Name: 著者スポットライト:ショウジョウバエの神経筋接合部における微小管ネットワークの理解
Uploaded: 2023-10-20T14:00:00
Description: Watch this Scientific Journal Video about Understanding Microtubule Network in Drosophila Neuromuscular Junctions at JoVE.com

論文に関する議論とコメントをしてくれたYing Xiong博士に感謝します。この研究は、中国国家科学基金会(NSFC)からC.M.(31500839)への助成金によって支援されています。

1.幼虫の解剖
注:解剖液の血リンパ様生理食塩水(HL3.1)18と固定液4%パラホルムアルデヒド(PFA)19,20は、温度が低すぎると微小管が解重合するため、室温で使用します。
<ol><li>長い鈍い鉗子を持つ徘徊する3番目の 幼虫を選びます。HL3.1で洗浄し、実体顕微鏡下の解剖皿に置きます。 注:徘徊...

ショウジョウバエの神経筋接合部および筋細胞における微小管ネットワークの表示

<ol>
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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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ここでは、 ショウジョウバエ の幼虫の神経筋接合部および筋肉細胞の解剖および免疫染色に関するプロトコルについて説明します。考慮すべき重要なポイントがいくつかあります。まず、解剖プロセスでは、観察された筋肉の損傷を避けることが重要です。鉗子と筋肉の直接接触を防ぐために、内臓を切除する前にフィレットを固定する価値があるかもしれません。筋肉の損傷や幼?...

微小管(MT)は、細胞骨格を構成する構成要素の1つとして、細胞分裂、細胞増殖と運動性、細胞内輸送、細胞形状の維持など、さまざまな生物学的プロセスにおいて重要な役割を果たしています。微小管の動態と機能は、MAP1、MAP2、タウ、カタニン、キネシンなどの他のタンパク質との相互作用によって調節されます1,2,3,4,5。
ニューロンでは、微小管は軸索と樹状突起の発達と維持に不可欠です。微小管の異常は、機能不全やニューロンの死さえも引き起こします。例えば、アルツハイマー病患者の脳では、タウタンパク質の過剰リン酸化により微小管ネットワークの安定性が低下し、神経学的不規則性が引き起こされます6。したがって、微小管ネットワークを調べることは、神経発達と神経疾患の病因の理解に貢献します。
神経筋接合部(NMJ)は、運動ニューロンの軸索末端と筋線維の間に形成される末梢シナプスであり、シナプスの構造と機能を研究するための優れた強力なモデルシステムです7。フッチはショウジョウバエのタンパク質で、哺乳類に見られる微小管結合タンパク質MAP1Bと相同である8。ニューロンにのみ発現し、NMJのシナプスボタンの発達に関与している8,9。野生型では、NMJ突起の中心に沿って走る糸状束を抗Futschで免疫染色することで可視化します。NMJの末端に達すると、この束は微小管からなるループを形成するか、またはその糸状構造を失い、びまん性および点状の外観をもたらす能力を有する10。微小管ループは一時停止した成長円錐と関連しており、微小管アレイが安定していることを示唆している11。そのため、フッチ染色によりNMJにおける微小管の安定な発生を間接的に決定することができます。ショウジョウバエの幼虫の筋肉細胞のサイズが大きいため、微小管ネットワークを明確に視覚化できます。微小管ネットワークの安定性に影響を与える要因は、微小管の密度と形状を分析することで見つけることができます。同時に、筋肉細胞の微小管ネットワークの状態をNMJの結果と交差検証して、より包括的な結論を得ることができます。
微小管のネットワークとダイナミクスを調べるために、多くのプロトコルが採用されています。しかし、これらの研究はしばしばin vitro研究に焦点を当てている12,13,14,15,16。或いは、いくつかのin vivo実験では、細胞骨格を検出するために電子顕微鏡法が用いられている17。蛍光標識抗体または化学色素のタンパク質またはDNAへの特異的結合によると、ここで紹介する方法により、NMJの微小管ネットワークをin vivoの個々のニューロンレベルで検出することができ、その結果は筋肉細胞での観察によって裏付けられています。このプロトコルは、ショウジョウバエのメラノガスターで利用可能な強力な遺伝ツールと組み合わせると、シンプルで安定しており、反復性があり、生体内の神経系における微小管ネットワーク調節タンパク質の役割について、多様な表現型検査および遺伝子スクリーニングを可能にします。

<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

微小管ネットワークは、神経系の不可欠な構成要素です。多くの微小管調節タンパク質の変異は、神経発達障害や神経疾患に関連しており、微小管関連タンパク質タウから神経変性疾患、微小管切断タンパク質スパスチンおよびカタニン60は、それぞれ遺伝性痙性対麻痺および神経発達異常を引き起こします。神経細胞内の微小管ネットワークの検出は、神経疾患の病態解明に有利である。しかし、ニューロンのサイズが小さく、軸索微小管束が密集しているため、微小管ネットワークの可視化は困難です。本研究では、ショウジョウバエの微小管ネットワークを可視化するために、幼虫の神経筋接合部と筋細胞を解剖し、α-チューブリンと微小管関連タンパク質Futschの免疫染色を行う方法について述べる。神経筋接合部はシナプス前部とシナプス後部の両方の微小管を観察することを可能にし、ショウジョウバエ幼虫の筋肉細胞のサイズは微小管ネットワークの明瞭な可視化を可能にします。本研究では、ショウジョウバエのカタニン60を変異させて過剰発現させ、神経筋接合部や筋細胞の微小管ネットワークを調べることで、神経発達におけるカタニン60の制御的役割を正確に明らかにしました。従って、ショウジョウバエのmelanogasterの強力な遺伝用具と結合されて、このプロトコルは神経系のmicrotubuleネットワークの規定する蛋白質の役割のための遺伝のスクリーニングそしてmicrotubuleの原動力の分析を非常に促進する。

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.

著者スポットライト:ショウジョウバエの神経筋接合部における微小管ネットワークの理解 

ショウジョウバエ幼虫の神経筋接合部と筋細胞を用いた微小管ネットワークの可視化

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.

私たちは、神経筋接合部(NMJ)と筋肉細胞の両方の微小管ネットワークを可視化するための段階的な手順を示しました。模式図(図1A-E)に従って解剖した後、免疫染色を行い、その後、レーザー共焦点顕微鏡または実体蛍光顕微鏡で画像を観察して収集します(図1F、G)。
NMJのシナプス前部とシナプス後部の...

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

ここでは、神経筋接合部と筋細胞の微小管ネットワークを可視化するための詳細なプロトコルを紹介します。ショウ ジョウバエのmelanogasterの強力な遺伝用具と結合されて、このプロトコルは神経系のmicrotubuleネットワークの規定蛋白質の役割のための遺伝のスクリーニングそしてmicrotubule原動力の分析を非常に促進する。

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

現在、微小管の動態はEB1などの微小管結合タンパク質を介して間接的に観察されています。アルファベットチューブリンヘテロ二量体は細胞質内に豊富に存在するため、隣接するチューブリンヘテロ二量体は生体内の微小管ネットワークのダイナミクスを欠いています。私たちのプロトコルは、神経筋シナプスと筋肉細胞の微小管を視覚化します。ショウジョウバエのメラノガスターで利用可能な強力な遺伝学的ツールと組み合わせることで、in vivoの神経系における微小管ネットワーク調節タンパク質の役割に関する多様な表現型検査と遺伝子スクリーニングが可能になります。私たちの研究は、細胞骨格と神経発達の関係、および神経疾患への影響に焦点を当てています。私たちは、細胞骨格制御タンパク質が神経細胞に及ぼす影響に着目することで、神経疾患の神経発達と単為生殖のメカニズムを解明したいと考えています。

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.

神経科学

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.

ショウジョウバエ 微小管可視化のための幼虫の解剖と固定

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.

ショウ ジョウバエ 幼虫の微小管可視化のための免疫組織化学

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.