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For decades, theDrosophilalarval neuromuscular junction (NMJ) has been a go-to model for synaptic development. This simple, accessible system is composed of a repeating pattern of 33 distinct neurons that stereotypically innervate 30 muscles. Fundamental mechanisms that underlie diverse aspects of axon pathfinding, synaptic form, and function have been uncovered at the NMJ, and new pathways continue to be uncovered. These discoveries are fueled by the ease of dissections and an extensive array of techniques. Chief among these techniques are various microscopy approaches, including super-resolution and electron microscopy. Functionally, theDrosophilaNMJ is glutamatergic, similar to the vertebrate central synapses, making it a great model to study normal development and neurological diseases. Here we provide a brief overview of the larval neuromuscular system, highlighting the connectivity patterns, development, and some of the mechanisms underlying these processes.
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Using the Proximity Ligation Assay to Visualize Colocalization of Proteins at the Drosophila Larval Neuromuscular Junction
In the nearly 50 years since the neuromuscular junction (NMJ) was first established as a model synapse, its molecular composition has been extensively characterized. Early work relied on fluorescent signals to determine whether proteins localized to the pre- and postsynaptic regions. As more synaptic molecules were identified, determining the localization of these proteins relative to each other became important. Conventional microscopy lacks the resolving power to assess whether two proteins are within an appropriate distance to bind directly or be part of a larger complex. Super-resolution and immunoelectron microscopies can improve spatial resolution, but these techniques can be difficult to execute and troubleshoot, and access to these instruments is limiting. However, another approach, proximity labeling, overcomes many of these limitations by using a DNA secondary label that can only be amplified if the two proteins of interest are within 40 nm of each other, which is ∼5× greater than the resolving power of conventional microscopy. In this protocol, we describe the use of the proximity ligation assay, which combines immunohistochemistry with DNA amplification, to reveal protein colocalization in theDrosophilaNMJ.
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- Award ID(s):
- 2048080
- PAR ID:
- 10564828
- Publisher / Repository:
- Cold Spring Harbor Laboratory Press
- Date Published:
- Journal Name:
- Cold Spring Harbor Protocols
- ISSN:
- 1940-3402
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1101/pdb.prot108502
