21;31(6):1379-1393.
2.
Figure 6

Figure 6. From: Hypoxia regulates glutamate receptor trafficking through an HIF-independent mechanism.

CDK-5 is required for oxygen and EGL-9 to regulate GLR-1 trafficking. GLR-1∷GFP ventral cord fluorescence for (A, B) wild-type, (C, D) cdk-5(ok626), (E, F) egl-9 cdk-5 double mutants, (G, H) wild type overexpressing wild-type CDK-5, and (I, J) egl-9(sa307) overexpressing wild-type CDK-5 under conditions of (A, C, E, G, I) normoxia or (B, D, F, H, J) hypoxia. Arrows indicate accumulations of GLR-1∷GFP in internalized compartments. (K) Quantification of the number of GLR-1∷GFP-containing elongated compartments in the indicated genotypes. (L) The mean spontaneous reversal frequency and (M) the mean nose touch mechanosensory response are plotted for the indicated genotypes. Red bars indicate normoxia, whereas blue bars indicate hypoxia. *P<0.01 compared with wild type, normoxia, and #P<0.01 compared with wild type, hypoxia, by ANOVA followed by Dunnett's multiple comparison. N=15–35 animals per condition and/or genotype. Error bars indicate s.e.m. Bar, 5 μm.

Eun Chan Park, et al. EMBO J. 2012 March 21;31(6):1379-1393.
3.
Figure 5

Figure 5. From: Hypoxia regulates glutamate receptor trafficking through an HIF-independent mechanism.

EGL-9 and CDK-5 regulate LIN-10 subcellular localization. LIN-10∷GFP ventral cord fluorescence for (A) wild type, normoxia; (B) wild type, hypoxia; (C) cdk-5(ok626), normoxia; (D) cdk-5(ok626), hypoxia; (E) egl-9(sa307), normoxia; and (F) egl-9 cdk-5 double mutants, normoxia. Quantification of LIN-10∷GFP (G) puncta number per length of ventral cord and (H) integrated optical density (IOD) per puncta per animal. IOD is the sum of the pixel values for each puncta, reflecting both puncta size and fluorescence intensity. Red bars indicate normoxia, whereas blue bars indicate hypoxia. ANOVA followed by Dunnett's multiple comparison with wild type, normoxia (*P<0.01). (I) Representative western blot detecting LIN-10∷GFP using anti-GFP antibodies (top panel) or actin using anti-actin antibodies (bottom panel) from nematode lysates of the given genotype. With the exception of the first lane, all nematodes carried the odIs22[Pglr-1∷LIN-10∷GFP] transgene. (J) Quantification of LIN-10∷GFP protein levels normalized to actin controls from the same blot. Means represent five independent blots. N=15–35 animals per condition and/or genotype. Error bars indicate s.e.m. Bar, 5 μm.

Eun Chan Park, et al. EMBO J. 2012 March 21;31(6):1379-1393.
4.
Figure 7

Figure 7. From: Hypoxia regulates glutamate receptor trafficking through an HIF-independent mechanism.

Proline-rich CDK-5 consensus sequences regulate LIN-10 localization. (A) Phos-tag western blot detecting in-vitro translated phosphorylated LIN-10 protein (upper bands) or unphosphorylated LIN-10 protein (lower band). Specific substrates (the first 574 amino acids of wild-type LIN-10 or LIN-10(Pmut), which contains proline to alanine substitutions in the CDK-5 consensus sequences) after incubation with CDK-5 kinase (indicated by ‘+') or buffer alone (indicated by ‘−'). Ventral cord fluorescence for (B, C) wild-type LIN-10∷GFP, (D, E) LIN-10(Pmut)∷GFP, with the proline mutations indicated above, or (F, G) LIN-10(S/Tmut)∷GFP, with the serine or threonine substituted to alanine in the CDK-5 consensus sequences. (H) Quantification of LIN-10∷GFP or LIN-10∷GFP(Pmut)∷GFP integrated optical density (IOD) per puncta for the indicated genotypes. IOD is the sum of the pixel values for each puncta, reflecting both puncta size and fluorescence intensity. Red bars indicate normoxia, whereas blue bars indicate hypoxia. ANOVA followed by Dunnett's multiple comparison with wild type, normoxia (*P<0.01). (I) Quantification of GLR-1∷GFP-containing compartments in the indicated genotypes, including lin-10 mutants expressing an untagged version of LIN-10(Pmut). N=15–30 animals per condition and/or genotype. Error bars indicate s.e.m. Bar, 5 μm.

Eun Chan Park, et al. EMBO J. 2012 March 21;31(6):1379-1393.
5.
Figure 8

Figure 8. From: Hypoxia regulates glutamate receptor trafficking through an HIF-independent mechanism.

Model for oxygen and EGL-9 regulation of GLR-1 trafficking. (A) Under normal oxygen, EGL-9 (red trapezoid) regulates two targets: HIF-1 (blue ellipse) and LIN-10 (yellow rectangle). EGL-9A transiently associates with HIF-1, hydroxylating key HIF-1 prolines (indicated by ‘P' and ‘P-OH') on it, resulting in HIF-1 ubiquitination by VHL-1 and turnover. By contrast, EGL-9E binds to LIN-10 and drives its localization to synaptic sites and endosomes, where it promotes GLR-1 (grey channel) recycling (indicated by the two large, red arrows). We speculate that EGL-9E might also hydroxylate key prolines (CDK-5 phosphorylation sites; indicated by ‘SP' and ‘SP-OH') on LIN-10, or otherwise obscure CDK-5 access to these sites. (B) Under hypoxic conditions, both EGL-9 isoforms are inactive due to lack of oxygen. This allows HIF-1 to enter the nucleus and regulate target gene expression, although this does not impact GLR-1 localization. In addition, LIN-10 is phosphorylated by CDK-5 (green rectangle), which prevents the localization of LIN-10 at synaptic sites and endosomes. Without LIN-10 present at synaptic sites, GLR-1 recycling is depressed, tipping the balance towards internalization of GLR-1 receptors (indicated by the large blue arrow), diminished GLR-1 activity, and a change in nematode foraging behaviour. As hypoxia can trigger massive synaptic vesicle exocytosis (indicated by multiple fused synaptic vesicles at the presynaptic terminus), we also speculate that this mechanism could help protect neurons from excitotoxicity.

Eun Chan Park, et al. EMBO J. 2012 March 21;31(6):1379-1393.
6.
Figure 4

Figure 4. From: Hypoxia regulates glutamate receptor trafficking through an HIF-independent mechanism.

EGL-9 interacts with LIN-10 to regulate GLR-1 trafficking. (A) Schematic of LIN-10 protein. Coloured boxes indicate the CID, PTB, PDZ1, and PDZ2 domains. Vertical lines indicate putative CDK-5 phosphorylation sites in the amino-terminus, with the number corresponding to the proline residue in the CDK-5 consensus sequence. Horizontal lines indicate the fragments of LIN-10 protein tested for interaction with the EGL-9 catalytic domain via yeast two-hybrid. ‘+++' Indicates a strong interaction (positive for all four reporters), whereas ‘+' indicates a weak interaction (positive for 1–3 reporters). GLR-1∷GFP fluorescence in (B) wild type, normoxia; (C) lin-10(e1439), normoxia; (D) wild type, hypoxia; and (E) lin-10(e1439), hypoxia. Arrows indicate accumulations of GLR-1∷GFP in elongated, internal compartments. (F) Quantification of the number of GLR-1∷GFP-containing elongated compartments in the indicated genotypes. *P<0.01 compared with wild type, normoxia, and #P<0.01 compared with wild type, hypoxia, by ANOVA followed by Dunnett's multiple comparison. (G, H) GFP∷EGL-9E fluorescence and (I, J) LIN-10∷mCherry fluorescence in (G, I, K) a neuron cell body (PVC) and (H, J, L) the ventral cord. (K, L) Merged images. (M) The mean spontaneous reversal frequency and (N) the mean nose touch mechanosensory response are plotted for the indicated genotypes. Red bars indicate normoxia, whereas blue bars indicate hypoxia. ANOVA followed by Dunnett's multiple comparison with wild type, normoxia (*P<0.01) or wild type, hypoxia (#P<0.01). N=20–30 animals per condition and/or genotype. Error bars indicate s.e.m. Bar, 5 μm.

Eun Chan Park, et al. EMBO J. 2012 March 21;31(6):1379-1393.
7.
Figure 3

Figure 3. From: Hypoxia regulates glutamate receptor trafficking through an HIF-independent mechanism.

A specific EGL-9 isoform regulates GLR-1 trafficking. (A) Schematic of egl-9 genomic DNA organization, the three predominant splice isoforms that are produced, and the coding sequences for the ‘catalytic domain' transgene that we generated. Boxes indicate coding sequences within exons. The arrows indicate the start sites of transcription for the three different isoforms. Brackets indicate regions that encode the indicated protein domains, the boxes for which have also been colour coded. The red lines indicate the regions of genomic DNA deleted in gk277 and sa307 mutants. The locations of amino-acid substitutions are indicated for n571 and the H487 residue mutated in the catalytically impaired EGL-9 transgene. GLR-1∷GFP fluorescence in (B) wild-type, (E) egl-9 mutants, and egl-9 mutants expressing the (F) EGL-9A, (G) EGL-9E, and (H) EGL-9E(H487A) isoforms via the glr-1 promoter. Arrows point to accumulations of GLR-1∷GFP in elongated compartments. (C) Quantification of the number of elongated, internal compartments of accumulated GLR-1∷GFP detected in the indicated genotypes. (D) The mean spontaneous reversal frequency as an indication of GLR-1 function is plotted for the indicated genotypes. For (C) and (D), ANOVA followed by Dunnett's multiple comparison with wild type (*P<0.01). (IN) Cell body (left panel) and ventral cord (right panel) fluorescence from animals expressing (I) an EGL-9A∷GFP chimera, (L) an EGL-9E∷GFP chimera, (M) an EGL-9C∷GFP chimera, and (N) a chimera containing GFP fused to just the catalytic domain of EGL-9. (J) Quantification of the number of fluorescent puncta along the ventral cord generated from the indicated transgene. ANOVA followed by Dunnett's multiple comparison with (J) GFP∷EGL-9A (*P<0.01). (K) Quantification of the number of GFP∷EGL-9E fluorescent puncta along the ventral cord under conditions of normoxia (red) and hypoxia (blue). Student's t-test, (***P<0.001). N=15–30 animals per condition and/or genotype. Error bars indicate s.e.m. Bar, 5 μm.

Eun Chan Park, et al. EMBO J. 2012 March 21;31(6):1379-1393.
8.
Figure 2

Figure 2. From: Hypoxia regulates glutamate receptor trafficking through an HIF-independent mechanism.

Oxygen levels and EGL-9 regulate GLR-1 function and locomotion behaviour. (A) The mean spontaneous reversal frequency (relative rate of spontaneous reversals in direction over a 5-min period) and (B) the mean nose touch mechanosensory response (percentage of reversal responses per animal to a train of 10 individual nose touch stimuli) are plotted for the indicated genotypes. Red bars indicate normoxia, whereas blue bars indicate hypoxia. ANOVA followed by Dunnett's multiple comparison with wild type, normoxia (*P<0.01) or wild type, hypoxia (#P<0.01). N=15–35 animals per condition and/or genotype. (C, D) Representative whole-cell recordings from AVA neurons are plotted. Example inward currents induced by glutamate application (bar) over time are plotted for (C) a wild-type neuron and (D) an egl-9(sa307) mutant neuron. (E) Mean peak current amplitudes for wild-type and egl-9(sa307) mutants. Student's t-test (***P<0.001). N=6–9 animals per genotype. (FH) Fluorescence from the ventral nerve cords of HA∷GLR-1∷GFP transgenic animals following injection of anti-HA antibodies (conjugated to Alexa 594) into the extracellular body cavity under non-membrane permeabilizing conditions. Injections were performed on (F) wild-type, (G) egl-9(sa307) mutants, and (H) lin-10(e1439) mutants. (FH) Total GFP fluorescence. (F′, G′, H′) Alexa 594 fluorescence detecting surface HA∷GLR-1∷GFP decorated with anti-HA antibodies. (F″, G″, H″) Merged images. Arrows point to elongated, internal compartments that accumulate HA∷GLR-1∷GFP and that cannot be decorated by anti-HA antibodies in egl-9 and lin-10 mutants. Error bars indicate s.e.m. Bar, 5 μm.

Eun Chan Park, et al. EMBO J. 2012 March 21;31(6):1379-1393.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk