Name: Autor destacado: Comprensión de la red de microtúbulos en las uniones neuromusculares de Drosophila
Uploaded: 2023-10-20T14:00:00
Description: Watch this Scientific Journal Video about Understanding Microtubule Network in Drosophila Neuromuscular Junctions at JoVE.com

Agradecemos al Dr. Ying Xiong por las discusiones y comentarios sobre el manuscrito. Este trabajo cuenta con el apoyo de una subvención de la Fundación Nacional de Ciencias de China (NSFC) a C. M. (31500839).

1. Disección de larvas
NOTA: La solución diseccionadora de solución salina similar a la hemolinfa (HL3.1)18 y la solución de fijación al 4% de paraformaldehído (PFA)19,20 se utilizan a temperatura ambiente porque los microtúbulos se despolimerizan cuando la temperatura es demasiado baja.
<ol><li>Escoge una larva errante de3er estadio con pinzas largas y romas. Lávelo con HL3...

Visualización de redes de microtúbulos en uniones neuromusculares y células musculares de Drosophila

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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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Aquí se describe un protocolo para la disección e inmunotinción de las uniones neuromusculares y las células musculares de las larvas de Drosophila . Hay varios puntos esenciales a tener en cuenta. En primer lugar, evitar lesiones en los músculos observados es crucial durante el proceso de disección. Puede valer la pena arreglar el filete antes de extraer los órganos internos para evitar el contacto directo entre las pinzas y los músculos. Para evitar daños musculares o la separación de la epidermis la...

Los microtúbulos (MT), como uno de los componentes estructurales del citoesqueleto, desempeñan un papel importante en diversos procesos biológicos, incluida la división celular, el crecimiento y la motilidad celular, el transporte intracelular y el mantenimiento de la forma celular. La dinámica y la función de los microtúbulos están moduladas por interacciones con otras proteínas, como MAP1, MAP2, Tau, Katanin y Kinesin 1,2,3,4,5.
En las neuronas, los microtúbulos son esenciales para el desarrollo y mantenimiento de axones y dendritas. Las anomalías en los microtúbulos conducen a la disfunción e incluso a la muerte de las neuronas. Por ejemplo, en el cerebro de los pacientes con Alzheimer, la hiperfosforilación de la proteína Tau reduce la estabilidad de la red de microtúbulos, causando irregularidades neurológicas6. Por lo tanto, el examen de las redes de microtúbulos contribuirá a la comprensión del neurodesarrollo y la patogénesis de las enfermedades neurológicas.
La unión neuromuscular (NMJ) es la sinapsis periférica formada entre el terminal axonal de una neurona motora y una fibra muscular, que es un excelente y potente sistema modelo para estudiar la estructura y las funciones sinápticas7. Futsch es una proteína de Drosophila que es homóloga a la proteína de unión a microtúbulos MAP1B que se encuentra en los mamíferos8. Se expresa solamente en las neuronas y desempeña un papel en el desarrollo de los botones sinápticos de la NMJ 8,9. En el tipo salvaje, los haces filamentosos que corren a lo largo del centro de los procesos NMJ se visualizan mediante inmunotinción con anti-Futsch. Al llegar al final de NMJ, este haz tiene la capacidad de formar un bucle formado por microtúbulos o de perder su estructura filamentosa, lo que da lugar a un aspecto difuso y punteado10. Los bucles de microtúbulos se asocian con conos de crecimiento en pausa, lo que sugiere que la matriz de microtúbulos es estable11. Por lo tanto, podemos determinar indirectamente el desarrollo estable de microtúbulos en NMJ mediante tinción de Futsch. El gran tamaño de las células musculares de la larva de Drosophila permite una visualización clara de la red de microtúbulos. Los factores que afectan la estabilidad de la red de microtúbulos se pueden encontrar analizando la densidad y la forma de los microtúbulos. Al mismo tiempo, el estado de la red de microtúbulos de las células musculares se puede verificar de forma cruzada con el resultado de la NMJ para obtener conclusiones más completas.
Se han empleado muchos protocolos para investigar la red y la dinámica de los microtúbulos. Sin embargo, estas investigaciones muchas veces se han centrado en estudios in vitro 12,13,14,15,16. Alternativamente, algunos experimentos in vivo han empleado microscopía electrónica para detectar el citoesqueleto17. De acuerdo con la unión específica de anticuerpos marcados con fluorescencia o colorantes químicos a proteínas o ADN, los métodos aquí presentados permiten la detección de redes de microtúbulos en NMJ a nivel de neuronas individuales in vivo, con resultados corroborados por observaciones en células musculares. Este protocolo es simple, estable y repetible cuando se combina con las poderosas herramientas genéticas disponibles en Drosophila melanogaster, lo que permite una amplia gama de exámenes fenotípicos y exámenes genéticos para el papel de las proteínas reguladoras de la red de microtúbulos en el sistema nervioso 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

La red de microtúbulos es un componente esencial del sistema nervioso. Las mutaciones en muchas proteínas reguladoras de los microtúbulos se asocian con trastornos del neurodesarrollo y enfermedades neurológicas, como la proteína Tau asociada a microtúbulos a enfermedades neurodegenerativas, la proteína cortante de microtúbulos Spastin y Katanin 60 causan paraplejia espástica hereditaria y anomalías del neurodesarrollo, respectivamente. La detección de redes de microtúbulos en las neuronas es ventajosa para dilucidar la patogénesis de los trastornos neurológicos. Sin embargo, el pequeño tamaño de las neuronas y la densa disposición de los haces de microtúbulos axonales dificultan la visualización de las redes de microtúbulos. En este estudio, describimos un método para la disección de la unión neuromuscular larvaria y las células musculares, así como la inmunotinción de α-tubulina y la proteína Futsch asociada a microtúbulos para visualizar redes de microtúbulos en Drosophila melanogaster. La unión neuromuscular nos permite observar los microtúbulos pre y postsinápticos, y el gran tamaño de las células musculares en la larva de Drosophila permite una visualización clara de la red de microtúbulos. Aquí, mutando y sobreexpresando Katanin 60 en Drosophila melanogaster, y luego examinando las redes de microtúbulos en la unión neuromuscular y las células musculares, revelamos con precisión el papel regulador de Katanin 60 en el neurodesarrollo. Por lo tanto, combinado con las poderosas herramientas genéticas de Drosophila melanogaster, este protocolo facilita enormemente el cribado genético y el análisis de la dinámica de los microtúbulos para determinar el papel de las proteínas reguladoras de la red de microtúbulos en el sistema nervioso.

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.

Autor destacado: Comprensión de la red de microtúbulos en las uniones neuromusculares de Drosophila 

Uso de la unión neuromuscular de las larvas de Drosophila y células musculares para visualizar la red de microtúbulos

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.

Demostramos un procedimiento paso a paso para visualizar la red de microtúbulos tanto en las uniones neuromusculares (NMJ) como en las células musculares. Después de la disección de acuerdo con el diagrama esquemático (Figura 1A-E), se realiza la inmunotinción y posteriormente se observan las imágenes y se recogen bajo un microscopio confocal láser o un microscopio de fluorescencia estereoscópica (Figura 1F, G).
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Watch this Scientific Journal Video about Understanding Microtubule Network in Drosophila Neuromuscular Junctions at JoVE.com

Aquí, presentamos un protocolo detallado para visualizar las redes de microtúbulos en las uniones neuromusculares y las células musculares. Combinado con las potentes herramientas genéticas de Drosophila melanogaster, este protocolo facilita enormemente el cribado genético y el análisis de la dinámica de los microtúbulos para determinar el papel de las proteínas reguladoras de la red de microtúbulos en el sistema nervioso.

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

Actualmente, la dinámica de los microtúbulos se observa indirectamente a través de proteínas de unión a microtúbulos como EB1. Dado que los heterodímeros de tubulina alfabéticos son abundantes en el citoplasma, se pliegan directamente, los heterodímeros de tubulina vecinos carecen de la dinámica de las redes de microtúbulos in vivo. Nuestro protocolo visualiza los microtúbulos en las sinapsis neuromusculares y las células musculares.Cuando se combina con las poderosas herramientas genéticas disponibles en Drosophila melanogaster, permite una amplia gama de exámenes fenotípicos y el cribado genético del papel de las proteínas reguladoras de la red de microtúbulos en el sistema nervioso in vivo. Nuestra investigación se centra en la relación entre el citoesqueleto y el neurodesarrollo, así como sus implicaciones para las enfermedades neurológicas. Esperamos descubrir el mecanismo del neurodesarrollo y la partenogénesis de las enfermedades neurológicas destacando el efecto de las proteínas reguladoras del citoesqueleto en las neuronas.

using-drosophila-larval-neuromuscular-junction-muscle-cells-to

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.

Investigación

Neurociencias

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 Disección y fijación de larvas para la visualización de microtúbulos

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.

Inmunohistoquímica de larvas de Drosophila para la visualización de microtúbulos

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.