Visualizing mRNA localization and local protein translation in neurons.
Fluorescent proteins (FPs) have been successfully used to study the localization and interactions of proteins in living cells. They have also been instrumental in analyzing the proteins involved in the localization of RNAs in different cell types, including neurons. With the development of methods that also tag RNAs via fluorescent proteins, researchers now have a powerful tool to covisualize RNAs and associated proteins in living neurons. Here, we review the current status of the use of FPs in the study of transport and localization of ribonucleoprotein particles (RNPs) in neurons and provide key protocols used to introduce transgenes into cultured neurons, including calcium-phosphate-based transfection and nucleofection. These methods allow the fast and efficient expression of fluorescently tagged fusion proteins in neurons at different stages of differentiation and form the basis for fluorescent protein-based live cell imaging in neuronal cultures. Additional protocols are given that allow the simultaneous visualization of RNP proteins and cargo RNAs in living neurons and aspects of the visualization of fluorescently tagged proteins in neurons, such as colocalization studies, are discussed. Finally, we review approaches to visualize the local synthesis of proteins in distal dendrites and axons.
D000818:Animals; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins; D017909:Levivirus / Q000235:genetics; D009474:Neurons / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D034443:RNA Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D014162:Transfection
D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D049452:Green Fluorescent Proteins