mounting and image acquisition of drosophila larval microtubules

观察 果蝇 神经肌肉接头和肌肉细胞中的微管网络

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.

作者聚焦：了解果蝇神经肌肉接头中的微管网络 

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

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 Mounting and Image Acquisition of Drosophila Larval Microtubules at JoVE.com

果蝇幼虫微管的安装和图像采集

首先将抗体标记的果蝇幼虫尸体置于载玻片上的0.2%PBST中。在立体显微镜下进行调整，确保幼虫胴体的内表面朝上。用擦拭布吸收多余的PBST溶液。然后轻轻添加一滴抗褪色封固剂。接下来，在载玻片上放置盖玻片，缓慢而轻柔地覆盖解剖的幼虫，避免气泡形成。通过在盖玻片边缘涂抹指甲油将盖玻片固定到位。将载玻片存放在黑暗的地方，以尽量减少荧光衰减。对于图像采集，请使用带有63倍油浸物镜的激光扫描共聚焦显微镜。然后调整波长和激光功率，以最好地满足实验的要求。为了识别神经肌肉接头并捕获 A 段 3 中肌肉四的图像，选择 488 纳米激光激活 α 微管蛋白或 Futsch，选择 546 纳米激光激活 HRP 成像轨迹。修改参数以实现 1， 024 x 1， 024 像素的帧大小、1.0 的数码变焦和 0.8 微米的成像间隔。为了可视化肌肉中的微管，请捕获 A 3 至 A 5 段中肌肉 2 的图像，因为它的气管分支较少。选择 488 纳米激光激活 α 微管蛋白，选择 633 纳米激光激活 T3605 成像轨道。将成像参数调整为 1， 024 x 1， 024 像素的帧大小、2.0 的数码变焦和 0.4 微米的成像间隔。神经肌肉接头的突触前和突触后微管组织均用抗α微管蛋白标记。Futsch染色反映了突触前神经元中稳定微管的丰度。当切断蛋白Katanin 60的微管在突触前神经元中过表达时，观察到抗Futsch的信号强度降低。轴突躯干的染色强度强于枝干。Katanin 60突变导致微管环增加。Katanin 60 的过表达导致末端按钮内出现短微管碎片。α 微管蛋白染色显示细胞核周围有清晰的微管网络。肌肉细胞在Katanin 60突变体中具有显着增加的核周微管强度，并表现出更强的束。过表达导致微管纤维碎裂。

mounting-and-image-acquisition-of-drosophila-larval-microtubules

Research

JoVE Journal

Neuroscience

Mounting and Image Acquisition of Drosophila Larval Microtubules

114 次观看.  Hubei University. 首先将抗体标记的果蝇幼虫尸体置于载玻片上的0.2%PBST中。在立体显微镜下进行调整，确保幼虫胴体的内表面朝上。用擦拭布吸收多余的PBST溶液。然后轻轻添加一滴抗褪色封固剂。接下来，在载玻片上放置盖玻片，缓慢而轻柔地覆盖解剖的幼虫，避免气泡形成。通过在盖玻片边缘涂抹指甲油将盖玻片固定到位。将载玻片存放在黑暗的地方，以尽量减少荧光衰减。对...

视频: 果蝇幼虫微管的安装和图像采集

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 ...

科研

神经科学

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.

果蝇 用于微管可视化的幼虫解剖和固定

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.

果蝇幼虫的免疫组织化学用于微管可视化

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.