Quantitative DNA-PAINT imaging of AMPA receptors in live neurons.
AMPAR
DNA-PAINT
live cell imaging
Journal
Cell reports methods
ISSN: 2667-2375
Titre abrégé: Cell Rep Methods
Pays: United States
ID NLM: 9918227360606676
Informations de publication
Date de publication:
27 02 2023
27 02 2023
Historique:
received:
09
05
2022
revised:
25
09
2022
accepted:
25
01
2023
entrez:
20
3
2023
pubmed:
21
3
2023
medline:
21
3
2023
Statut:
epublish
Résumé
DNA-point accumulation for imaging at nanoscale topography (DNA-PAINT) can image fixed biological specimens with nanometer resolution and absolute stoichiometry. In living systems, however, the usage of DNA-PAINT has been limited due to high salt concentration in the buffer required for specific binding of the imager to the docker attached to the target. Here, we used multiple binding motifs of the docker, from 2 to 16, to accelerate the binding speed of the imager under physiological buffer conditions without compromising spatial resolution and maintaining the basal level homeostasis during the measurement. We imaged endogenous α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) in cultured neurons-critical proteins involved in nerve communication-by DNA-PAINT in 3-dimensions using a monovalent single-chain variable fragment (scFv) to the GluA1 subunit of AMPAR. We found a heterogeneous distribution of synaptic AMPARs: ≈60% are immobile, primarily in nanodomains, defined as AMPARs that are within 0.3 μm of the Homer1 protein in the postsynaptic density; the other ∼40% of AMPARs have restricted mobility and trajectory.
Identifiants
pubmed: 36936077
doi: 10.1016/j.crmeth.2023.100408
pii: S2667-2375(23)00019-X
pmc: PMC10014303
doi:
Substances chimiques
Receptors, AMPA
0
Carrier Proteins
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Pagination
100408Subventions
Organisme : NINDS NIH HHS
ID : R01 NS038631
Pays : United States
Organisme : NINDS NIH HHS
ID : R01 NS100019
Pays : United States
Informations de copyright
© 2023 The Authors.
Déclaration de conflit d'intérêts
The authors declare no competing interests.
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