| Annotated protein: | Protein VAC14 homolog. Gene symbol: VAC14. Taxonomy: Mus musculus (Mouse). Uniprot ID: Q80WQ2 |
| antibody wiki: | |
| SynGO gene info: | SynGO data @ VAC14 |
| Ontology domain: | Biological Process |
| SynGO term: | regulation of postsynaptic neurotransmitter receptor endocytosis (GO:0099149) |
| Synapse type(s): | hippocampus |
| Annotated paper: | Zhang Y, et al. "Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P(2) and PI(5)P" EMBO J. 2012 Aug 15;31(16):3442-56 PMID:22842785 |
| Figure(s): | Fig 3, 6, 7, 8, S8 and S9 |
| Annotation description: | Fig 3A-B: mEPSCs from Vac14-/- neurons displayed a significant increase (24±6%) in amplitude relative to wild-type mEPSCs. Fig S8: Western Blot analysis shows that the total GluA2 between wild-type and Vac14-/- neurons was similar. To test if the level of GluA2 at the cell surface was different, intact cultured neurons were incubated with an antibody against an extracellular epitope of GluA2 (Mouse IgG2a, MAB397; Chemicon), followed by fixation and incubation with a fluorescent secondary antibody under non-permeabilizing conditions. Surface GluA2 puncta were quantified using immunofluorescence microscopy. Fig6A-B: In Vac14-/- neurons, the average and median surface GluA2 intensities were 34 and 17% higher, respectively, relative to wild-type neurons. Fig 6C-D: The ratio of surface to total GluA2 was increased in Vac14-/- dendrites, relative to wild type. Fig7A: To measure the rate of endocytosis of AMPA receptors, live labelling of surface GluA2 with anti-GluA2 antibody and stimulated endocytosis via the addition of NMDA was performed. After 10 min, surface-bound GluA2 antibodies were stripped by a brief wash in low pH solution, such that only internalized GluA2 antibodies were detected after fixation and permeabilization. Leupeptin was present throughout to prevent lysosomal degradation. Fig7B-E: The number of internalized GluA2 puncta was decreased by 30% in dendrites in Vac14-/- neurons. Total levels of internalized GluA2 puncta in the soma and dendrites were reduced to 71 and 56%, respectively, compared to wild type. Fig S9: To determine whether internalized GluA2 still enters the degradation pathway in Vac14-/- neurons, the proportion of internalized GluA2 puncta that colocalized with the late endosomal and lysosomal marker LAMP1 was measured. Though fewer internalized GluA2 puncta were observed in Vac14-/- neurons, a similar proportion exhibited colocalization with LAMP1 (27% in wild type versus 26% in Vac14-/-) suggesting that the transport of AMPA receptors late in the endocytic pathway is normal in Vac14-/- neurons. Fig 8A-E:To test whether the reduction in internalized puncta was due to enhanced recycling back to the surface, rate of internalization and recycling of pHluorin-GluA1 following NMDA stimulation were measured. The magnitude of NMDA-dependent internalization of GluA1 was diminished in Vac14-/- neurons suggesting reduced receptor endocytosis in these neurons. Fig 8F: To measure the rate of recycling, the time point after NMDA stimulation at which fluorescence intensity recovered to 50% of the pre-NMDA baseline was calculated. Whereas GluA1 internalization was reduced in Vac14-/- neurons, no difference was observed in the rate of recycling relative to wild type suggesting that the initial steps in AMPA receptor endocytosis, rather than post-endocytic sorting, represent the most prominent trafficking defect accompanying loss of VAC14. |
| Evidence tracking, Biological System: | Cultured neurons |
| Evidence tracking, Protein Targeting: | Genetic transformation (eg; knockout, knockin, mutations) Antibody (detection) |
| Evidence tracking, Experiment Assay: | Confocal Whole-cell patch clamp |
| Annotator(s): | Dnyanada Sahasrabudhe (ORCID:0000-0003-2916-7616) Guus Smit (ORCID:0000-0002-2286-1587) Matthijs Verhage (ORCID:0000-0002-2514-0216) |
| Lab: | Department of Functional Genomics, Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands |
| SynGO annotation ID: | 4106 |
| Dataset release (version): | 20231201 |
| View annotation as GO-CAM model: |  |