Monitoring Synaptic Vesicle Protein Sorting with Enhanced Horseradish Peroxidase in the Electron Microscope.
Methods Mol Biol
; 1474: 327-41, 2016.
Article
em En
| MEDLINE
| ID: mdl-27515091
ABSTRACT
Protein sorting is the fundamental cellular process that creates and maintains cell organelles and subcellular structures. The synaptic vesicle (SV) is a unique cell organelle that contains a plethora of specific SV proteins and its protein composition is crucial for its function. Thus understanding the mechanisms that sort proteins to SVs and other cell organelles is central to neuroscience and cell biology.While in the past protein sorting was studied in the fluorescence and confocal microscope, we here present a protocol that reveals SV protein trafficking and sorting in the electron microscope (EM). The protocol exploits tagging SV proteins with a new genetically encoded label for EM enhanced horseradish peroxidase (eHRP). eHRP gained its high sensitivity through direct evolution of its catalytic activity and is detectable in the EM and LM after expression in neurons and other mammalian cells. The protocol describes the use of eHRP, labeling of SVs in cultured hippocampal neurons, and analysis via serial section reconstruction.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Coloração e Rotulagem
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Vesículas Sinápticas
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Microscopia Eletrônica
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Astrócitos
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Peroxidase do Rábano Silvestre
Limite:
Animals
Idioma:
En
Revista:
Methods Mol Biol
Ano de publicação:
2016
Tipo de documento:
Article