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Direct fluorescent labeling of NF186 and NaV1.6 in living primary neurons using bioorthogonal click chemistry: tools and resources

The axon initial segment (AIS) is a highly specialized neuronal compartment that regulates the generation of action potentials and maintenance of neuronal polarity. Live imaging of the AIS is challenging due to the limited number of suitable labeling methods. To overcome this limitation, we establis...

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Hauptverfasser: Stajković, Nevena (VerfasserIn) , Liu, Yuanyuan (VerfasserIn) , Arsić, Aleksandra (VerfasserIn) , Meng, Ning (VerfasserIn) , Lyu, Hang (VerfasserIn) , Zhang, Nan (VerfasserIn) , Grimm, Dirk (VerfasserIn) , Lerche, Holger (VerfasserIn) , Nikić-Spiegel, Ivana (VerfasserIn)
Dokumenttyp: Article (Journal)
Sprache:Englisch
Veröffentlicht: June 2023
In: Journal of cell science
Year: 2023, Jahrgang: 136, Heft: 12, Pages: 1-23
ISSN:1477-9137
DOI:10.1242/jcs.260600
Online-Zugang:Verlag, kostenfrei, Volltext: https://doi.org/10.1242/jcs.260600
Verlag, kostenfrei, Volltext: https://journals.biologists.com/jcs/article/136/12/jcs260600/320411
Volltext
Verfasserangaben:Nevena Stajković, Yuanyuan Liu, Aleksandra Arsić, Ning Meng, Hang Lyu, Nan Zhang, Dirk Grimm, Holger Lerche and Ivana Nikić-Spiegel
Beschreibung
Zusammenfassung:The axon initial segment (AIS) is a highly specialized neuronal compartment that regulates the generation of action potentials and maintenance of neuronal polarity. Live imaging of the AIS is challenging due to the limited number of suitable labeling methods. To overcome this limitation, we established a novel approach for live labeling of the AIS using unnatural amino acids (UAAs) and click chemistry. The small size of UAAs and the possibility of introducing them virtually anywhere into target proteins make this method particularly suitable for labeling of complex and spatially restricted proteins. Using this approach, we labeled two large AIS components, the 186 kDa isoform of neurofascin (NF186; encoded by Nfasc) and the 260 kDa voltage-gated Na+ channel (NaV1.6, encoded by Scn8a) in primary neurons and performed conventional and super-resolution microscopy. We also studied the localization of epilepsy-causing NaV1.6 variants with a loss-of-function effect. Finally, to improve the efficiency of UAA incorporation, we developed adeno-associated viral (AAV) vectors for click labeling in neurons, an achievement that could be transferred to more complex systems such as organotypic slice cultures, organoids, and animal models.
Beschreibung:Im Titel ist "V" bei NaV1.6 tiefgestellt
Online veröffentlicht: 28. Juni 2023
Gesehen am 14.11.2023
Beschreibung:Online Resource
ISSN:1477-9137
DOI:10.1242/jcs.260600

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