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

Summary

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.

Abstract

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.

Introduction

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 Kinesin^1,2,3,4,5.

In neurons, microtubules are essential for the development and maintenance of axons and dendrites. Abnormalities in microtubules lead to dysfunction and even the death of neurons. For instance, in the brain of Alzheimer's patients, Tau protein hyperphosphorylation reduces the stability of the microtubule network, causing neurological irregularities⁶. Thus, examining microtubule networks will contribute to a comprehension of neurodevelopment and the pathogenesis of neurological diseases.

The neuromuscular junction (NMJ) is the peripheral synapse formed between a motor neuron axon terminal and a muscle fiber, which is an excellent and powerful model system for studying synaptic structure and functions⁷. Futsch is a protein in Drosophila that is homologous to the microtubule-binding protein MAP1B found in mammals⁸. It is expressed only in neurons and plays a role in the development of the NMJ's synaptic buttons^8,9. In wild-type, filamentous bundles that run along the center of NMJ processes are visualized by immunostaining with anti-Futsch. When reaching NMJ's end, this bundle has the ability to either form a loop consisting of microtubules or to lose its filamentous structure, resulting in a diffuse and punctate appearance¹⁰. Microtubule loops are associated with paused growth cones, which suggests the microtubule array is stable¹¹. Therefore, we can indirectly determine the stable microtubule development in NMJ by Futsch staining. The large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. The factors affecting the stability of the microtubule network can be found by analyzing the density and shape of microtubules. Simultaneously, the microtubule network status of muscle cells can be cross-verified with the result of NMJ to obtain more comprehensive conclusions.

Many protocols have been employed for investigating the network and dynamics of microtubules. However, these researches have often focused on in vitro studies^{12,13,14,15,16}. Alternatively, some in vivo experiments have employed electron microscopy to detect the cytoskeleton¹⁷. According to the specific binding of fluorescently labeled antibodies or chemical dyes to proteins or DNA, the methods presented here allow the detection of microtubule networks in NMJ at the level of individual neurons in vivo, with results corroborated by observations in muscle cells. This protocol is simple, stable, and repeatable when combined with the powerful genetic tools available in Drosophila melanogaster, enabling a diverse range of phenotypic examinations and genetic screenings for the role of microtubule network regulatory proteins in the nervous system in vivo.

Protocol

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 3^rd instar larva with long blunt forceps. Wash it with HL3.1 and place it on the dissection dish under the stereomicroscope.
   NOTE: Wandering 3^rd instar larva is identified by branched anterior spiracle and crawls around the tube at approximately 96 h (25 °C)²¹.
2. Position the larva with the dorsal or ventral side up. Identify the dorsal side by the two long tracheas and the ventral by the abdominal denticle belts.
   1. Depending on the tissue to observe, opt for dorsal or ventral dissection. This will allow for a more precise and detailed view of the specific area of interest. For NMJ and muscle cell observation, cut open the larval dorsal and ventral regions, respectively.
3. Pin the mouth hooks and the tail. Adjust the pins to keep the larva in an extended state. Add a drop of HL3.1 to the larva to prevent it from drying.
   NOTE: Ca²⁺ -free HL 3.1 can be used to minimize contraction of the muscles during dissection.
4. Use the dissecting scissors to make a small transverse cut close to the posterior end, but do not cut off the posterior end. Then cut along the ventral midline towards the anterior end.
5. Insert four insect pins on the four corners of the larva. Readjust insect pins such that the larva is maximally stretched in all directions.
6. Use the forceps to remove internal organs while not damaging the muscles.

2. Fixation

1. Add 100 µL of PFA (4%) to immerse carcasses for 40 min while the carcasses are still pinned on the dissecting dish.
   CAUTION: Effective protection measures are taken to avoid direct contact with skin or inhalation, as PFA is a hazard.
2. Dismount the pins and transfer the sample to a 2 mL microcentrifuge tube. Rinse off PFA with 1x phosphate buffered saline containing 0.2% Triton X-100 (0.2% PBST) to fill 2 mL microcentrifuge tube for 10 min on the decoloring shaker at 15 rpm. Repeat the wash process 5x.

3. Immunocytochemistry

1. Immerse the carcasses in blocking agent (5% goat serum in 0.2% PBST) and block for 40 min at room temperature.
2. Remove the blocking agent and replace it with 200 µL of the primary antibody (e.g., anti-α-tubulin, 1:1000; anti-Futsch, 1:50) diluted with 0.2% PBST at 4 °C overnight. Visualize muscle microtubules by immunostaining with monoclonal anti-α-tubulin. Anti-Futsch can indirectly reflect the microtubule morphology in NMJ.
3. After incubation of the primary antibody, wash larvae with 0.2% PBST for 10 min. Repeat 5x.
4. Incubate the larvae with 200 µL of secondary antibody (e.g., goat anti-Mouse-488, 1:1000) diluted with 0.2% PBST at room temperature for 1.5 h in the dark.
5. Next, add a nuclear dye such as TO-PRO(R) 3 iodide (T3605) at a concentration of 1:1000 into the incubating tube for 30 min when staining the microtubules in the dark^20,22.
6. Rinse off the secondary antibody and the nuclear dye with 0.2% PBST for 10 min. Repeat 5x in the dark.

4. Mounting

1. Place the larval carcass in 0.2% PBST on a glass slide and adjust it under the stereomicroscope. Make sure the inner surface of the larval carcass is facing up and all larval carcasses are arranged as desired.
2. Absorb excess PBST solution with a wipe and gently add a drop of antifade mounting medium.
3. Place a coverslip on the slide to cover the dissected larvae slowly and gently to avoid bubbles.
4. Apply fingernail polish around the coverslip. Place the slide in the dark space to reduce fluorescent attenuation.

5. Image acquisition

1. To acquire the images, use a laser scanning confocal microscope, select the 60x oil immersion objective (numerical aperture 1.42) or similar, and adjust the laser power and wavelength based on the experiment.
2. To identify the NMJ, capture images in muscle 4 in segment A3 (as shown in the location in Figure 1F). Select a 488 nm laser to activate the α-tubulin or Futsch and 543 nm to activate the HRP imaging track. Adjust the parameters to a frame size of 800 pixels x 800 pixels, a digital zoom of 2.0, and an imaging interval of 0.8 µm in NMJs (Figure 2).
3. To identify the microtubules in muscle, capture images of muscle 2 in segment A3-A5 (as shown in the location in Figure 1G) as it has fewer tracheal branches. Choose a 488 nm laser for activating α-tubulin and a 635 nm laser for activating the T3605 imaging track. Adjust the parameters to a frame size of 1024 pixels x 1024 pixels, a digital zoom of 3.0, and an imaging interval of 0.4 µm in muscle cells (Figure 3).

Representative Results

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-synaptic microtubule organization of NMJ could be labeled with anti-α-tubulin, and by selecting the layer thickness of the laser scanning confocal microscope, the microtubule morphology of different slices would be displayed (Figure 2A-C). Futsch has also been used for neural tracing by revealing the microtubule organization in the axons of neurons. Anti-HRP is used to label the neuronal membrane^10,23,24. Co-staining with anti-Futsch and anti-HRP antibodies is performed to detect the microtubule status in axons of neurons. Futsch staining can reflect the abundance of stable microtubules in the presynaptic neurons of NMJ⁹. Tubulin-specific chaperone E (TBCE) plays a crucial role in the development of the MT cytoskeleton. When TBCE is knocked down in presynaptic neurons, the signal of anti-Futsch becomes weaker and thinner, and the staining in the terminal boutons is either unobvious or absent²³. Tau is a microtubule-associated protein that has an important role in microtubule assembly and stabilization^25,26. In Drosophila with ectopic expression of human wild-type and mutant human Tau protein, a decrease in the intensity of Futsch signal at the NMJ terminals is revealed²⁰. According to the results of Futsch staining, the staining intensity of the axon trunk was stronger than that of the branches (Figure 2D-F). Microtubules at the end of branches are less stable and more prone to morphological changes, so the dynamics of microtubules can be reflected by staining the terminal microtubules.

Morphological changes of microtubules can also be easily visualized²². Microtubule loops can be stained with anti-Futsch and anti-α-tubulin. Furthermore, the shape of a single loop can be clearly displayed, which facilitates quantitative analysis. For instance, Katanin is a microtubule-severing protein that plays an important role in the regulation of microtubule dynamics. The catalytic subunit Katanin 60 has been reported to have an effect on the morphology of microtubules²². Mao has constructed Katanin 60 mutant by P-element-mediated excision and uas-Katanin 60 by inserting pUAST-attB-Katanin 60 in the second chromosome at 51D. The increase in microtubule loops caused by Katanin 60^17A mutations was clearly demonstrated (Figure 2A,B,D,E). In addition, in the overexpression of Katanin 60, short MT fragments could also be significantly observed within the terminal boutons (Figure 2C).

The microtubule network can be observed by staining with α-tubulin antibodies in muscle cells. A network of microtubules extending around the nucleus was clearly visible (Figure 3A). The microtubule severing protein Katanin regulates the distribution of the microtubule network²². In Katanin 60^17A mutant, muscle cells had a significantly increased perinuclear MT intensity and exhibited stronger bundles compared with the wild type (Figure 3B). Fragmentation of microtubule fibers caused by overexpression of Katanin 60 was represented (Figure 3C). Thus, the protocol enables the microtubule network visualization in both neurons and muscle cells.

Figure 1. Dorsal dissection procedure of Drosophila larva. (A-E) The procedure for preparing larval carcasses (modified from²¹). (A) Insert two pins each into the mouth hooks and tail of a larva. (B) Use the dissecting scissors to make a small transverse cut close to the posterior end. (C) Cut along the ventral midline towards the anterior end. (D) Insert four insect pins on the four corners of the larva. (E)Use forceps to remove internal organs and readjust the position of the pins to place the larva in an appropriate position for photography. (F) Phalloidin is used to label the cytoskeleton to visualize the muscle, and HRP is used to label the cell membrane of neurons. The observed region of the NMJ is restricted to muscle 4 in segment A3, co-stained with anti-HRP (green) and phalloidin (magenta). The right panel shows a schematic representation of the local muscle structure. The frame size of 1024 pixels x 1024 pixels, digital zoom of 0.6, and an imaging interval of approximately 4 µm using a 10x objective lens (numerical aperture 0.45). (G) The observed region of the muscle cells is restricted to muscle 2 in segments A3 to A5, stained with phalloidin (magenta). The single section is captured by a stereoscopic fluorescence microscope. Scale bar: 500 µm. Please click here to view a larger version of this figure.

Figure 2. Confocal images of microtubules within NMJ by anti-α-tubulin or anti-Futsch. NMJ terminals of w¹¹¹⁸ control (A), Katanin 60^17A mutant (B) and neuronal overexpression of Katanin 60 (C), co-stained with anti-HRP (red) and anti-α-tubulin (green) are shown in the left column. The images are projections from complete z-stacks through the entire muscle 4 NMJ of the abdominal segment A3. The middle column shows a clearer image of microtubules in the NMJ terminals by removing the microtubules from the muscle. The right column depicts the morphology of microtubules in synaptic boutons using the analysis software. Scale bar: 1 µm. NMJ terminals of different genotypes co-stained with anti-HRP (red) and anti-Futsch (green) (D-F). MT loops were indicated by arrows. Scale bar: 5 µm. This figure has been adapted from²². Please click here to view a larger version of this figure.

Figure 3. Perinuclear microtubule network staining in the Drosophila larval muscle cells. (A-C) Larval muscles are co-stained with anti-α-tubulin (green) to show the microtubule network and T3605 (blue) to show the nucleus for w¹¹¹⁸ control (A), Katanin 60^17A mutant (B) and Katanin 60 overexpression in muscle system (C). The images are projections from complete z-stacks from the appearance of microtubules to the center of the nuclear. Scale bar: 10 µm. Please click here to view a larger version of this figure.

Discussion

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 does not exceed 15 rpm during washing and incubation. Additionally, precise control over skin extension is necessary to ensure clear and complete photography of NMJ morphology. It is advisable to conduct preliminary experiments to optimize the antibody concentration, fixation, blocking, and incubation times. The fixed duration is limited to about 40 min. Too short or too long is not conducive to the immunostaining of the microtubule network.

Microtubules in neurons are densely packed and challenging to be visualized clearly using conventional microscopy. Compared to neurons, the muscle cells of Drosophila are notably large, with muscle 2 of segment A3 measuring up to 40 µm x 150 µm, rendering it amenable to high-resolution imaging. Therefore, utilizing Drosophila muscle cells for investigating the regulation of microtubule network-associated proteins is an intuitive and lucid approach that holds significant value in validating NMJ phenotypes^20,22,23. Compared to the previous method²⁷, we have changed the dissection direction from the dorsal to the abdomen in order to obtain an unobstructed object. The elution buffer of the muscle with 0.2% PBST is relatively gentle and helps to preserve muscle integrity.

There are still some limitations to this approach. As a microtubule-binding protein, Futsch is expressed exclusively in neurons and can indirectly represent polymerized microtubules. However, if only Futsch is used to visualize the microtubule network during interaction screening, some candidate proteins may be missed. α-tubulin or β-tubulin can directly represent the microtubule network, however, these two proteins present both pre-synaptic and postsynaptic, and it is sometimes difficult to distinguish. Moreover, a large number of α/β-tubulin heterodimers are distributed in the cytoplasm, which means that labeling either α-tubulin or β-tubulin may not be suitable for live imaging, as both polymerized and non-polymerized tubulin can be observed at the same time.

Observing microtubules from motor neurons to muscle cells can provide a comprehensive understanding of the movement mechanisms from the perspective of neural circuits, facilitating the study of neurodevelopmental processes and the pathogenesis of neurological diseases. Visualizing the microtubule network in different model systems is beneficial for verifying gene function from multiple perspectives using this protocol. This method can also be used to observe the postsynaptic receptors of NMJ, which is beneficial to the study of communication between synapses and synaptic plasticity.

Materials

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