title: Monitoring Synaptic Vesicle Protein Sorting with Enhanced Horseradish Peroxidase in the Electron Microscope.
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.
authors: Department of Anatomy, Universidad Central del Caribe, Avenida Laurel, Santa Juanita, Bayamon, PR, 00956, USA. ts.uccaribe@tutamail.com.
authors: Thomas
authors: T
authors: Schikorski
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000378:metabolism* / Q000648:ultrastructure; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism / Q000648:ultrastructure; D006735:Horseradish Peroxidase / Q000235:genetics* / Q000378:metabolism; D007091:Image Processing, Computer-Assisted / Q000379:methods; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D008854:Microscopy, Electron / Q000379:methods*; D008867:Microtomy; D061251:Primary Cell Culture; D021381:Protein Transport; D051381:Rats; D013194:Staining and Labeling / Q000379:methods*; D013572:Synaptic Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D019076:Transgenes
chemical_list: D006735:Horseradish Peroxidase
keywords: Electron microscope; Gene transfer; Protein sorting; Synaptic vesicle; Viral vectors