Name: quantitative cell biology of neurodegeneration in drosophila through unbiased analysis of fluorescently tagged proteins using imagej
Uploaded: 2018-08-03T14:00:00.000+00:00
Duration: 8 min 44 s
Description: Watch this Scientific Journal Video about Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ at JoVE.com

这项工作是支持希拉和大卫恩特神经病理性疼痛研究计划研究生奖学金 (J.M.B.), 露易丝教皇生活研究员计划 (铁匠, Y.Z., 和 J.M.B.), 斯奈德-鲁滨逊基金会 Predoctoral 奖学金 (铁匠), 约翰博士 T.麦克唐纳基金会 (铁匠), 合同, 国家卫生研究院 (NIH) HHSN268201300038C, R21GM119018 和 R56NS095893 (R.G.Z.) 的赠款, 并由泰山学者项目 (山东省, 中华人民共和国) (R.G.Z.)。

1. 图像分析实验设计的思考与准备
<ol><li>预先确定合适的解剖, 细胞, 或亚型标记, 以作为标志, 以标准化的兴趣区域 (ROI) 横跨不同的样本, 例如 4 ', 6-diamidino 2-苯基吲哚 (DAPI), 膜标记, 局部荧光蛋白等.
</li>
	<li>选择一个荧光标记, 它可以将离散 puncta 划分为兴趣结构以外的背景层。 注: 本协议是为蛋白质结构 (如错误折叠蛋白聚集) 和膜结合器 (如自噬记者) 进行优化的。为了可视化蛋白质聚集, 使用了一个 RFP 标记的 n-末端片段的人 Htt 蛋白与致病 polyQ 道138重复 (RFP-hHttQ138)5。突变 Htt 蛋白在神经元特异驱动物 (如elav-GAL45、6) 下表达时形成细胞质团聚体。为了形象化 autophagosome 溶酶体的通量, actin-GAL4被用来无所不在串联 mCherry-GFP-Atg8a 记者的驱动表达。mCherry 和 GFP 阳性 puncta 表明自噬体, 当 GFP 荧光在溶酶体酸性环境中被淬火时, 通量被监测, 酸不敏感的 mCherry 一直保持到蛋白质降解7。对于比例分析, 作为结构的一致标记的单个通道可以用于分割, 或者如果适当, 可以使用两个或多个通道的投影来描绘结构, 尽管本协议没有明确描述。在这个例子中, mCherry 是用于 Atg8-positive 粒子的分割, 因为它是酸不敏感的, 并将保持在车厢贯穿整个通路通量。</li>
	<li>下载并安装开放源码图像分析平台 ImageJ 或斐济-ImageJ 的首选分布与捆绑的插件2,3。</li>
	<li>为 h 极大交互分水岭安装插件。 注: 本议定书使用斐济;ImageJ 可能需要额外的插件。Benoit Lombardot 为超临界-MPI-CBG 开发的插件可在线提供 (https://imagej.net/Interactive_Watershed)。</li>
	<li>导航到 "帮助 |更新 ", 选择" 管理更新站点 "从" ImageJ 更新程序 ", 从列表中选择" 超临界-MPI-CBG 更新站点 ", 然后关闭并重新启动斐济。</li>
</ol>2. 脑解剖和免疫荧光染色
<ol><li>
做硅胶衬里的解剖碟。
	<ol>
<li>将硅胶元件倒入烧杯中, 由制造商指示, 搅拌良好, 直至彻底混合。</li>
		<li>用吸管填充5厘米组织培养皿1/3 至半满 (大约10毫升的弹性体混合物)。允许任何气泡上升到表面, 并用纸巾轻轻地移除。把盘子盖好, 放在一层表面, 在室温下 48–72 h。</li>
	</ol>
</li>
	<li>
解剖果蝇的大脑和视觉系统, 如果需要的话。
	<ol>
<li>执行果蝇大脑解剖, 如前所述8,9。在弹性衬里的解剖皿中进行解剖, 使用弹性体底部稳定大脑以避免损伤组织或镊子。</li>
		<li>为了在解剖过程中保持叶片完好无损, 在视网膜下滑动两个钳, 轻轻地撕裂眼睛的中间, 以去除视网膜。用与椎板表面平行的镊子拔出附着在叶片上的任何视网膜组织。 注: 残留色素将在以下步骤洗涤, 通常不会干扰抗体染色和成像。</li>
		<li>稀释37% 甲醛在磷酸盐缓冲盐水 (PBS) 使最后的集中3.7% 新鲜之前使用在500µL 离心管。用固定溶液将解剖过的大脑转移到离心管, 在 RT 中孵育15分钟, 并进行轻柔的摇摆。 注: 甲醛具有天然的氧化倾向, 可产生甲酸。因此, 建议整除37% 甲醛储存, 并稀释到3.7% 新鲜之前, 每次使用。过度固定或不固定会影响组织的完整性和荧光10。</li>
		<li>洗涤3次与 PBTx (PBS 与0.4% 海卫 X-100) 为15分钟每。</li>
	</ol>
</li>
	<li>
执行免疫组化和装标本。
	<ol>
<li>如果需要抗体染色, 去除 PBTx, 添加原抗体稀释在 PBTx 与5% 正常山羊血清, 并孵化在一个整除搅拌机过夜4摄氏度。</li>
		<li>去除原抗体, 用 PBTx 洗3次, 每次15分钟。添加二次抗体稀释在 PBTx 与5% 正常山羊血清和孵育1小时在 RT 或隔夜在4摄氏度。用 PBTx 洗3次, 每人15分钟。</li>
		<li>如果需要 DAPI 染色, 去除 PBTx, 添加 DAPI 溶液 (股票溶液: 5 毫克/毫升, 稀释到工作浓度的15µg/毫升在 PBTx), 并孵化10分钟在 RT. 每15分钟洗3次。</li>
		<li>要清除组织, 请将一滴安装介质放到显微镜幻灯片的中心, 两侧有一层透明胶带的间隔。把大脑转移到安装培养基的下落上, 等到大脑变得透明时再等待。用吸管小心地取下安装介质。</li>
		<li>要装入, 请将一滴新鲜的安装介质放到幻灯片上的大脑上。小心地覆盖一个盖子玻璃避免气泡。用橡胶水泥封边, 在 RT 上风干20分钟. 进行图像采集以获得最佳效果。</li>
	</ol>
</li>
</ol>3. 图像采集
<ol><li>优化显微镜采集参数, 包括空间分辨率、目标、变焦、扫描速度和步长, 以最大程度地解决感兴趣的结构, 以便在图像分析过程中实现半自动化分割。 注意: 在对所有实验样本进行成像之前, 捕获一个样本图像并测试半自动分割 (在本协议的步骤5中) 可能很有用。这样, 用户就可以调整成像设置, 以便分析工具可以很容易地辨别出感兴趣的生物结构。</li>
	<li>
调整图像检测器控制, 捕捉最高的信噪比和最高的动态范围的标本。
	<ol>
<li>在调整设置时, 请使用查找表 (查找表格), 指示像素饱和度 (曝光过大) 或钻井 (偏移过低) (图 3B, 红色表示由高/低的查找像素饱和度)。</li>
		<li>验证增益和偏移设置适用于所有实验组, 因为实验操作可能会改变兴趣结构。 注意: 所有组都必须使用相同的设置进行量化比较。</li>
	</ol>
</li>
	<li>图像通过组织捕获所有数据。 注意: 建议在一个映像会话期间捕获所有可用数据, 并在需要时在步骤4中的 ROI 选择过程中定义更精确的区域。</li>
	<li>将图像保存为影像软件支持的文件类型, 以保留元数据 (如采集参数和空间校准)。保存图像的文件名包括实验日期、遗传学背景、荧光记者、抗体或与扫描的通道相对应的染料, 如 [实验组名称] _ [标本名] _ [实验日期] _Ch1. [荧光-目标/染料] _Ch2 [荧光靶/染料]等。
</li>
</ol>4. 斐济/ImageJ 图像导入和 ROI 选择
<ol><li>在斐济打开图像。使用 "生物格式导入选项" 对话框, 选择 "查看堆栈: Hyperstack" 并设置 "颜色模式: 灰度"。 注: 建议打开显微镜文件类型, 如空间标定等元数据可由斐济阅读。</li>
	<li>
从单个 z 平面或基于标记通道 (可用作跨样本的标准 roi) 的投影标识区域 (图 1, ROI 选择)。
	<ol>
<li>使用 "C" 滚动条查看用于捕获步骤1.1 中指定的标记的通道。</li>
		<li>使用 "Z" 滚动条在焦点平面中移动。选择单个切片或通过单击 "图像 |" 来创建多个切片的投影。栈 |Z 项目 "并设置" 开始切片 "和" 停止切片 "以包含 ROI。 注意: 将 " 投影类型" 设置为 "Z 项目" 对话框中的 "最大强度" 通常最适合强调5节中的细分结构, 尽管这可能不适用于所有应用程序。如果分析需要另一种类型的投影, 请注意保存并记录此文件, 以便在6和/或7节中提取特征。</li>
		<li>生成包含感兴趣的信道和焦点平面的新图像, 以简化图像分析协议中的以下步骤。选择 "图像 |重复 ", 设置所需的" 通道 "(c) 和" 切片 "(z), 并更改文件名以反映所选内容 (例如, [实验组名称] _ [样本名称] _ [实验日期] _ [通道] _ [z 平面])。</li>
	</ol>
</li>
	<li>使用斐济工具栏上的 "选择工具" 之一, 根据步骤4.2 中确定的选择标准手动选择标准化的 ROI。</li>
	<li>单击 "分析", 将 roi 添加到 roi 管理器中。工具 |roi 管理器 ", 然后单击" roi 管理器 "菜单上的" 添加 "。单击 "更多 |", 从 "roi 管理器" 菜单中保存 roi保存 ", 然后命名该文件以反映 ROI (例如, [实验组名称] _ [样本名称] _ [实验日期] _ [频道] _ [z 面] _ [ROI 说明])。 注: 这可以在斐济重新开放, 以便以后进行分析, 也可以应用于5.3 节中的其他渠道或图像。</li>
</ol>5. h 极大 Watershedding 的预处理与分割
<ol><li>
对用于捕获感兴趣结构的通道执行预处理。
	<ol>
<li>如果图像文件由多个通道组成, 请通过单击 "图像 |" 来分隔通道。颜色 |分割渠道 ", 以隔离的渠道利益。</li>
		<li>应用一个过滤器, 如中值过滤器, 以减少噪音或高斯模糊的平滑通过单击 "过程 |过滤器 |过滤器类型 "(例如," 中值滤波 "或" 高斯模糊 ")。<ol>
<li>在每个实验组的图像上取样不同的过滤器和过滤器参数。从每个组中的一个典型示例开始步骤 6.2, 以检查分割性能。选择便于细分利益结构的筛选器和设置。</li>
		</ol>
</li>
		<li>记录筛选器和设置。在实验组中应用这些参数。</li>
		<li>将图像的副本另存为 tiff 文件, 包括应用的筛选器以供参考。使用详细名称, 如 [实验组名称] _ [样本名称] _ [实验日期] _ [通道] _ [z 面] _ [带参数的过滤器]。</li>
	</ol>
</li>
	<li>
在步骤5.1 中生成并保存的预处理图像上, 使用交互式的 h 极大值分水岭工具对感兴趣的结构进行分割。
	<ol>
<li>利用在斐济的预处理图像, 通过点击 "超临界值" 来启动 h 极大交互分水岭工具;标签 |交互式 H_Watershed "。 注意: 这个插件首先计算分水岭, 然后允许用户通过一个即时更新的输出图像来探索局部极大值和门限选项对分割的影响。</li>
		<li>从控制面板, 调整种子动力学 (h), 强度阈值 (T), 峰值洪水 (%), 以优化分割。 注: 在4.2 节预处理前, 请始终参考原始图像, 以手动检查感兴趣结构的细分性能。</li>
		<li>当对分割结果满意时, 选择 "导出区域掩码" 并选择 "导出"。这将生成分水岭结果的二进制图像 (图 1, 分割)。</li>
		<li>记录分割参数;这些参数需要在实验组中应用以进行定量比较。保存二进制结果掩码 (如果需要参考), 并在名称 (如 [实验组名称] _ [样本名称] _ [实验日期] _ [通道] _ [z 面] _ [H_Watershed 参数] _ 中详细描述了细分参数。</li>
	</ol>
</li>
	<li>
从分水岭结果中提取出感兴趣的结构。
	<ol>
<li>在 ROI 管理器中打开步骤4.4 中保存的 roi。使用步骤5.2.4 活动中的二进制区域掩码, 从 roi 管理器中选择要应用于图像的 roi, 以将特征提取限制为标准化的 ROI。</li>
		<li>利用 ROI 在二进制分水岭掩码上活动, 选择 "分析 |在对话框中选择 "添加到 Manager" 来分析粒子以提取特征。 注意: 这将为细分期间为 ROI 管理器划分的每个结构添加一个粒子标识符。此菜单上的选项可在需要时根据大小或循环来排除特征, 以细化分析中包含的结构。</li>
		<li>通过单击 "更多 |" 来保存粒子从 ROI 管理器菜单中保存 "。确保取消选择标识符, 所有粒子都将保存在 zip 文件中。使用一个详细的名称, 如 [实验组名称] _ [样本名称] _ [实验日期] _ [通道] _ [z 面] _ [用参数过滤] _ [H_Watershed 参数] _ [结构]。</li>
	</ol>
</li>
</ol>6. 数量、面积和强度的量化和分析
<ol><li>打开生成并保存在步骤4.2 中的原始映像。滚动到用于捕获感兴趣结构的通道。 注意: 从原始图像 (预处理前) 进行强度测量非常关键, 因为在步骤5.1 中应用滤镜会更改像素值。</li>
	<li>打开步骤5.3 中从特征提取获得的粒子, 然后从 ROI 管理器中选择 "显示全部"。 注意: 此操作将将 "ROI 管理器" 上每个粒子的轮廓叠加到图像上, 并应匹配感兴趣结构的边缘 (图 1, 特征提取)。</li>
	<li>通过单击 "分析 |" 来设置所需的度量值设置测量 "。 注意: 粒子的面积、强度和密度可以通过选择 "区域" 和 "集成密度" 来测量。当选择集成密度时, 斐济将产生两个值: "集成密度", 计算为区域的乘积和平均值的灰度值和 "原始集成密度", 测量为像素值的总和。荧光蛋白在粒子中的密度被计算为除以区域的像素值的总和。步骤5.3 中生成的粒子数表明步骤4.4 中定义的组织 ROI 中的粒子数量。</li>
	<li>从 ROI 管理器菜单中选择 "度量"。只要测量是从图像软件中从斐济获得的文件中提取的, 结果就用校准单位表示。从结果窗口复制结果并粘贴到电子表格软件中进行编译和进一步计算。</li>
</ol>7. 比例量化和数据分析
<ol><li>按照步骤6.1 和 6.2, 打开感兴趣的第一个原始通道上的粒子标识符。</li>
	<li>通过单击 "分析 |" 来设置所需的度量值设置测量 "。 注: 通过选择 "平均灰度值" 可以测量每个粒子的平均强度。</li>
	<li>从 ROI 管理器中选择 "度量"。从结果窗口复制数据并粘贴到电子表格软件中。</li>
	<li>将 "C" 滚动条移至感兴趣的第二个原始通道, 此示例中的 GFP (图 4A), 然后重复步骤7.2。和7.3。 注意: 在步骤5.3 中保存的粒子将默认为生成它的通道和切片。注意确保粒子被应用到正确的感兴趣的通道上。</li>
	<li>计算电子表格软件中每个粒子的强度与第二个通道强度的比值。</li>
	<li>
使用散点图可视化和量化来自显影的比例数据, 它们反映了潜在的生物过程的个人变化。
	<ol>
<li>在图形软件中生成散点图。 注意: 每个数据点表示一个感兴趣的结构, 其中第一个荧光的强度是 "x 值", 第二个荧光的强度是从每个粒子测量的 "y 值"。</li>
		<li>为每个荧光设置浇口阈值以定义象限。</li>
	</ol>
</li>
	<li>
使用线条轮廓可视化的荧光强度跨结构的兴趣。
	<ol>
<li>选择在步骤5.3 中生成的 ROI, 并使用工具栏上的直线工具通过 roi 绘制一条直线。将行 roi 添加到 roi 管理器中。</li>
		<li>对于每个感兴趣的频道, 用行 ROI 活动, 选择 "分析 |剧情简介 "。 注意: 剧情剖面函数将生成一个直方图图和一个表的强度值沿线。复制每个通道测量的结果并粘贴到电子表格软件中, 以生成显示沿线的两个通道的图。</li>
	</ol>
</li>
</ol>

<ol>
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G., 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=NAD+synthase+NMNAT+acts+as+a+chaperone+to+protect+against+neurodegeneration.">NAD synthase NMNAT acts as a chaperone to protect against neurodegeneration.</a> Nature. 452 (7189), 887 (2008).</li><li>Li, C., 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=Spermine+synthase+deficiency+causes+lysosomal+dysfunction+and+oxidative+stress+in+models+of+Snyder-Robinson+syndrome.">Spermine synthase deficiency causes lysosomal dysfunction and oxidative stress in models of Snyder-Robinson syndrome.</a> Nat Commun. 8 (1), 1257 (2017).</li><li>Prokop, A., Meinertzhagen, I. 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作者没有什么可透露的。

此处概述的协议可用于重现性定量细胞生物学过程的荧光成像。必须仔细考虑生物环境和技术限制, 以指导实验设计。荧光标记的兴趣, 无论是免疫组织化学, 染料基础, 或基因表达, 需要区分以上背景的形态学和强度。UAS/GAL4系统广泛应用于果蝇的靶向基因表达。这里的例子使用了携带 GAL4 驱动程序的转基因线激活荧光标记蛋白的转录在无人驾驶增强器的控制下, 以调查蛋白质聚集和?...

神经退行性疾病每年影响上百万人, 发病率随着人口老龄化1而增加。尽管每种神经退行性疾病都有一个独特的病因, 错误折叠蛋白的聚集和 proteostasis 网络的崩溃是许多这些疾病的常见病理特征。阐明这些基本和相互关联的过程的中断如何导致神经元功能障碍, 细胞死亡对于理解神经退行性疾病以及指导治疗干预是至关重要的。荧光成像技术允许对神经元的这些复杂和动态过程进行调查, 并极大地促进了我们对神经细胞生物学的理解。荧光标记蛋白的分析具有挑战性, 尤其是在体内进行实验时, 由于组织高度紧凑, 细胞类型多样, 形态学异质性。人工辅助量化是负担得起和直接的, 但往往是费时的, 并受人的偏见。因此, 需要有无偏见的、可重复的和易于获取的方法来从成像研究中提取量化数据。
我们概述了一个简单和适应性的工作流程使用斐济/ImageJ, 一个强大的和自由访问的图像处理软件2,3, 从荧光成像研究中提取定量数据的实验模型变性使用果蝇。通过遵循这一协议来量化蛋白质聚集和自噬性通量-两个细胞生物学特征, 这是高度相关的神经退行性疾病病理学-我们展示了敏感性和再现这种方法。荧光标记突变体杭丁顿蛋白 (Htt) 蛋白在果蝇视神经叶中的分析揭示了蛋白质聚集的数量、大小和强度。我们在果蝇视觉系统中可视化了自噬性通量的串联荧光记者, 它根据区划环境4显示不同的发射信号。比例分析的串联记者允许从 autophagosome 的形成, 成熟和运输到降解溶酶体的自噬溶酶通量的定量和全面的看法, 并进一步强调脆弱神经退行性疾病的车厢中断。重要的是, 在这两种分析中, 我们在协议中实现了半自动阈值和分割步骤, 以最大限度地减少无意识偏差, 增加采样功率, 并提供一个标准来促进类似研究之间的比较。简单的工作流旨在使强大的斐济/ImageJ 插件 (由计算机科学家根据数学算法开发) 更容易被 neurobiologists 和生命科学社区访问。

<table><tbody><tr><td>SYLGARD(R) 184 Silicone Elastomer Kit</td><td>Dow Corning Corporation</td><td>PF184250</td><td>Dissection dish</td></tr><tr><td>Falcon 35 mm Not TC-Treated Easy-Grip Style Bacteriological Petri Dish</td><td>Corning</td><td>351008</td><td>Dissection dish</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-20</td><td>Dissection tool</td></tr><tr><td>Sodium Chloride</td><td>Sigma</td><td>S3014</td><td>PBS solution</td></tr><tr><td>Sodium Phosphate Dibasic&amp;nbsp;</td><td>Sigma</td><td>S5136</td><td>PBS solution</td></tr><tr><td>Potassium Phosphate Monobasic</td><td>Sigma</td><td>P5655</td><td>PBS solution</td></tr><tr><td>Triton&amp;nbsp;X-100</td><td>Sigma</td><td>T9284</td><td>Washing and antibody incubaton solution.&amp;nbsp;</td></tr><tr><td>37% Formaldehyde</td><td>VWR</td><td>10790-710</td><td>Fixation</td></tr><tr><td>Disposable Microcentrifuge Tubes (0.5mL, blue)</td><td>VWR</td><td>89000-022</td><td>Fixation, washing, and antibody incubaton.&amp;nbsp;</td></tr><tr><td>Plain and Frosted Micro Slides (25&amp;times;75mm)</td><td>VWR</td><td>48312-004</td><td>Slides for confocal imaging</td></tr><tr><td>Micro Cover Glasses, rectangular (22&amp;times;40mm)</td><td>VWR</td><td>48393-172</td><td>Slides for confocal imaging</td></tr><tr><td>Rubber Cement</td><td>Slime</td><td>1051-A</td><td>Mounting</td></tr><tr><td>VECTASHIELD Antifade Mounting Medium</td><td>Vector Laboratories, Inc.</td><td>H-1000</td><td>Mounting</td></tr><tr><td>Scotch Magic 810 Invisible Tape (19mm&amp;times;25.4m)</td><td>3M Company</td><td>810</td><td>Mounting</td></tr><tr><td>Normal Goat Serum</td><td>Thermo Fisher Scientific</td><td>PCN5000</td><td>Antibody incubaton</td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>D1306</td><td>Nucleic acid staining. Dissolve in deionized water to make a 5 mg/mL stock solution and store at -80&amp;deg;C. Dilute to a working concentration of 10-20 &amp;mu;g/mL in PBTx.</td></tr><tr><td>3.5X-90X Stereo Zoom Inspection Industrial Microscope</td><td>AmScope</td><td>SM-1BNZ</td><td>Dissection scope. Equipped with 6W LED Dual Gooseneck Illuminator</td></tr><tr><td>ImageJ/Fiji</td><td>NIH</td><td>v1.51u</td><td>With SCF_MPI_CBG plugins (version 1.1.2)</td></tr><tr><td>FV1000-IX81 Confocal-laser Scanning Microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Recombinant Construct</td><td>Bloomington Drosophila Stock Center</td><td>BL37749</td><td></td></tr></tbody></table>

quantitative cell biology of neurodegeneration in drosophila through unbiased analysis of fluorescently tagged proteins using imagej

随着神经退行性疾病患病率的上升, 了解导致神经元功能障碍和丧失的潜在病理生理学越来越重要。基于荧光的成像工具和技术能够对亚细胞神经生物学过程进行空前的分析, 但仍然需要有无偏见、可重现和易于获取的方法来从成像研究中提取量化数据。.我们开发了一个简单而适应的工作流程, 从荧光成像研究中提取定量数据, 利用果蝇模型变性。具体来说, 我们描述了一个易于跟踪, 半自动化的方法, 使用斐济/ImageJ 分析两个细胞过程: 首先, 我们用荧光标记突变体量化果蝇光学叶中蛋白质的总含量和剖面。杭丁顿蛋白蛋白;第二, 我们评估果蝇视觉系统的自噬溶酶通量, 并以比例为基础, 对一个串联荧光报告器进行定量化。重要的是, 这里概述的协议包括一个半自动的分割步骤, 以确保所有的荧光结构进行分析, 以尽量减少选择偏差, 并增加微妙比较的分辨率。这种方法可以推广到分析其他细胞生物学结构和过程中涉及变性, 如蛋白质 puncta (应力颗粒和突触复合物), 以及膜约束的车厢 (线粒体和膜贩运囊泡)。该方法为图像分析和量化提供了一个标准化的、可适应的参考点, 并能促进整个领域的可靠性和重现性, 最终增强了对变性的机械理解。

果蝇荧光标记突变 Htt 集的数量、面积和强度的量化
为了研究亨廷顿氏病果蝇模型的中枢神经系统中的错误折叠蛋白聚集, 用 RFP 标记的突变体 Htt 非病理 (UAS-RFP-hHttQ15) 或病理扩张 (UAS-RFP-hHttQ138) 和膜 GFP (UAS-mCD8::GFP)11是共同表达下一个泛神经元驱动elav-GAL4。根据《议定书?...

Watch this Scientific Journal Video about Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ at JoVE.com

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

我们开发了一个简单且适应性强的工作流, 从荧光成像的细胞生物学研究中提取定量数据自噬性果蝇模型中中枢神经系统蛋白质聚集和通量变性.

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

quantitative-cell-biology-neurodegeneration-drosophila-through

Research

JoVE Journal

Neuroscience

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

9.1K 次观看.  University of Miami Miller School of Medicine. 我们开发了一个简单且适应性强的工作流, 从荧光成像的细胞生物学研究中提取定量数据自噬性果蝇模型中中枢神经系统蛋白质聚集和通量变性.

视频: Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

单果蝇 Ommatidium解剖和影像

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

科研

神经科学

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

发育生物学

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

评估神经退行性表型<em&gt;果蝇</em&gt;爬坡试验和全脑多巴胺能神经元免疫组化染色

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

攀登缺陷的神经退行性疾病的果蝇模型定量分析

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

大众组织学量化在神经退行性变<em&gt;果蝇</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

监测果蝇中朊病毒样蛋白聚集物的细胞间传输

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused on neuromuscular junctions (NMJs) in Drosophila larvae, assessing synaptic integrity in adult Drosophila has received much less attention. Here we provide a straightforward method for dissection of the dorsal longitudinal muscles (DLMs), which are required for flight ability. In addition to flight as a behavioral readout, this dissection allows for the both DLM synapses and muscle tissue to be amenable to structural analysis using fluorescently labeled antibodies for synaptic markers or proteins of interest. This protocol allows for the evaluation of the structural integrity of synapses in adult Drosophila during aging to model the progressive, age-dependent nature of most neurodegenerative diseases.

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

The goal of this procedure is to dissect the dorsal longitudinal muscle (DLM) tissue to assess the structural integrity of DLM neuromuscular junctions (NMJs) in neurodegenerative disease models using Drosophila melanogaster.

与神经退化有关的成人 果 蝇的可视化突触变性

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused ...

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700&#181;&#948;6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700&#181;&#948;6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700&#181;&#948;6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Glucose uptake is increased in Drosophila motor neurons affected by TAR DNA binding protein (TDP-43) proteinopathy, as indicated by a FRET-based, genetically encoded glucose sensor.

测量TDP-43蛋白病果 蝇 模型中的葡萄糖摄取

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical ...

Direct Cryosectioning of Drosophila Heads for Enhanced Brain Fluorescence Staining and Immunostaining

Immunostaining Drosophila melanogaster brains is essential for exploring the mechanisms behind complex behaviors, neural circuits, and protein expression patterns. Traditional methods often involve challenges such as performing complex dissection, maintaining tissue integrity, and visualizing specific expression patterns during high-resolution imaging. We present an optimized protocol that combines cryosectioning with fluorescence staining and immunostaining. This method improves tissue preservation and signal clarity and reduces the need for laborious dissection for Drosophila brain imaging. The method entails rapid dissection, optimal fixation, cryoprotection, and cryosectioning, followed by fluorescent staining and immunostaining. The protocol significantly reduces tissue damage, enhances antibody penetration, and yields sharp, well-defined images. We demonstrate the effectiveness of this approach by visualizing specific neural populations and synaptic proteins with high fidelity. This versatile method allows for the analysis of various protein markers in the adult brain across multiple z-planes and can be adapted for other tissues and model organisms. The protocol provides a reliable and efficient tool for researchers conducting high-quality immunohistochemistry in Drosophila neurobiology studies. This method's detailed visualization facilitates comprehensive analysis of neuroanatomy, pathology, and protein localization, making it particularly valuable for neuroscience research.

This study presents a simplified protocol for tissue processing involving decapitation, fixation, cryosectioning, fluorescence staining, immunostaining, and imaging, which can be extended to confocal and multiphoton imaging. The method maintains efficacy comparable to complex dissections, bypassing the need for advanced motor skills. Quantitative image analysis provides extensive investigative potential.

果 蝇 头部的直接冷冻切片，用于增强脑荧光染色和免疫染色

Immunostaining Drosophila melanogaster brains is essential for exploring the mechanisms behind complex behaviors, neural circuits, and protein expression ...

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally important for cell function and cell integrity of neurons. Drosophila photoreceptor cells have become a model for studying membrane protein trafficking. Besides rhodopsin, which upon illumination becomes internalized from the photoreceptor membrane and is degraded, the transient receptor potential-like (TRPL) ion channel in Drosophila exhibits a light-dependent translocation between the rhabdomeral photoreceptor membrane (where it is located in the dark) and the photoreceptor cell body (to which it is transported upon illumination). This intracellular transport of TRPL can be studied in a simple and non-invasive way by expressing eGFP-tagged TRPL in photoreceptor cells. The eGFP fluorescence can then be observed either in the deep pseudopupil or by water immersion microscopy. These methods allow detection of fluorescence in the intact eye and are therefore useful for high-throughput assays and genetic screens for Drosophila mutants defective in TRPL translocation. Here, the preparation of flies, the microscopic techniques, as well as quantification methods used to study this light-triggered translocation of TRPL are explained in detail. These methods can be applied also for trafficking studies on other Drosophila photoreceptor proteins, for example, rhodopsin. In addition, by using eGFP-tagged rhabdomeral proteins, these methods can be used to assess the degeneration of photoreceptor cells.

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Here, non-invasive methods are described for localization of photoreceptor membrane proteins and assessment of retinal degeneration in the Drosophila compound eye using eGFP fluorescence.

使用eGFP标记的蛋白质研究 果蝇 光感受器细胞中的膜蛋白运输

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally ...

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

摘要

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此视频中的章节

单果蝇 Ommatidium解剖和影像

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

评估神经退行性表型

攀登缺陷的神经退行性疾病的果蝇模型定量分析

大众组织学量化在神经退行性变

监测果蝇中朊病毒样蛋白聚集物的细胞间传输

与神经退化有关的成人果蝇的可视化突触变性

测量TDP-43蛋白病果蝇模型中的葡萄糖摄取

果蝇头部的直接冷冻切片，用于增强脑荧光染色和免疫染色

使用eGFP标记的蛋白质研究果蝇光感受器细胞中的膜蛋白运输

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

摘要

探索更多视频

此视频中的章节

单果蝇 Ommatidium解剖和影像

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

评估神经退行性表型

攀登缺陷的神经退行性疾病的果蝇模型定量分析

大众组织学量化在神经退行性变

监测果蝇中朊病毒样蛋白聚集物的细胞间传输

与神经退化有关的成人 果 蝇的可视化突触变性

测量TDP-43蛋白病果 蝇 模型中的葡萄糖摄取

果 蝇 头部的直接冷冻切片，用于增强脑荧光染色和免疫染色

使用eGFP标记的蛋白质研究 果蝇 光感受器细胞中的膜蛋白运输

与神经退化有关的成人果蝇的可视化突触变性

测量TDP-43蛋白病果蝇模型中的葡萄糖摄取

果蝇头部的直接冷冻切片，用于增强脑荧光染色和免疫染色

使用eGFP标记的蛋白质研究果蝇光感受器细胞中的膜蛋白运输