Name: 3d mitochondrial ultrastructure of drosophila indirect flight muscle revealed by serial-section electron tomography
Uploaded: 2017-12-19T13:00:00.000+00:00
Duration: 6 min 45 s
Description: Watch this Scientific Journal Video about 3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography at JoVE.com

这项研究是在细胞和有机体生物学研究所和台湾台北中央研究院的低温 em 核心进行的。这项工作得到了中央研究院和大多数的支持。

1. 使用振动叶片切片的切片果蝇组织
<ol>
	<li>麻醉果蝇在冰上, 将每只苍蝇浸泡在1毫升的4% 低熔点琼脂糖的磷酸盐缓冲液中。允许琼脂糖凝固在冰上。通常, 4-6 只苍蝇被处理。</li>
	<li>使用振动刀片切片的部分琼脂糖凝胶嵌入果蝇到切片100µm 厚度和浸泡在定影液中含有2.5% 戊二醛在0.1 米磷酸缓冲液。 注: Vibratome 切片是首选, 因为组织结构保持更完整的比其他方法。另外, 解剖镊子可用于解剖 IFM 的固定液中含有2.5% 戊二醛在0.1 米磷酸缓冲液。</li>
</ol>
2. 用高压冷冻和冷冻置换 (HPF/FS) 法制备 EM 试样
<ol>
	<li>在磷酸盐缓冲液的3滴 (150 µL) 中洗涤组织切片, 其次是2滴 (100 µL) 的磷酸盐缓冲液和 20% BSA。然后在金载体中放置部分, HPF 填充有缓冲液和 20% BSA。</li>
	<li>根据用户手册将载有携带者的样品装入高压冷藏箱。</li>
	<li>冷冻后, 在液氮下释放托架, 并将其转移到冷冻替代装置, 冷却到-140 ° c。</li>
	<li>执行表 1中所示的冻结替代协议, FS 鸡尾酒含有2% 戊二醛、2% 锇氧化和0.1% 铀丙酮乙酸酯。</li>
	<li>小心地从携带针的载体上取出标本, 并在室温下将试样嵌入树脂中。聚合树脂在65° c 为16小时。 注意: 应修改 FS 协议以准备其他类型的示例。HPF/FS 是保存超微结构和减少细胞含量损失的首选。<ol>
		<li>或者, 应用化学固定协议。用2.5% 戊二醛固定标本过夜, 用缓冲器冲洗, 然后用1% 锇氧化 2 h. 洗涤和脱水与上升的乙醇浓度, 然后渗透和嵌入在 Spurr 的树脂在65° c 聚合前16h</li>
	</ol>
	</li>
</ol>
3. 制备电子断层扫描标本的系列切片
<ol>
	<li>修剪标本块, 露出所需的块面, 其中含有组织。</li>
	<li>预处理金颗粒 (直径 10 nm) 与 1% BSA, 30 分钟洗涤和悬浮在 PBS 缓冲区中的金粒子。覆盖在铜槽网格上涂上碳膜的金颗粒, 以创建基准标记。</li>
	<li>在透射电镜下检查, 以获得足够的基准标记 (至少 5-10 标记) 在层析成像的视野。</li>
	<li>切割串行部分, 200-250 nm 的厚度, 使用 ultramicrotome。</li>
	<li>在插槽网格上使用一个完美的环形截面来收集串行部分。</li>
	<li>染色的部分与雷诺的铅柠檬酸10分钟。</li>
	<li>在剖面顶部覆盖第二层的基准金粒子。</li>
</ol>
4. 收集双倾斜电子断层扫描
<ol>
	<li>将网格加载到双轴层析成像支架上, 并插入到 200 kV 的透射电子显微镜中。</li>
	<li>将显微镜对准 eucentric 焦点。</li>
	<li>设置自动数据收集软件。调整和对齐电子束在多尺度成像设置。有关操作详细信息, 请参阅用户手册10 (图 2)。</li>
	<li>获取相机黑暗和明亮的参考在一个空白的区域没有碳膜下的断层扫描收集设置。</li>
	<li>在低放大率下收集栅格地图集。选择序列切片上的线粒体作为断层扫描收集的目标。</li>
	<li>获取一个倾斜系列, 从-60 °到 + 60 °, 每个目标的轴上2递增。 注意: 倾斜角度将被机械约束的标本持有人的设计。支架将在高倾角下阻挡光束。</li>
	<li>要收集第二个倾斜系列, 请旋转试样托架90°。获得一个新的地图集。选择相应的位置, 并获取每个目标轴-b 上的倾斜系列。 注: 其他软件包 (如 SerialEM 和 Xplore3D) 可用于自动数据收集。</li>
</ol>
5. 使用软件重构3D 断层和分段子卷
<ol>
	<li>使用 IMOD 软件11重构双轴倾斜图像的断层。
	<ol>
		<li>有关操作细节, 请参阅用户手册。</li>
		<li>将单个倾斜级数与黄金基准标记的位置对齐。通过后向投影方法或由轴 a 和轴 B 的同时迭代重建技术 (SIRT) 方法 (图 3) 重新构造断层。</li>
		<li>结合轴 a 和轴 B 的断层, 生成 double-tilt 图, 减少缺少楔形工件。</li>
		<li>联合 double-tilt 断层的序列部分, 以获得覆盖整个线粒体体积的重建。通过 IMOD 程序对串行段之间的间隙进行建模。</li>
		<li>将连接的断层和纸盒修剪为所需的卷分割大小。</li>
	</ol>
	</li>
	<li>使用3D 软件分析连接的串行断层。
	<ol>
		<li>有关操作细节, 请参阅用户手册。</li>
		<li>用高斯滤镜 (或其他所需方法) 过滤断层, 以改善特征对比度, 降低背景密度。</li>
		<li>对线粒体的超微结构进行人工或自动分割。</li>
		<li>显示细分模型以允许在3D 中进行检查。</li>
		<li>使用3D 中可用的工具生成影片。</li>
	</ol>
	</li>
</ol>

<ol>
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作者声明他们没有竞争的金融利益。

在本协议中, 我们描述了一个优化的工作流程, 应用序列切片电子断层扫描研究的3D 线粒体超微结构的果蝇间接飞行肌肉。在样品中保存超微结构是这类分析的主要技术挑战。为了更好地保存超微结构, 包括两个方法步骤。首先, 用振动叶片切片切片对组织进行取样, 以尽可能地保持组织结构。其次, 对 HPF/FS 协议进行了优化, 以保留细胞的超微结构, 在准备嵌入标本块。试样在高压下被冻结, 降低了水的冰点, 减少了冰晶的形成, 破坏了超微结构的1。厚度为0.1 毫米的试样可以瞬间被玻璃化, 然后进行冷冻置换, 生成标本块进行 EM 分析。与化学固定方法相比, HPF/FS 对超微结构的保存有明显改善。利用这一系列步骤进行样品制备, 线粒体双层膜和嵴膜的保存得到了明显的改善。
获取试样的连续截面是该方法最具挑战性的步骤。由于电子束的穿透力有限, 因此该截面的厚度分别限制为 250 nm 或 500 nm, 使用 TEM 运行在200伏或300伏。由于线粒体的厚度可能超过2µm, 因此需要连续的部分才能获得完整的体积重建。但是, 恢复足够数量的串行部分来跨越整个细胞是一个技术难题。在梯形的基座之间尽可能平整地修剪块面, 可以使插槽网格容纳更多的截面, 从而覆盖较大的卷。此外, 对于精简部分, 使用一个完美的循环会增加将串行部分转移到网格的成功率。
串联电子断层扫描可以用标准的 EM 核心设备来完成。然而, 由于技术的限制, 该方法有一些不可避免的局限性。一是材料不可避免地在连续的区段之间丢失, 在联接的重建留下空白。第二是缺少的楔形工件, 这是由于有限的倾斜角度, 是可以实现的。这种限制发生的原因是, 试样持有者不能完全旋转而不堵塞电子束。尽管有这些限制, 连续切片电子断层扫描提供了足够的分辨率, 以揭示细胞和器官的超微结构在3D。
对于较小规模的成像, 低温电子断层扫描是一种新兴的技术, 可用于获得结构的大分子复合体和程序集原位在 nm 或 sub-angstrom 分辨率与 sub-tomographic 结合重建3。在这种应用中, 细胞通过切片或聚焦离子束在液氮下铣削而变薄。断层是在低温条件下收集的, 在这种情况下, 分子结构保存在靠近原生状态的地方, 没有化学固定、脱水或嵌入。在天平的另一端, 以牺牲分辨率为代价来分析大的组织体积, 串口扫描电子显微镜是一种吸引人的方式, 尽管它需要特定的仪器4。

电子显微镜是一个有价值的工具, 研究的结构上下文的亚细胞组装和设备, 执行蜂窝过程。方法已经开发, 以保持组织或细胞的超微结构, 无论是通过化学固定醛或高压冷冻 (HPF) 后, 冷冻置换 (FS)1,2。嵌入的标本块, 然后可以切片, 染色, 并观察用透射电子显微镜 (TEM)。HPF 标本也可以在低温条件下处理, 如冷冻切片或聚焦离子束 (纤维蛋白原) 铣削, 并通过低温 EM3,4观察。
虽然薄片 EM 提供了丰富的形态学洞察力, 结果2D 图像只能揭示特定截面的超微结构。在3D 体积中组织超微结构的方法仍然模糊不清。为了使三维的细胞超微结构可视化, 在获得了一系列倾斜图像并将其投影到生成层析重建5 (图 1) 的情况下, 开发了一种电子断层扫描方法。双倾斜系列可通过旋转样品90°并获得第二倾斜系列来收集。这将减少因取样角度有限而导致的缺少楔形工件, 并提高图的分辨率。
在这里, 我们描述了用于研究果蝇间接飞行肌 (IFM)6、7、8、9的连续切片电子断层扫描的应用。.为了获得覆盖整个线粒体 (大约2.5 µm 厚) 的3D 重建, 序列切片从果蝇IFM 组织块获得。每个部分的断层使用自动数据收集软件单独收集。层析重建和串行断层与 IMOD 包, 以获得重建体积的整个线粒体。通过3D 软件对加入的断层进行了分析。对线粒体嵴的密度进行了细分, 产生了一个在三维度中揭示组织的分割模型。

<table><tbody><tr><td>vibrating blade microtome</td><td>Leica</td><td>VT1200S</td><td>Tissue sectioning</td></tr><tr><td>high-pressure freezer</td><td>Leica</td><td>EM HPM100</td><td>Specimen preparation</td></tr><tr><td>freeze-substitution device</td><td>Leica</td><td>EM AFS2</td><td>Specimen preparation</td></tr><tr><td>ultramicrotome</td><td>Leica</td><td>EM UC7</td><td>Ultra-thin sectioning</td></tr><tr><td>dual-axis tomography holder</td><td>Fischione</td><td>Model 2040</td><td>tomography collection</td></tr><tr><td>transmission electron microscope</td><td>FEI</td><td>Tecnai F20</td><td>tomography collection</td></tr><tr><td>CCD</td><td>Gatan</td><td>UltraScan 1000</td><td>tomography collection</td></tr><tr><td>Leginon</td><td>NRAMM/AMI</td><td>tomography collection</td><td></td></tr><tr><td>IMOD</td><td>Boulder Laboratory for 3-D Electron Microscopy of Cells</td><td>Tomography reconstruction&amp;nbsp;</td><td></td></tr><tr><td>Avizo 3D</td><td>FEI</td><td></td><td>Tomography analysis</td></tr></tbody></table>

3d mitochondrial ultrastructure of drosophila indirect flight muscle revealed by serial-section electron tomography

线粒体是细胞的动力, 产生 ATP, 脂质和代谢物, 以及调节钙稳态和细胞死亡。独特的嵴丰富的双膜超微结构的这个细胞是优雅的安排, 以执行多种功能的划分生物分子。线粒体超微结构与各种功能密切相关;然而, 这些结构-函数关系的细微细节才刚刚开始被描述。在这里, 我们展示了在果蝇间接飞行肌肉中应用的序列切片电子断层扫描来阐明线粒体结构。串联电子断层扫描可用于研究三维的任何细胞结构。

应用序列切片电子断层扫描技术分析线粒体嵴的结构特征, 反映其能量状态和衰老。我们发现,果蝇IFM 的线粒体在 3D (图 4)7中形成一个集成的嵴和矩阵网络。此外, 突变蝇与线粒体 DNA 复制缺陷和加速衰老表型积累线粒体, 其中包含洋葱状旋转核心 (图 5)7的分部。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56567/56567fig1.jpg"> 
图 1: 从倾斜级数中重建电子断层的图示.在实验中, 当物体倾斜到不同程度时, 电子束通过3D 物体传递。对于每个倾斜条件, 将生成一个2D 投影并用照相机捕获。然后将2D 投影投影到基于中心切片定理的3D 对象重建中。在插图中, 对于在不同倾角下投射到单个尺寸中的原始2D 图像, 将显示相同的过程。然后使用1D 投影重建2D 图像。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56567/56567fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56567/56567fig2.jpg"> 
图 2: 自动数据收集一个图像查看器显示的软件显示的地图集的一个槽网包含串行部分, 多尺度目标的线粒体和获得倾斜图像。缩放栏 = 500 µm (左上面板), 100 µm (左下面板), 1 µm (右面板)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56567/56567fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56567/56567fig3.jpg"> 
图 3: 在果蝇中的单个线粒体的序列剖面电子断层扫描间接飞行肌肉。() 2D 显微和 (B) 3D 断层从覆盖整个线粒体体积的串行部分。(C) 连接的串行节断层被投影以创建纵向剖面, z-axis 垂直显示。值得注意的是, 组织切片导致材料丢失, 在连接的断层之间留下间隙。缩放条形图 = 500 nm。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56567/56567fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56567/56567fig4.jpg"> 
图 4: 3D 中显示的集成 intra-mitochondria 嵴和矩阵网络.(A) 线粒体电子断层重建切片, 显示通过 z-axis 的层状膜之间的开关。(B) 右手和/或 left-handed 螺旋的观察到的嵴开关模式的图示。(C) 层析分割, 在 3D (D) 中说明了 left-handed 螺旋的嵴开关模式, 并对分割模型 (E, F) 渲染了颜色。层析切片显示横向基质 70-100 (暗密度, 标记红色) 横跨嵴膜 (白色密度)。(G) 图的分割模型 (E) 显示横向矩阵 70-100 (以红色表示) 和具有代表性的嵴 (灰色)。标尺 = 50 毫微米 (A), 200 毫微米 (C) 和300毫微米 (D, E, F, G)。图为转载于江et al.7 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56567/56567fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56567/56567fig5.jpg"> 
图 5: 在老化期间线粒体 DNA 复制缺陷的果蝇中积累的具有洋葱状旋转芯的线粒体.具有代表性的层析切片和相应的分割 (右面板) 显示一个横截面视图 (顶部) 和纵向剖面视图 (底部) 的漩涡核心。通过任意颜色渲染来表示体积分割, 以突出旋涡的核心。缩放栏: 200 nm。图为转载于江et al.7 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56567/56567fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>步</td>
			<td colspan="2">临时</td>
			<td colspan="2">时间</td>
			<td colspan="2">解决</td>
		</tr>
		<tr>
			<td>1</td>
			<td colspan="2">-140 ° c 到-9 ° c</td>
			<td colspan="2">30分钟</td>
			<td colspan="2">液氮</td>
		</tr>
		<tr>
			<td>2</td>
			<td colspan="2">-90 ° c</td>
			<td colspan="2">96小时</td>
			<td colspan="2">FS 鸡尾酒</td>
		</tr>
		<tr>
			<td>3</td>
			<td colspan="2">-90 ° c 到-60 ° c</td>
			<td colspan="2">6小时 (5 ° c/小时)</td>
			<td colspan="2">FS 鸡尾酒</td>
		</tr>
		<tr>
			<td>4</td>
			<td colspan="2">-60 ° c</td>
			<td colspan="2">12小时</td>
			<td colspan="2">FS 鸡尾酒</td>
		</tr>
		<tr>
			<td>5</td>
			<td colspan="2">-60 ° c 到-25 ° c</td>
			<td colspan="2">7小时 (5 ° c/小时)</td>
			<td colspan="2">FS 鸡尾酒</td>
		</tr>
		<tr>
			<td>6</td>
			<td colspan="2">-25 ° c</td>
			<td colspan="2">12小时</td>
			<td colspan="2">FS 鸡尾酒</td>
		</tr>
		<tr>
			<td>7</td>
			<td colspan="2">-25 ° c 到0° c</td>
			<td colspan="2">5小时 (5 ° c/小时)</td>
			<td colspan="2">FS 鸡尾酒</td>
		</tr>
		<tr>
			<td>8</td>
			<td colspan="2">0° c</td>
			<td colspan="2">1小时 x 3 次</td>
			<td colspan="2">丙酮</td>
		</tr>
		<tr>
			<td>9</td>
			<td colspan="2">房间温度</td>
			<td colspan="2"></td>
			<td colspan="2">树脂渗入</td>
		</tr>
	</tbody>
</table>
表 1: 冻结替代议定书。

Watch this Scientific Journal Video about 3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography at JoVE.com

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

连续切片电子断层扫描显示的 3D果蝇间接飞行肌的线粒体超微结构研究

在本协议中, 我们展示了在果蝇间接飞行肌肉中应用的序列切片电子断层扫描来阐明线粒体结构。

Mitochondria are cellular powerhouses that produce ATP, lipids, and metabolites, as well as regulate calcium homeostasis and cell death. The unique ...

3d-mitochondrial-ultrastructure-drosophila-indirect-flight-muscle

Nanyang Technological University

Research

JoVE Journal

Biology

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

8.5K 次观看.  National Taiwan University. 在本协议中, 我们展示了在果蝇间接飞行肌肉中应用的序列切片电子断层扫描来阐明线粒体结构。

视频: 3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (&#60;48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (&#62;48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis.

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development.

解剖果蝇黑色素组飞行肌肉为奥米克斯方法

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and ...

科研

遗传学

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

在体内果蝇蛹肌肌腱形态的影像学研究

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

发育生物学

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

使用微计算断层扫描的 果蝇黑色素加斯特 全动物成像

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

生物学

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at the Drosophila NMJ enables researchers to acquire synaptic information, encompassing both quantitative data on synapse abundance and detailed insights into their morphology. However, the diffuse distribution and limited visual range of the TEM present challenges for the ultrastructural analysis. This study introduces an innovative and efficient sample preparation method that surpasses the conventional approach. The procedure begins by placing a metal mesh at the base of a flat-bottomed bottle or test tube, followed by positioning fixed larvae samples onto the mesh. An additional mesh is placed over the samples, ensuring that they are positioned between the two meshes. The fixed samples are thoroughly dehydrated and infiltrated before proceeding with the embedding procedure. Then embedding of the samples in epoxy resin is performed in a flat sheet manner, which allows for the preparation of muscles for positioning and sectioning. Benefiting from these steps, all the muscles of Drosophila larvae can be visualized under light microscopy, therey facilitating subsequent positioning and sectioning. Excess resin is removed after locating the 6th and 7th muscles of body segments A2 and A3. Serial ultra-thin sectioning of the 6th or 7th muscle is performed.

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

This report provides a new sample preparation procedure for visualizing neuromuscular junctions in Drosophila Larvae. This method is more effective in preventing the curling of the samples compared to the traditional method and is particularly useful for Drosophila neuromuscular junction ultrastructural analysis.

优化 果蝇 幼虫神经肌肉接头透射电子显微镜样品制备工艺

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at ...

神经科学

观察 果蝇 神经肌肉接头和肌肉细胞中的微管网络

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.

作者聚焦:了解果蝇神经肌肉接头中的微管网络 

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.

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

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

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

研究线粒体结构与功能<em&gt;果蝇</em&gt;卵巢

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

连续切片电子断层扫描显示的 3D果蝇间接飞行肌的线粒体超微结构研究

摘要

探索更多视频

此视频中的章节

解剖果蝇黑色素组飞行肌肉为奥米克斯方法

在体内果蝇蛹肌肌腱形态的影像学研究

使用微计算断层扫描的果蝇黑色素加斯特全动物成像

石蜡包埋及冷冻的部分果蝇成人肌肉

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

优化果蝇幼虫神经肌肉接头透射电子显微镜样品制备工艺

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

研究线粒体结构与功能

连续切片电子断层扫描显示的 3D果蝇间接飞行肌的线粒体超微结构研究

摘要

探索更多视频

此视频中的章节

解剖果蝇黑色素组飞行肌肉为奥米克斯方法

在体内果蝇蛹肌肌腱形态的影像学研究

使用微计算断层扫描的 果蝇黑色素加斯特 全动物成像

石蜡包埋及冷冻的部分果蝇成人肌肉

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

优化 果蝇 幼虫神经肌肉接头透射电子显微镜样品制备工艺

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

研究线粒体结构与功能

使用微计算断层扫描的果蝇黑色素加斯特全动物成像

优化果蝇幼虫神经肌肉接头透射电子显微镜样品制备工艺

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络