Počet záznamů: 1
Subunit-Dependent Surface Mobility and Localization of NMDA Receptors in Hippocampal Neurons Measured Using Nanobody Probes
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SYSNO ASEP 0574116 Druh ASEP J - Článek v odborném periodiku Zařazení RIV J - Článek v odborném periodiku Poddruh J Článek ve WOS Název Subunit-Dependent Surface Mobility and Localization of NMDA Receptors in Hippocampal Neurons Measured Using Nanobody Probes Tvůrce(i) Kortus, Štěpán (UEM-P) RID
Řeháková, Kristýna (UEM-P)
Klíma, Martin (UOCHB-X) RID, ORCID
Kolcheva, Marharyta (UEM-P) ORCID
Ladislav, Marek (UEM-P) RID, ORCID
Langore, Emily (UEM-P)
Baráčková, Petra (UEM-P)
Netolický, Jakub (UEM-P)
Misiachna, Anna (UEM-P)
Hemelíková, Katarína (UEM-P)
Humpolíčková, Jana (UOCHB-X) ORCID
Chalupská, Dominika (UOCHB-X) ORCID
Šilhán, Jan (UOCHB-X) ORCID
Kaniaková, Martina (UEM-P)
Hrčka Krausová, Barbora (UEM-P) RID, ORCID
Bouřa, Evžen (UOCHB-X) ORCID
Zápotocký, Martin (FGU-C) RID, ORCID
Horák, Martin (UEM-P) RID, ORCIDZdroj.dok. Journal of Neuroscience. - : Society for Neuroscience - ISSN 0270-6474
Roč. 43, č. 26 (2023), s. 4755-4774Poč.str. 20 s. Jazyk dok. eng - angličtina Země vyd. US - Spojené státy americké Klíč. slova GluN subunit ; excitatory synapse ; glutamate receptor ; lateral diffusion ; live microscopy ; mammalian neuron Obor OECD Neurosciences (including psychophysiology CEP GA20-12420S GA ČR - Grantová agentura ČR LX22NPO5107 GA MŠMT - Ministerstvo školství, mládeže a tělovýchovy Výzkumná infrastruktura Czech-BioImaging III - 90250 - Ústav molekulární genetiky AV ČR, v. v. i.
Czech-BioImaging II - 90129 - Ústav molekulární genetiky AV ČR, v. v. i.Způsob publikování Open access Institucionální podpora UEM-P - RVO:68378041 ; UOCHB-X - RVO:61388963 ; FGU-C - RVO:67985823 UT WOS 001033553700002 EID SCOPUS 85164063271 DOI 10.1523/JNEUROSCI.2014-22.2023 Anotace NMDA receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs are regulated at several levels, including their externalization, internalization, and lateral diffusion between the synaptic and extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either the smallest commercially available quantum dot 525 (QD525) or the several nanometer larger (and thus brighter) QD605 (referred to as nanoGFP-QD525 and nanoGFP-QD605, respectively). Targeting the yellow fluorescent protein-tagged GluN1 subunit in rat hippocampal neurons, we compared these two probes to a previously established larger probe, a rabbit anti-GFP IgG together with a secondary IgG conjugated to QD605 (referred to as antiGFP-QD605). The nanoGFP-based probes allowed faster lateral diffusion of the NMDARs, with several-fold increased median values of the diffusion coefficient (D). Using thresholded tdTomato-Homer1c signals to mark synaptic regions, we found that the nanoprobe-based D values sharply increased at distances over 100 nm from the synaptic edge, while D values for antiGFP-QD605 probe remained unchanged up to a 400 nm distance. Using the nanoGFP-QD605 probe in hippocampal neurons expressing the GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits, we detected subunit-dependent differences in the synaptic localization of NMDARs, D value, synaptic residence time, and synaptic-extrasynaptic exchange rate. Finally, we confirmed the applicability of the nanoGFP-QD605 probe to study differences in the distribution of synaptic NMDARs by comparing to data obtained with nanoGFPs conjugated to organic fluorophores, using universal point accumulation imaging in nanoscale topography and direct stochastic optical reconstruction microscopy.SIGNIFICANCE STATEMENT Our study systematically compared the localization and mobility of surface NMDARs containing GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits expressed in rodent hippocampal neurons, using anti-green fluorescent protein (GFP) nanobodies conjugated to the quantum dot 605 (nanoGFP-QD605), as well as nanoGFP probes conjugated with small organic fluorophores. Our comprehensive analysis showed that the method used to delineate the synaptic region plays an important role in the study of synaptic and extrasynaptic pools of NMDARs. In addition, we showed that the nanoGFP-QD605 probe has optimal parameters for studying the mobility of NMDARs because of its high localization accuracy comparable to direct stochastic optical reconstruction microscopy and longer scan time compared with universal point accumulation imaging in nanoscale topography. The developed approaches are readily applicable to the study of any GFP-labeled membrane receptors expressed in mammalian neurons. Pracoviště Ústav experimentální medicíny Kontakt Lenka Koželská, lenka.kozelska@iem.cas.cz, Tel.: 241 062 218, 296 442 218 Rok sběru 2024 Elektronická adresa https://www.jneurosci.org/content/43/26/4755
Počet záznamů: 1