Name: 저자 스포트라이트: Understanding Microtubule Network in Drosophila Neuromuscular Junctions
Uploaded: 2023-10-20T14:00:00
Description: Watch this Scientific Journal Video about Understanding Microtubule Network in Drosophila Neuromuscular Junctions at JoVE.com

원고에 대한 토론과 논평을 해주신 Ying Xiong 박사님께 감사드립니다. 이 연구는 중국 국립과학재단(NSFC)이 C. M.(31500839)에 제공한 보조금으로 지원됩니다.

1. 유충 해부
알림: 해부 용액 혈림프 유사 식염수(HL3.1)18 및 고정 용액 4% 파라포름알데히드(PFA)19,20은 온도가 너무 낮을 때 미세소관이 해중합되기 때문에 실온에서 사용됩니다.
<ol><li>길고 뭉툭한 집게로 방황하는 3번째 인스타 유충을 골라냅니다. HL3.1로 세척하고 실체현미경 아래의 해부 접...

Observation Microtubule networks in Drosophila Neuromuscular junctions and muscle cells(초파리 신경근 접합부 및 근육 세포의 미세소관 네트워크 관찰)

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+essential+role+for+katanin+in+severing+microtubules+in+the+neuron.">An essential role for katanin in severing microtubules in the neuron.</a> The Journal of cell biology. 145 (2), 305-315 (1999).</li><li>Hu, Z., 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=Fidgetin+regulates+cultured+astrocyte+migration+by+severing+tyrosinated+microtubules+at+the+leading+edge.">Fidgetin regulates cultured astrocyte migration by severing tyrosinated microtubules at the leading edge.</a> Molecular biology of the cell. 28 (4), 545-553 (2017).</li><li>Feizabadi, M. S., Castillon, V. 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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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여기에서는 초파리 유충 신경근 접합부 및 근육 세포의 해부 및 면역염색을 위한 프로토콜이 설명되어 있습니다. 고려해야 할 몇 가지 필수 사항이 있습니다. 첫째, 관찰된 근육의 부상을 피하는 것은 해부 과정에서 매우 중요합니다. 집게와 근육 사이의 직접적인 접촉을 방지하기 위해 내부 장기를 제거하기 전에 필렛을 고정하는 것이 좋습니다. 근육 손상이나 유충 표피로부터의 분리를 ?...

미세소관(MT)은 세포골격의 구조적 구성 요소 중 하나로서 세포 분열, 세포 성장 및 운동성, 세포 내 수송, 세포 모양 유지 등 다양한 생물학적 과정에서 중요한 역할을 합니다. 미세소관의 역학 및 기능은 MAP1, MAP2, Tau, Katanin 및 Kinesin 1,2,3,4,5와 같은 다른 단백질과의 상호 작용에 의해 조절됩니다.
뉴런에서 미세소관은 축삭돌기와 수상돌기의 발달과 유지에 필수적입니다. 미세소관의 이상은 기능 장애와 심지어 뉴런의 죽음으로 이어집니다. 예를 들어, 알츠하이머 환자의 뇌에서 타우 단백질 과인산화는 미세소관 네트워크의 안정성을 감소시켜 신경학적 불규칙성을 유발한다6. 따라서 미세소관 네트워크를 검사하는 것은 신경 발달과 신경 질환의 발병 기전을 이해하는 데 기여할 것입니다.
신경근 접합부(NMJ)는 운동 뉴런 축삭 말단과 근육 섬유 사이에 형성된 말초 시냅스로, 시냅스 구조와 기능을 연구하기 위한 우수하고 강력한 모델 시스템이다7. Futsch는 포유류에서 발견되는 미세소관 결합 단백질 MAP1B와 상동적인 초파리의 단백질입니다8. 그것은 뉴런에서만 발현되며 NMJ의 시냅스 버튼 8,9의 발달에 중요한 역할을합니다. 야생형에서 NMJ 공정의 중심을 따라 흐르는 사상 다발은 anti-Futsch를 사용한 면역염색을 통해 시각화됩니다. NMJ의 말단에 도달하면, 이 다발은 미세소관으로 구성된 고리를 형성하거나 사상 구조를 잃게 되어, 확산 및 반점 모양(10)을 형성할 수 있다. 미세소관 고리는 일시 중지된 성장 원뿔과 관련이 있으며, 이는 미세소관 배열이 안정적임을 시사한다11. 따라서 Futsch 염색을 통해 NMJ에서 안정적인 미세소관 발달을 간접적으로 확인할 수 있습니다. 초파리 유충의 근육 세포의 크기가 크기 때문에 미세소관 네트워크를 명확하게 시각화할 수 있습니다. 미세소관 네트워크의 안정성에 영향을 미치는 요인은 미세소관의 밀도와 모양을 분석하여 찾을 수 있습니다. 동시에 근육 세포의 미세소관 네트워크 상태를 NMJ 결과와 교차 검증하여 보다 포괄적인 결론을 얻을 수 있습니다.
미세소관의 네트워크와 역학을 조사하기 위해 많은 프로토콜이 사용되었습니다. 그러나, 이러한 연구들은 종종 시험관 내 연구(in vitro studies)12,13,14,15,16에 초점을 맞추었다. 대안적으로, 일부 생체내 실험은 세포골격(17)을 검출하기 위해 전자 현미경을 이용하였다. 형광 표지된 항체 또는 화학 염료가 단백질 또는 DNA에 특이적으로 결합하는 것에 따르면, 여기에 제시된 방법을 사용하면 생체 내 개별 뉴런 수준에서 NMJ의 미세소관 네트워크를 검출할 수 있으며, 결과는 근육 세포의 관찰에 의해 확증됩니다. 이 프로토콜은 Drosophila melanogaster에서 사용할 수 있는 강력한 유전 도구와 결합할 때 간단하고 안정적이며 반복 가능하여 생체 내 신경계에서 미세소관 네트워크 조절 단백질의 역할에 대한 다양한 표현형 검사 및 유전자 스크리닝을 가능하게 합니다.

<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

미세소관 네트워크는 신경계의 필수 구성 요소입니다. 많은 미세소관 조절 단백질의 돌연변이는 신경퇴행성 질환에 대한 미세소관 관련 단백질 타우(Tau), 미세소관 절단 단백질 스파스틴(Spastin) 및 카타닌 60(Katanin 60)과 같은 신경 발달 장애 및 신경 발달 질환과 관련이 있습니다. 뉴런에서 미세소관 네트워크를 검출하는 것은 신경 장애의 발병 기전을 밝히는 데 유리합니다. 그러나 뉴런의 크기가 작고 축삭 미세소관 다발이 조밀하게 배열되어 있어 미세소관 네트워크를 시각화하는 것이 어렵습니다. 이 연구에서는 유충 신경근 접합부와 근육 세포의 해부 방법과 α-튜불린 및 미세소관 관련 단백질 Futsch의 면역염색을 통해 Drosophila melanogaster의 미세소관 네트워크를 시각화하는 방법을 설명합니다. 신경근 접합부는 시냅스 전후 미세소관을 모두 관찰할 수 있게 해주며, 초파리 유충의 근육 세포는 크기가 커서 미세소관 네트워크를 명확하게 시각화할 수 있습니다. 여기에서는 초파리 멜라노가스터(Drosophila melanogaster)에서 카타닌 60을 돌연변이시키고 과발현시킨 다음 신경근 접합부와 근육 세포의 미세소관 네트워크를 조사하여 신경 발달에서 카타닌 60의 조절 역할을 정확하게 밝힙니다. 그러므로, Drosophila melanogaster의 강력한 유전 도구와 결합해, 이 의정서는 매우 신경계에 있는 microtubule 네트워크 통제 단백질의 역할을 위한 유전자 검열 그리고 microtubule 역동성 분석을 촉진합니다.

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.

저자 스포트라이트: Understanding Microtubule Network in Drosophila Neuromuscular Junctions 

초파리 유충 신경근 접합부 및 근육 세포를 사용하여 미세소관 네트워크 시각화

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.

우리는 신경근 접합부(NMJ)와 근육 세포 모두에서 미세소관 네트워크를 시각화하기 위한 단계별 절차를 시연했습니다. 개략도(그림 1A-E)에 따라 해부한 후 면역염색을 수행하고 레이저 컨포칼 현미경 또는 입체 형광 현미경(그림 1F,G)으로 이미지를 관찰 및 수집합니다.
NMJ의 시냅스 전후 미세소관 조직 모두...

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

여기에서는 신경근 접합부와 근육 세포의 미세소관 네트워크를 시각화하기 위한 자세한 프로토콜을 제시합니다. Drosophila melanogaster의 강력한 유전 도구와 결합된 이 프로토콜은 신경계에서 미세소관 네트워크 조절 단백질의 역할에 대한 유전자 스크리닝 및 미세소관 역학 분석을 크게 용이하게 합니다.

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

현재 미세소관 역학은 EB1과 같은 미세소관 결합 단백질을 통해 간접적으로 관찰됩니다. 알파벳 튜불린 헤테로이디머는 세포질에 풍부하기 때문에 직접 접히기 때문에 인접한 튜불린 헤테로다이머는 생체 내 미세소관 네트워크의 역학이 부족합니다. 당사의 프로토콜은 신경근 시냅스와 근육 세포의 미세소관을 시각화합니다.Drosophila melanogaster에서 사용할 수 있는 강력한 유전 도구와 결합하면 다양한 표현형 검사 및 생체 내 신경계에서 미세소관 네트워크 조절 단백질의 역할에 대한 유전자 스크리닝이 가능합니다. 우리의 연구는 세포골격과 신경 발달 사이의 관계와 신경 질환에 대한 영향에 중점을 둡니다. 우리는 세포골격 조절 단백질이 뉴런에 미치는 영향을 강조함으로써 신경 발달의 메커니즘과 신경 질환에 대한 단형성을 발견하기를 희망합니다.

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.

연구

신경과학

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.

초파리 미세소관 시각화를 위한 유충 해부 및 고정

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.

미세소관 시각화를 위한 초파리 유충의 면역조직화학(Immunohistochemistry of Drosophila Larva for Microtubule Visualization)

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.