drosophila larval dissection and fixation for microtubule visualization

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

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.

Watch this Scientific Journal Video about Drosophila Larval Dissection and Fixation for Microtubule Visualization at JoVE.com

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

徘徊するショウジョウバエの3番目の幼虫を長い鈍い鉗子を使って選ぶことから始めます。幼虫を血リンパ様生理食塩水で洗い、ワイプで乾かします。次に、実体顕微鏡の下の解剖皿に幼虫を背側または腹側に配置します。観察する組織に応じて、背側または腹側の解剖を選択します。幼虫の口のフックと尾を固定して、長い位置を維持します。次に、幼虫が乾燥するのを防ぐために、血リンパ様の生理食塩水を一滴加えます。解剖ハサミを使用して、完全に切断せずに後端に近い小さな横切開を行います。腹側正中線に沿って切断し、前端に向かって移動します。次に、4つの昆虫ピンをそれぞれ幼虫の角に挿入します。幼虫の伸びを最大化するために、昆虫ピンに必要な調整を行います。鉗子を使用して、筋肉への害を避けながら内臓を優しく切除します。幼虫を固定するには、死骸を解剖皿に貼り付けたまま、100Lの4%パラホルムアルデヒドに浸します。この後、ピンを取り外します。次に、0.2%PBSTで満たされた2 mLの微量遠心チューブにサンプルを移します。チューブをシェーカーの上に15RPMで10分間置きます。

drosophila-larval-dissection-fixation-for-microtubule

Research

JoVE Journal

Neuroscience

Drosophila Larval Dissection and Fixation for Microtubule Visualization

217 ビュー.  Hubei University. 徘徊するショウジョウバエの3番目の幼虫を長い鈍い鉗子を使って選ぶことから始めます。幼虫を血リンパ様生理食塩水で洗い、ワイプで乾かします。次に、実体顕微鏡の下の解剖皿に幼虫を背側または腹側に配置します。観察する組織に応じて、背側または腹側の解剖を選択します。幼虫の口のフックと尾を固定して、長い位置を維持します。次に、幼虫が乾燥す...

ビデオ: 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

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.

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

神経科学

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.

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.

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

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