Live imaging of synapse development and measuring protein dynamics using two-color fluorescence recovery after photo-bleaching at Drosophila synapses.
Here we describe how to anesthetize and image Drosophila larvae as to follow 'the life history' of identified synapses and synaptic components. This protocol is sensitive, for example, the distribution of glutamate receptors expressed at physiological levels can be monitored. Typically, 2-20 time points can be recorded in the intact organism. Finally, we discuss how to extract the kinetic information on protein dynamics from two-color fluorescence recovery after photo-bleaching (FRAP) measurements and give advice how to keep the in vivo imager's five arch enemies--limited temporal and spatial resolution, injury of the animal, inactivation of proteins and movement artifacts--in check. While we focus on synapses, as model structure, the protocol can easily be adapted to study other developmental processes such as muscle growth, gut development or tracheal branching.
Füger
Petra
P
Department of Cellular Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen, Otfried-Müller Strasse 27, Tübingen D-72076, Germany.
Behrends
Laila B
LB
Mertel
Sara
S
Sigrist
Stephan J
SJ
Rasse
Tobias M
TM
D000818:Animals; D004330:Drosophila / Q000254:growth & development*; D005453:Fluorescence; D007814:Larva; D038761:Photobleaching; D011506:Proteins / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D013569:Synapses / Q000378:metabolism*
D011506:Proteins