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Neuroscience

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

Published: October 20th, 2023

DOI:

10.3791/65774

1National & Local Joint Engineering Research Center of High-throughput Drug Screening Technology, School of life science, Hubei University

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of α-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster, and then examining the microtubule networks in the neuromuscular junction and muscle cells, we accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Microtubules (MTs), as one of the structural components of the cytoskeleton, play an important role in diverse biological processes, including cell division, cell growth and motility, intracellular transport, and the maintenance of cell shape. Microtubule dynamics and function are modulated by interactions with other proteins, such as MAP1, MAP2, Tau, Katanin, and Kinesin1,2,3,4,5.

In neurons, microtubules are essential for the development and maintenance of axons and dendrites. ....

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1. Dissection of larvae

NOTE: The dissecting solution hemolymph-like saline (HL3.1)18 and the fixing solution 4% paraformaldehyde (PFA)19,20are used at room temperature because the microtubules depolymerize when the temperature is too low.

  1. Pick out a wandering 3rd instar larva with long blunt forceps. Wash it with HL3.1 and place it on the dissection dish under the stereom.......

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We demonstrated a step-by-step procedure for visualizing the microtubule network in both neuromuscular junctions (NMJs) and muscle cells. Following dissection according to the schematic diagram (Figure 1A-E), immunostaining is performed, and images are subsequently observed and collected under a laser confocal microscope or a stereoscopic fluorescence microscope (Figure 1F,G).

Both pre-and post-synapt.......

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Here a protocol is described for the dissection and immunostaining of Drosophila larval neuromuscular junctions and muscle cells. There are several essential points to consider. Firstly, avoiding injury to the observed muscles is crucial during the dissection process. It may be worth fixing the fillet before removing internal organs to prevent direct contact between the forceps and the muscles. To avoid muscle damage or separation from the larval epidermis, it is important to ensure that the speed of the shaker .......

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We thank Dr. Ying Xiong for discussions and comments on the manuscript. This work is supported by a grant from the National Science Foundation of China (NSFC) to C. M. (31500839).

....

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Name Company Catalog Number Comments
Alexa Fluor Plus 405 phalloidin invitrogen A30104 dilute 1:200
Enhanced Antifade Mounting Medium Beyotime P0128M
FV10-ASW confocal microscope Olympus
Goat anti-Mouse antibody, Alexa Fluor 488 conjugated Thermo Fisher A-11001 dilute 1:1,000
Laser confocal microscope LSM 710 Zeiss
Micro Scissors 66vision 54138B
Mouse anti-Futsch antibody Developmental Studies Hybridoma Bank   22C10 dilute 1:50
Mouse anti-α-tubulin antibody Sigma T5168 dilute 1:1,000
Paraformaldehyde Wako 168-20955 Final concentration: 4% in PB Buffer
Stainless Steel Minutien Pins Entomoravia 0.1mm Diam
Stereomicroscope SMZ161 Motic
stereoscopic fluorescence microscope BX41 Olympus
Texas Red-conjugated goat anti-HRP Jackson ImmunoResearch dilute 1:100
TO-PRO(R) 3 iodide Invitrogen T3605 dilute 1:1,000
Transfer decoloring shaker TS-8 Kylin-Bell lab instruments E0018
TritonX-100 BioFroxx 1139
Tweezers  dumont 500342

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