Name: Forfatter Spotlight: Forståelse af mikrotubuli-netværk i Drosophila neuromuskulære kryds
Uploaded: 2023-10-20T14:00:00
Description: Watch this Scientific Journal Video about Understanding Microtubule Network in Drosophila Neuromuscular Junctions at JoVE.com

Vi takker Dr. Ying Xiong for diskussioner og kommentarer til manuskriptet. Dette arbejde er støttet af en bevilling fra National Science Foundation of China (NSFC) til CM (31500839).

1. Dissektion af larver
BEMÆRK: Den dissekerende opløsning hæmolymfelignende saltvand (HL3.1)18 og fikseringsopløsningen 4% paraformaldehyd (PFA)19,20 anvendes ved stuetemperatur, fordi mikrotubuli depolymeriserer, når temperaturen er for lav.
<ol><li>Vælg en vandrende 3. stjernelarve med lange stumpe tang. Vask det med HL3.1 og læg det på dissektionsskålen under stereomikr...

Visning af mikrotubuli-netværk i Drosophila neuromuskulære kryds og muskelceller

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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. D., Lockwood, A. H., Hwo, S. Y., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protein+factor+essential+for+microtubule+assembly.">A protein factor essential for microtubule assembly.</a> Proceedings of the National Academy of Sciences of the United States of America. 72 (5), 1858-1862 (1975).</li><li>Kadavath, H., 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=Tau+stabilizes+microtubules+by+binding+at+the+interface+between+tubulin+heterodimers.">Tau stabilizes microtubules by binding at the interface between tubulin heterodimers.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (24), 7501-7506 (2015).</li><li>Trotta, N., Orso, G., Rossetto, M. 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Her beskrives en protokol til dissektion og immunfarvning af Drosophila-larve , neuromuskulære krydsninger og muskelceller. Der er flere vigtige punkter at overveje. For det første er det afgørende at undgå skade på de observerede muskler under dissektionsprocessen. Det kan være værd at fastgøre fileten, før du fjerner indre organer for at forhindre direkte kontakt mellem tang og muskler. For at undgå muskelskader eller adskillelse fra larveepidermis er det vigtigt at sikre, at rysterens hastighed ikke...

Mikrotubuli (MT'er), som en af cytoskeletets strukturelle komponenter, spiller en vigtig rolle i forskellige biologiske processer, herunder celledeling, cellevækst og motilitet, intracellulær transport og vedligeholdelse af celleform. Mikrotubulus dynamik og funktion moduleres af interaktioner med andre proteiner, såsom MAP1, MAP2, Tau, Katanin og Kinesin 1,2,3,4,5.
I neuroner er mikrotubuli afgørende for udvikling og vedligeholdelse af axoner og dendritter. Abnormiteter i mikrotubuli fører til dysfunktion og endda neuronernes død. For eksempel reducerer Tau-proteinhyperphosphorylering i hjernen hos Alzheimers patienter stabiliteten af mikrotubulusnetværket og forårsager neurologiske uregelmæssigheder6. Således vil undersøgelse af mikrotubulusnetværk bidrage til en forståelse af neuroudvikling og patogenesen af neurologiske sygdomme.
Det neuromuskulære kryds (NMJ) er den perifere synaps dannet mellem en motorneuronaxonterminal og en muskelfiber, som er et fremragende og kraftfuldt modelsystem til undersøgelse af synaptisk struktur og funktioner7. Futsch er et protein i Drosophila, der er homologt med det mikrotubulusbindende protein MAP1B, der findes hos pattedyr8. Det udtrykkes kun i neuroner og spiller en rolle i udviklingen af NMJ's synaptiske knapper 8,9. I vildtype visualiseres filamentøse bundter, der løber langs midten af NMJ-processer, ved immunfarvning med anti-Futsch. Når NMJ's ende nås, har dette bundt evnen til enten at danne en løkke bestående af mikrotubuli eller at miste sin trådformede struktur, hvilket resulterer i et diffust og punkteret udseende10. Mikrotubulussløjfer er forbundet med pausede vækstkegler, hvilket tyder på, at mikrotubulusarrayet er stabilt11. Derfor kan vi indirekte bestemme den stabile mikrotubulusudvikling i NMJ ved Futsch-farvning. Den store størrelse af muskelceller i Drosophila larve giver mulighed for klar visualisering af mikrotubulusnetværket. De faktorer, der påvirker stabiliteten af mikrotubulusnetværket, kan findes ved at analysere tætheden og formen af mikrotubuli. Samtidig kan muskelcellernes mikrotubulusnetværksstatus krydsverificeres med resultatet af NMJ for at opnå mere omfattende konklusioner.
Mange protokoller er blevet anvendt til at undersøge mikrotubulis netværk og dynamik. Imidlertid har disse undersøgelser ofte fokuseret på in vitro-undersøgelser 12,13,14,15,16. Alternativt har nogle in vivo-eksperimenter anvendt elektronmikroskopi til at detektere cytoskelettet17. Ifølge den specifikke binding af fluorescerende mærkede antistoffer eller kemiske farvestoffer til proteiner eller DNA tillader de metoder, der præsenteres her, påvisning af mikrotubulusnetværk i NMJ på niveauet af individuelle neuroner in vivo, med resultater bekræftet af observationer i muskelceller. Denne protokol er enkel, stabil og gentagelig, når den kombineres med de kraftfulde genetiske værktøjer, der er tilgængelige i Drosophila melanogaster, hvilket muliggør en bred vifte af fænotypiske undersøgelser og genetiske screeninger for rollen som mikrotubulusnetværksregulerende proteiner i nervesystemet 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

Mikrotubulusnetværket er en væsentlig bestanddel af nervesystemet. Mutationer i mange mikrotubuli regulatoriske proteiner er forbundet med neurodevelopmental lidelser og neurologiske sygdomme, såsom mikrotubulus-associeret protein Tau til neurodegenerative sygdomme, mikrotubulus skæreprotein Spastin og Katanin 60 forårsager henholdsvis arvelige spastiske paraplegi og neurodevelopmental abnormiteter. Påvisning af mikrotubulusnetværk i neuroner er fordelagtigt til belysning af patogenesen af neurologiske lidelser. Imidlertid gør den lille størrelse af neuroner og det tætte arrangement af aksonale mikrotubulusbundter visualisering af mikrotubulusnetværkene udfordrende. I dette studie beskriver vi en metode til dissektion af larvens neuromuskulære kryds og muskelceller samt immunfarvning af α-tubulin og mikrotubulusassocieret protein Futsch for at visualisere mikrotubulusnetværk i Drosophila melanogaster. Det neuromuskulære kryds tillader os at observere både præ- og postsynaptiske mikrotubuli, og den store størrelse af muskelceller i Drosophila-larven muliggør klar visualisering af mikrotubulusnetværket. Her, ved at mutere og overudtrykke Katanin 60 i Drosophila melanogaster og derefter undersøge mikrotubulusnetværkene i det neuromuskulære kryds og muskelceller, afslører vi nøjagtigt Katanin 60's regulerende rolle i neuroudvikling. Derfor, kombineret med de kraftfulde genetiske værktøjer af Drosophila melanogaster, letter denne protokol i høj grad genetisk screening og mikrotubulusdynamikanalyse for rollen som mikrotubulusnetværksregulerende proteiner i nervesystemet.

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.

Forfatter Spotlight: Forståelse af mikrotubuli-netværk i Drosophila neuromuskulære kryds 

Brug af Drosophila larve neuromuskulær junction og muskelceller til at visualisere mikrotubulus netværk

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.

Vi demonstrerede en trin-for-trin procedure til visualisering af mikrotubulusnetværket i både neuromuskulære kryds (NMJ'er) og muskelceller. Efter dissektion i henhold til det skematiske diagram (figur 1A-E) udføres immunfarvning, og billeder observeres efterfølgende og indsamles under et laserkonfokalmikroskop eller et stereoskopisk fluorescensmikroskop (figur 1F, G).
Både præ- og postsynaptis...

Watch this Scientific Journal Video about Understanding Microtubule Network in Drosophila Neuromuscular Junctions at JoVE.com

Her præsenterer vi en detaljeret protokol til visualisering af mikrotubulusnetværkene i neuromuskulære kryds og muskelceller. Kombineret med de kraftfulde genetiske værktøjer i Drosophila melanogaster letter denne protokol i høj grad genetisk screening og mikrotubulus-dynamikanalyse for mikrotubulusnetværksregulerende proteiners rolle i nervesystemet.

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

I øjeblikket observeres mikrotubulusdynamik indirekte gennem mikrotubulusbindende proteiner såsom EB1. Da alphabeta tubulin heterodimerer er rigelige i cytoplasmaet, foldes direkte, mangler nærliggende tubulinheterodimerer dynamikken i mikrotubulusnetværk in vivo. Vores protokol visualiserer mikrotubuli i neuromuskulære synapser og muskelceller.Når det kombineres med de kraftfulde genetiske værktøjer, der er tilgængelige i Drosophila melanogaster, muliggør en bred vifte af fænotypiske undersøgelser og genetisk screening for mikrotubulusnetværkets regulerende proteiners rolle i nervesystemet in vivo. Vores forskning fokuserer på forholdet mellem cytoskelettet og neuroudviklingen, samt dets implikationer for neurologiske sygdomme. Vi håber at opdage mekanismen for neuroudvikling og parthenogenese for neurologiske sygdomme ved at fremhæve effekten af cytoskeletale regulatoriske proteiner på neuroner.

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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 Larvedissektion og fiksering til mikrotubulusvisualisering

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.

Immunohistokemi af Drosophila larve til mikrotubulus visualisering

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.