Neuronal subtype-specific growth cone and soma purification from mammalian CNS via fractionation and fluorescent sorting for subcellular analyses and spatial mapping of local transcriptomes and proteomes.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
02 2022
02 2022
Historique:
received:
29
12
2020
accepted:
24
09
2021
pubmed:
14
1
2022
medline:
24
2
2022
entrez:
13
1
2022
Statut:
ppublish
Résumé
During neuronal development, growth cones (GCs) of projection neurons navigate complex extracellular environments to reach distant targets, thereby generating extraordinarily complex circuitry. These dynamic structures located at the tips of axonal projections respond to substrate-bound as well as diffusible guidance cues in a neuronal subtype- and stage-specific manner to construct highly specific and functional circuitry. In vitro studies of the past decade indicate that subcellular localization of specific molecular machinery in GCs underlies the precise navigational control that occurs during circuit 'wiring'. Our laboratory has recently developed integrated experimental and analytical approaches enabling high-depth, quantitative proteomic and transcriptomic investigation of subtype- and stage-specific GC molecular machinery directly from the rodent central nervous system (CNS) in vivo. By using these approaches, a pure population of GCs and paired somata can be isolated from any neuronal subtype of the CNS that can be fluorescently labeled. GCs are dissociated from parent axons using fluid shear forces, and a bulk GC fraction is isolated by buoyancy ultracentrifugation. Subtype-specific GCs and somata are purified by recently developed fluorescent small particle sorting and established FACS of neurons and are suitable for downstream analyses of proteins and RNAs, including small RNAs. The isolation of subtype-specific GCs and parent somata takes ~3 h, plus sorting time, and ~1-2 h for subsequent extraction of molecular contents. RNA library preparation and sequencing can take several days to weeks, depending on the turnaround time of the core facility involved.
Identifiants
pubmed: 35022617
doi: 10.1038/s41596-021-00638-7
pii: 10.1038/s41596-021-00638-7
pmc: PMC9751848
mid: NIHMS1849342
doi:
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Review
Langues
eng
Sous-ensembles de citation
IM
Pagination
222-251Subventions
Organisme : NINDS NIH HHS
ID : R01 NS049553
Pays : United States
Organisme : NIA NIH HHS
ID : T32 AG000222
Pays : United States
Organisme : NINDS NIH HHS
ID : F31 NS103262
Pays : United States
Organisme : NINDS NIH HHS
ID : R01 NS045523
Pays : United States
Organisme : NINDS NIH HHS
ID : R01 NS104055
Pays : United States
Organisme : NINDS NIH HHS
ID : R01 NS075672
Pays : United States
Organisme : NINDS NIH HHS
ID : DP1 NS106665
Pays : United States
Informations de copyright
© 2022. The Author(s), under exclusive licence to Springer Nature Limited.
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