Name: Auteur in de schijnwerpers: inzicht in het netwerk van microtubuli in neuromusculaire knooppunten van Drosophila
Uploaded: 2023-10-20T14:00:00
Description: Watch this Scientific Journal Video about Understanding Microtubule Network in Drosophila Neuromuscular Junctions at JoVE.com

We danken Dr. Ying Xiong voor de discussies en commentaren op het manuscript. Dit werk wordt ondersteund door een subsidie van de National Science Foundation of China (NSFC) aan C. M. (31500839).

1. Dissectie van larven
OPMERKING: De ontleedoplossing hemolymfe-achtige zoutoplossing (HL3.1)18 en de fixeeroplossing 4% paraformaldehyde (PFA)19,20 worden bij kamertemperatuur gebruikt omdat de microtubuli depolymeriseren wanneer de temperatuur te laag is.
<ol><li>Kies een zwervende larve van de 3einstar met een lange stompe pincet. Was het met HL3.1 en plaats het op de dissecties...

Bekijken van microtubuli-netwerken in Drosophila neuromusculaire knooppunten en spiercellen

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+Tau+and+taxol+on+polymerization+of+MCF7+microtubules+in+vitro.">The effect of Tau and taxol on polymerization of MCF7 microtubules in vitro.</a> International journal of molecular sciences. 23 (2), 677 (2022).</li><li>Velasco, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+depolymerization+contributes+to+spontaneous+neurotransmitter+release+in+vitro.">Microtubule depolymerization contributes to spontaneous neurotransmitter release in vitro.</a> Communications biology. 6 (1), 488 (2023).</li><li>H&#246;&#246;g, J. 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S., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+embryonic+neuromuscular+synapse+of+Drosophila+melanogaster.">Development of the embryonic neuromuscular synapse of Drosophila melanogaster.</a> The Journal of neuroscience: the official journal of the Society for Neuroscience. 13 (1), 144-166 (1993).</li><li>Xiong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDAC6+mutations+rescue+human+tau-induced+microtubule+defects+in+Drosophila.">HDAC6 mutations rescue human tau-induced microtubule defects in Drosophila.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (12), 4604-4609 (2013).</li><li>Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila Protocols. , (2000).</li><li>Mao, C. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule-severing+protein+Katanin+regulates+neuromuscular+junction+development+and+dendritic+elaboration+in+Drosophila.">Microtubule-severing protein Katanin regulates neuromuscular junction development and dendritic elaboration in Drosophila.</a> Development. 141 (5), 1064-1074 (2014).</li><li>Jin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Tubulin-specific+chaperone+E+functions+at+neuromuscular+synapses+and+is+required+for+microtubule+network+formation.">Drosophila Tubulin-specific chaperone E functions at neuromuscular synapses and is required for microtubule network formation.</a> Development. 136 (9), 1571-1581 (2009).</li><li>Sarthi, J., Elefant, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dTip60+HAT+activity+controls+synaptic+bouton+expansion+at+the+Drosophila+neuromuscular+junction.">dTip60 HAT activity controls synaptic bouton expansion at the Drosophila neuromuscular junction.</a> PloS one. 6 (10), 26202 (2011).</li><li>Weingarten, M. 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Hier wordt een protocol beschreven voor de dissectie en immunokleuring van Drosophila larvale neuromusculaire juncties en spiercellen. Er zijn verschillende essentiële punten om rekening mee te houden. Ten eerste is het voorkomen van letsel aan de waargenomen spieren cruciaal tijdens het dissectieproces. Het kan de moeite waard zijn om de filet te fixeren voordat u inwendige organen verwijdert om direct contact tussen de tang en de spieren te voorkomen. Om spierbeschadiging of scheiding van de larvale epidermis...

Microtubuli (MT's), als een van de structurele componenten van het cytoskelet, spelen een belangrijke rol in diverse biologische processen, waaronder celdeling, celgroei en -motiliteit, intracellulair transport en het behoud van de celvorm. De dynamiek en functie van microtubuli worden gemoduleerd door interacties met andere eiwitten, zoals MAP1, MAP2, Tau, Katanin en Kinesin 1,2,3,4,5.
In neuronen zijn microtubuli essentieel voor de ontwikkeling en het onderhoud van axonen en dendrieten. Afwijkingen in microtubuli leiden tot disfunctie en zelfs de dood van neuronen. In de hersenen van Alzheimerpatiënten vermindert hyperfosforylering van het Tau-eiwit bijvoorbeeld de stabiliteit van het microtubuli-netwerk, waardoor neurologische onregelmatigheden ontstaan. Het onderzoeken van microtubuli-netwerken zal dus bijdragen aan een beter begrip van de neurologische ontwikkeling en de pathogenese van neurologische aandoeningen.
De neuromusculaire junctie (NMJ) is de perifere synaps die wordt gevormd tussen een axonterminal van een motorneuron en een spiervezel, wat een uitstekend en krachtig modelsysteem is voor het bestuderen van synaptische structuur en functies7. Futsch is een eiwit in Drosophila dat homoloog is aan het microtubuli-bindende eiwit MAP1B dat wordt aangetroffen bij zoogdieren8. Het komt alleen tot expressie in neuronen en speelt een rol bij de ontwikkeling van de synaptische knoppen van het NMJ 8,9. In wild-type worden filamenteuze bundels die langs het centrum van NMJ-processen lopen, gevisualiseerd door immunokleuring met anti-Futsch. Bij het bereiken van het einde van NMJ heeft deze bundel het vermogen om ofwel een lus te vormen die bestaat uit microtubuli of om zijn filamenteuze structuur te verliezen, wat resulteert in een diffuus en puntvormig uiterlijk10. Microtubuli-lussen worden geassocieerd met gepauzeerde groeikegels, wat suggereert dat de microtubuli-array stabiel is11. Daarom kunnen we indirect de stabiele ontwikkeling van microtubuli in NMJ bepalen door Futsch-kleuring. De grote omvang van spiercellen in de larve van Drosophila zorgt voor een duidelijke visualisatie van het netwerk van microtubuli. De factoren die van invloed zijn op de stabiliteit van het netwerk van microtubuli kunnen worden gevonden door de dichtheid en vorm van microtubuli te analyseren. Tegelijkertijd kan de netwerkstatus van spiercellen in microtubuli worden geverifieerd met het resultaat van NMJ om uitgebreidere conclusies te verkrijgen.
Er zijn veel protocollen gebruikt voor het onderzoeken van het netwerk en de dynamiek van microtubuli. Deze onderzoeken hebben zich echter vaak gericht op in vitro studies 12,13,14,15,16. Als alternatief hebben sommige in vivo experimenten elektronenmicroscopie gebruikt om het cytoskelet te detecteren17. Volgens de specifieke binding van fluorescerend gelabelde antilichamen of chemische kleurstoffen aan eiwitten of DNA, maken de hier gepresenteerde methoden de detectie van microtubuli-netwerken in NMJ op het niveau van individuele neuronen in vivo mogelijk, met resultaten die worden bevestigd door waarnemingen in spiercellen. Dit protocol is eenvoudig, stabiel en herhaalbaar in combinatie met de krachtige genetische hulpmiddelen die beschikbaar zijn in Drosophila melanogaster, waardoor een breed scala aan fenotypische onderzoeken en genetische screenings mogelijk is voor de rol van regulerende eiwitten van microtubuli-netwerken in het zenuwstelsel in vivo.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor Plus 405 phalloidin</td>
			<td>invitrogen</td>
			<td>A30104</td>
			<td>dilute 1:200</td>
		</tr>
		<tr>
			<td>Enhanced Antifade Mounting Medium</td>
			<td>Beyotime</td>
			<td>P0128M</td>
			<td></td>
		</tr>
		<tr>
			<td>FV10-ASW confocal microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse antibody, Alexa Fluor 488 conjugated</td>
			<td>Thermo Fisher</td>
			<td>A-11001</td>
			<td>dilute 1:1,000</td>
		</tr>
		<tr>
			<td>Laser confocal microscope LSM 710</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Scissors</td>
			<td>66vision</td>
			<td>54138B</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-Futsch antibody</td>
			<td>Developmental Studies Hybridoma Bank&#160;&#160;</td>
			<td>22C10</td>
			<td>dilute 1:50</td>
		</tr>
		<tr>
			<td>Mouse anti-&#945;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T5168</td>
			<td>dilute 1:1,000</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Wako</td>
			<td>168-20955</td>
			<td>Final concentration: 4% in PB Buffer</td>
		</tr>
		<tr>
			<td>Stainless Steel Minutien Pins</td>
			<td>Entomoravia</td>
			<td></td>
			<td>0.1mm Diam</td>
		</tr>
		<tr>
			<td>Stereomicroscope SMZ161</td>
			<td>Motic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>stereoscopic fluorescence microscope BX41</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Texas Red-conjugated goat anti-HRP</td>
			<td>Jackson ImmunoResearch</td>
			<td></td>
			<td>dilute 1:100</td>
		</tr>
		<tr>
			<td>TO-PRO(R) 3 iodide</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td>dilute 1:1,000</td>
		</tr>
		<tr>
			<td>Transfer decoloring shaker TS-8</td>
			<td>Kylin-Bell lab instruments</td>
			<td>E0018</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>BioFroxx</td>
			<td>1139</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers&#160;</td>
			<td>dumont</td>
			<td>500342</td>
			<td></td>
		</tr>
	</tbody>
</table>

using drosophila larval neuromuscular junction and muscle cells to visualize microtubule network

Het netwerk van microtubuli is een essentieel onderdeel van het zenuwstelsel. Mutaties in veel regulerende eiwitten van microtubuli worden geassocieerd met neurologische ontwikkelingsstoornissen en neurologische aandoeningen, zoals microtubuli-geassocieerd eiwit Tau tot neurodegeneratieve ziekten, microtubuli-afsnijdend eiwit Spastin en Katanin 60 veroorzaken respectievelijk erfelijke spastische dwarslaesie en neurologische ontwikkelingsafwijkingen. Detectie van microtubuli-netwerken in neuronen is voordelig voor het ophelderen van de pathogenese van neurologische aandoeningen. De kleine omvang van neuronen en de dichte rangschikking van axonale microtubulibundels maken het visualiseren van de microtubuli-netwerken echter een uitdaging. In deze studie beschrijven we een methode voor dissectie van de larvale neuromusculaire junctie en spiercellen, evenals immunokleuring van α-tubuline en microtubuli-geassocieerd eiwit Futsch om microtubuli-netwerken in Drosophila melanogaster te visualiseren. De neuromusculaire junctie stelt ons in staat om zowel pre- als postsynaptische microtubuli te observeren, en de grote omvang van spiercellen in Drosophila-larven zorgt voor een duidelijke visualisatie van het microtubuli-netwerk. Hier, door Katanin 60 te muteren en tot overexpressie te brengen in Drosophila melanogaster, en vervolgens de microtubuli-netwerken in de neuromusculaire junctie en spiercellen te onderzoeken, onthullen we nauwkeurig de regulerende rol van Katanin 60 in de neurologische ontwikkeling. Daarom, in combinatie met de krachtige genetische hulpmiddelen van Drosophila melanogaster, vergemakkelijkt dit protocol de genetische screening en de analyse van de dynamiek van microtubuli aanzienlijk voor de rol van regulerende eiwitten van het microtubuli-netwerk in het zenuwstelsel.

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. Abnormalities in microtubules lead to dysfunction and even the death of neurons. For instance, in the brain of Alzheimer&#39;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&#39;s synaptic buttons8,9. In wild-type, filamentous bundles that run along the center of NMJ processes are visualized by immunostaining with anti-Futsch. When reaching NMJ&#39;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 studies12,13,14,15,16. 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.

Auteur in de schijnwerpers: inzicht in het netwerk van microtubuli in neuromusculaire knooppunten van Drosophila 

Drosophila-larvale neuromusculaire junctie en spiercellen gebruiken om het netwerk van microtubuli te visualiseren

Currently, microtubule dynamics are observed indirectly through microtubule-binding proteins such as EB1. Since alphabeta tubulin heterodimers are abundant in the cytoplasm, fold directly, neighboring tubulin heterodimers are lacking the dynamics of microtubule networks in vivo. Our protocol visualizes microtubules in neuromuscular synapses and muscle cells.When combined with the powerful genetic tools available in Drosophila melanogaster, enabling a diverse range of phenotypic examinations and the genetic screening for the role of microtubule network regulatory proteins in the nervous system in vivo. Our research focuses on the relationship between the cytoskeleton and the neurodevelopment, as well as its implications for neurological diseases. We hope to discover the mechanism of neurodevelopment and the parthenogenesis for neurological diseases by highlighting the effect of cytoskeletal regulatory proteins on neurons.

We demonstreerden een stap-voor-stap procedure voor het visualiseren van het microtubuli-netwerk in zowel neuromusculaire juncties (NMJ's) als spiercellen. Na dissectie volgens het schematische diagram (Figuur 1A-E) wordt immunokleuring uitgevoerd en worden vervolgens beelden waargenomen en verzameld onder een confocale lasermicroscoop of een stereoscopische fluorescentiemicroscoop (Figuur 1F,G).
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Watch this Scientific Journal Video about Understanding Microtubule Network in Drosophila Neuromuscular Junctions at JoVE.com

Hier presenteren we een gedetailleerd protocol om de microtubuli-netwerken in neuromusculaire knooppunten en spiercellen te visualiseren. In combinatie met de krachtige genetische hulpmiddelen van Drosophila melanogaster, vergemakkelijkt dit protocol de genetische screening en de analyse van de microtubuli-dynamiek aanzienlijk voor de rol van regulerende eiwitten van het microtubuli-netwerk in het zenuwstelsel.

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with ...

Momenteel wordt de dynamiek van microtubuli indirect waargenomen via microtubuli-bindende eiwitten zoals EB1. Aangezien alphabeta tubuline-heterodimeren overvloedig aanwezig zijn in het cytoplasma, vouw direct, missen naburige tubuline-heterodimeren de dynamiek van microtubuli-netwerken in vivo. Ons protocol visualiseert microtubuli in neuromusculaire synapsen en spiercellen.In combinatie met de krachtige genetische hulpmiddelen die beschikbaar zijn in Drosophila melanogaster, waardoor een breed scala aan fenotypische onderzoeken en de genetische screening op de rol van regulerende eiwitten van microtubuli-netwerken in het zenuwstelsel in vivo mogelijk wordt. Ons onderzoek richt zich op de relatie tussen het cytoskelet en de neurologische ontwikkeling, evenals de implicaties ervan voor neurologische aandoeningen. We hopen het mechanisme van neurologische ontwikkeling en de parthenogenese voor neurologische aandoeningen te ontdekken door het effect van regulerende eiwitten van het cytoskelet op neuronen te benadrukken.

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Research

JoVE Journal

Neuroscience

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

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 &#945;-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,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

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.

Drosophila Larval Dissection and Fixation for Microtubule Visualization

Begin by picking out a wandering Drosophila third instar larva using long blunt forceps. Wash the larva with hemolymph-like saline, then dry it with a wipe. Next, position the larva dorsally or ventrally on a dissection dish under the stereo microscope.Depending on the tissue to be observed, opt for dorsal or ventral dissection. Pin the larva's mouth hooks and tail to maintain extended positioning. Next, add a drop of the hemolymph-like saline to prevent the larva from drying.Using dissection scissors, make a small transverse incision close to the posterior end without severing it completely. Proceed to cut along the ventral midline, moving toward the anterior end. Now insert four insect pins each into the corners of the larva.Make necessary adjustments to the insect pins to maximize the stretching of the larva. Use forceps to gently remove the internal organs while avoiding harm to the muscles. To fix the larva, immerse the carcasses in 100 L of 4%paraformaldehyde, while still affixed to the dissecting dish.After this, dismount the pins. Then transfer the sample into a 2 mL microcentrifuge tube filled with 0.2%PBST. Place the tube on a shaker for 10 minutes at 15 RPM.

Drosophila Larvale dissectie en fixatie voor visualisatie van microtubuli

Immunohistochemistry of Drosophila Larva for Microtubule Visualization

Remove the PBST and immerse the paraformaldehyde-fixed drosophila larval carcasses in a blocking agent for 40 minutes at room temperature. Replace the blocking agent with 200 microliters of the primary antibody diluted with 0.2%PBST. Leave the larvae to incubate at four degrees Celsius overnight.The next day, wash the larvae with 0.2%PBST for 10 minutes. Next, remove the PBST from the tube and add 200 microliters of diluted secondary antibody. Incubate it at ambient temperature for 1.5 hours in the dark.Introduce a nuclear dye such as TO-PRO R 3 iodide into the incubating tube for 30 minutes while staining the microtubules in the dark. Finally, rinse off the secondary antibody and the nuclear dye with 0.2%PBST for 10 minutes.

Immunohistochemie van Drosophila-larve voor visualisatie van microtubuli

Mounting and Image Acquisition of Drosophila Larval Microtubules

Begin by placing the antibody labeled drosophila larval carcasses in 0.2%PBST on a glass slide. Adjusted under the stereo microscope, ensuring that the inner surface of the larval carcass is facing up. Absorb the excess PBST solution using a wipe.Then delicately add a drop of Anti-Fade mounting medium. Next place a cover slip on the slide covering the dissected larvae slowly and gently, avoiding bubble formation. Secure the cover slip in place by applying fingernail polish around its edges.Store the slide in a dark location to minimize fluorescent attenuation. For image acquisition, use the laser scanning confocal microscope with the 63x Oil immersion objective. Then adjust the wavelength and laser power to fit the requirements of the experiment best.To identify the neuromuscular junctions and capture images of muscle four in segment A three, select a 488 nanometer laser to activate alpha tubulin or Futsch, and 546 nanometers to activate the HRP imaging track. Modify the parameters to achieve a frame size of 1, 024 by 1, 024 pixels, a digital zoom of 1.0, and an imaging interval of 0.8 micrometers. To visualize microtubules in the muscle, capture images of muscle two in the segments A three to A five as it has fewer tracheal branches.Choose a 488 nanometer laser for activating alpha tubulin and a 633 nanometer laser for activating the T3605 imaging track. Adjust the imaging parameters to a frame size of 1, 024 by 1, 024 pixels, a digital zoom of 2.0, and an imaging interval of 0.4 micrometers. Both pre-and post-synaptic microtubule organizations of the neuromuscular junction were labeled with anti-alpha tubulin.Futsch staining reflected the abundance of stable microtubules in the pre-synaptic neurons. Decreased signal intensity of anti-Futsch was observed when microtubules severing protein Katanin 60 was overexpressed in the presynaptic neurons. The staining intensity of the axon trunk was stronger than that of the branches.Katanin 60 mutations resulted in increased microtubule loops. Overexpression of Katanin 60 caused short microtubule fragments within the terminal buttons. Alpha tubulin staining demonstrated a clear network of microtubules around the nucleus.Muscle cells had a significantly increased perinuclear microtubular intensity and exhibited stronger bundles in the Katanin 60 mutant. Overexpression resulted in fragmentation of the microtubule fibers.