Name: in situ labeling of mitochondrial dna replication in drosophila adult ovaries by edu staining
Uploaded: 2016-10-15T14:00:00.000+00:00
Duration: 10 min 31 s
Description: Watch this Scientific Journal Video about In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining at JoVE.com

We thank K. Delaney for comments on the manuscript. This work was supported by the National, Heart, Lung, and Blood Institute (NHLBI) Intramural Program.

1.组织收集和解剖<ol><li>在含干酵母每个小瓶，文化10只成年雌性苍蝇与10名男性2-3天。 注意:保持良好的喂养雌性果蝇会提高整体素质和卵巢的生产产量和促进清扫。 </li><li>麻醉雌性蝇上的二氧化碳（CO 2）的飞垫。 </li><li>下的立体镜，放置逆转录介质上的解剖垫（补充有10％胎牛血清（FBS）的施耐德的果蝇介质）数滴。所有进行解剖过程中的媒介，以保持组织健康地活着。 </li><li>抓住一个女养肥飞在其下端胸部精尖鼻钳。使用另一套镊子，直到腹部组织暴露在苍蝇的极端后轻轻拉扯。从分离其他组织（ 如胆）的两个卵巢。 注:每个卵巢显示一个不透明的结构，是撰写16-20连接卵巢管Ð。 </li><li>为了提高试剂的渗透，通过拉开他们，并传递钳提示在每间卵巢管几次打开卵巢。保留卵巢内连接时下列程序，以尽量减少组织损失的卵巢管。 </li><li>马上转移卵巢含有500微升施耐德的果蝇培养基含10％FBS的1.5ml微量管中。 </li><li>重复清扫，收集每个离心管中10-15卵巢。 </li></ol> 2.标签的EdU <ol><li>吸在每个管的介质，并用500μl含有7微米阿非迪霉素10％FBS的施耐德的果蝇介质的替换。 注意:阿非迪霉素用于通过抑制DNA聚合酶α，而不影响线粒体DNA复制7,12到方框核DNA的合成。它是可以提高阿非迪霉素浓度最多到70微米，以达到更好的抑制作用。 3-30毫阿非迪霉素的DMSO储备溶液可以储存在黑暗中在-20℃下长达6个星期。 </li><li>保持卵巢健康，确保它们下溶液浸渍，同时改变介质。通过步2.6使用带有10％FBS施耐德的果蝇网上平台。 </li><li>孵育卵巢在RT上轻轻旋转台式摇杆3小时。 <ol><li>进行药物治疗， 例如，线粒体解偶联剂羰氰化物4-三氟甲氧基苯腙（FCCP），添加药物的适当的浓度（ 例如，10μMFCCP）到培养基中之后阿非迪霉素治疗的2小时。继续孵育另外1小时。 </li></ol></li><li>有或没有药物删除含有阿非迪霉素网上平台。简要地冲洗中等卵巢（阿非迪霉素是没有必要的）的两倍。 </li><li>加入1ml含有10μM的EdU和7微米阿非迪霉素，继续incubating的室温2小时。储存在-20℃下的10毫米的EdU原液（在DMSO中）。 </li><li>删除含有的EdU和阿非迪霉素的媒介。用培养基（无阿非迪霉素）洗涤两次，每次3分钟。 </li></ol> 3.组织固定和透<ol><li>制备在磷酸盐缓冲盐水（PBS，pH7.4）中4％的多聚甲醛溶液中。 注:我们使用存储在预刻划安瓿市售的多聚甲醛。打开新的安瓿并在使用前稀释至4％。 注意:甲醛是有毒的;它应与皮肤和眼睛的保护在通风橱处理。 </li><li>用4％多聚甲醛的卵巢在室温20分钟，轻轻转动。 </li><li>除去固定剂并且在1ml的PBS中的3％BSA的清洗卵巢两次，每次5分钟。 </li><li>通透的组织，取出洗净解决方案，并在PBS加入1毫升中含0.5％的Triton X-100。孵育在室温20分钟。 </li><li>除去透化溶液并在1ml洗两次的在PBS中的3％BSA中。 </li></ol> 4.检测的EdU 注意:的EdU检测是基于"点击"化学，铜（Ⅰ）催化[3 + 2]环加成13，这增加了荧光叠氮化物向的EdU的末端炔基，并且荧光分子进行后续检测。由于铜（I）中，这是很容易氧化成非催化铜（II）物种，需要以催化反应，建议减少铜（II）硫酸盐原位得到的Cu（I）。即，铜（II）硫酸盐是在还原剂的存在下使用，例如抗坏血酸（这里的"缓冲添加剂"），以生成铜（I）。 <ol><li>在实验之前，准备的Alexa Fluor叠氮化物的工作溶液（的Alexa Fluor 488叠氮化物，的Alexa Fluor 555叠氮化物或其他人，这取决于优选的荧光团，其中被命名为"染料叠氮化物"以下），埃杜反应缓冲液和的EdU缓冲添加剂根据制造商的说明。 <ol><li>每瓶70微升DMSO染料叠氮化物。储存在-20℃下长达1年的工作溶液中。 </li><li>通过用去离子水稀释10倍的EdU反应缓冲液制备新鲜的EdU反应缓冲液。 注意事项:使用后，存放在4℃的任何剩余1X解决方案。 1倍溶液为长达6个月是稳定的。 </li><li>通过在2ml去离子水将粉末充分溶解使的EdU缓冲添加剂的10倍的原液。 注意:此原液是长达1年，在-20℃的稳定。如果该溶液开发了棕褐色，它已经降低，并应被丢弃。 </li></ol></li><li>准备这些EDU反应鸡尾酒新鲜每次和准备的15分钟内使用。实现可重复的结果，确保反应组分保持在相同的比率。 <ol><li>使新鲜的1X的EdU稀释在去离子水中的10倍的原液（在步骤4.1.3的方法制备）1:10缓冲添加剂溶液。使用这个Solution在同一天。 </li><li>由下列成分以组合制备1毫升的EdU反应鸡尾酒:860微升1×的EdU反应缓冲液，40微升硫酸铜 ，2.5微升染料叠氮化物，将100μl1×的EdU缓冲添加剂。 注意:该成分以确保最佳性能增加是重要的。 </li></ol></li><li>用3％BSA的除去PBS中，加入0.5毫升的EdU反应鸡尾酒到每个管中。孵育在室温下，轻轻旋转30分钟。保持避光样本。 </li><li>取出的EdU反应鸡尾酒并用1ml的PBS中的3％BSA的洗一次。 </li><li>用1毫升的PBS洗一次样品。 </li></ol> 5.抗体标记注:的EdU染色后进行抗体标记。如果没有额外的染色需要，可以进行直接安装和成像。重要的是在样品中所有以下程序避光。 <ol><li>除去洗涤溶液。在1毫升含有0.2％BSA和0.1％的Triton X-100的PBS 30分钟阻断溶液阻断卵巢。 </li><li>除去封闭溶液，并用封闭液稀释的第一抗体（ 例如，小鼠ATP合酶亚基α抗体，1:1000稀释）代替。在4°CO / N孵育在黑暗中。 </li><li>用1ml封闭溶液3次，每次10分钟的洗样品。为了最大限度地减少的背景下，洗两次，每30分钟。除去封闭溶液。 </li><li>在RT 2小时:（200稀释例如，山羊抗小鼠的Alexa Fluor 568二级抗体，1）与封闭溶液稀释的二级抗体孵育。 注:使用不同的颜色比一个耦合到的EdU。 </li><li>重复步骤5.3执行的洗涤步骤。 </li><li>用1毫升的PBS冲洗一次，以去除去污剂。 </li></ol> 6.安装和影像<ol><li>小心取出所有的PBS和IMMediately覆盖用50μl固定培养基（有或没有DAPI）卵巢。 注意:使用而卵巢管被彼此分离不变硬的安装平台。卵巢可以存储在安装介质在4℃下进行长达一周。 </li><li>切枪头的末端，用它小心卵巢转移到一个载玻片。 </li><li>完全独立的每个卵巢管的体视显微镜下精尖鼻钳。在后端和舞台14或成熟的卵子取出室连接的组织。年轻的蛋商会是在前端和透明。 </li><li>对准镊子卵腔室，使它们不相互重叠。 </li><li>慢慢降低对样品的顶部上的＃1.5玻璃盖玻片。允许安装介质聚合在室温数小时。用透明的指甲油密封。 </li><li>图像滑动第二天，或储存于4℃之前成像。 </li><li>共聚焦MI下可视化具有63X油浸物镜croscope。捕获三维的z堆栈的图像。 注意:有较强的教育栏目后期蛋商会和背景荧光信号在上皮鞘。当检查在特定germarium的EdU标记，应避免由重叠或靠近其他组织或后期蛋商会germariums。 </li></ol>

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The authors have nothing to disclose.

的EdU标记是一种新颖的和有效的方法来检测在增殖细胞，它是基于在新合成的DNA的掺入和核苷类似物的染色DNA的合成。此方法优于在于它是更快和高度敏感的BrdU标记方法。更重要的是，它允许良好的结构保存和高效率的EdU染料渗透整个安装组织9,10。在历史上，以BrdU标记一个更好的选择，使用期间的S相学习核DNA复制的EdU标记细胞周期。阿非迪霉素是DNA聚合酶α的抑制剂，它是用于在S期12 15核DNA复制的主要聚合酶。线粒体DNA复制是通过DNA聚合酶γ，这是不敏感的阿非迪霉素处理进行。因此，与前和的EdU孵育期间阿非迪霉素治疗显著抑制的EdU掺入核DNA。这应该另外，阿非迪霉素可以是数周稳定的，如果适当地存储，并且阿非迪霉素在抑制核DNA复制的效果是在我们手中变量。用强大的核DNA标记的卵巢管和蛋商会应排除进一步的数据分析。 线粒体DNA复制可以容易形象化为在细胞质中，这也提供了一个简单的方法通过计数的EdU泪点的数量归一化到细胞质的总体积来定量线粒体DNA复制水平泪点。成像软件可以应用到自动识别在显微图像，这对大型数据集计算分析特别有用的EdU泪点。然而，应采取预防措施，因为核DNA复制的不完全抑制可以导致在染色体上不同基因座的的EdU掺入和显示为在细胞核内泪点。另外，在其他情况下，埃杜的强度掺入REPLIcating线粒体DNA可能是微弱的，而背景和噪声可能会很高。因此，对于自动图像分析的各个参数应仔细限定。它也建议，图像应经过培训的眼睛进行检查，以确保适当的EdU泪点被确定。 果蝇卵子发生期间新合成的线粒体DNA的可视化提供了一个机会来研究线粒体DNA复制如何生理或病理条件下的监管，通过开展在经受各种药理或遗传扰动的苍蝇实验。 COI T300I 11:在先前的研究中，在一个线粒体DNA突变体果蝇公吨进行的EdU掺入法。此外，为了破坏线粒体膜电位，卵巢用各种浓度的线粒体解偶联剂FCCP或2,4-二硝基苯酚（DNP）的EdU之前掺入处理。根据实验的目的和药物的特点，不同的方法可能是有效的传递采纳。对于成年果蝇，药物可以作为蒸气（例如，乙醇和可卡因）16,17或药物可以注射到腹部，在那里它迅速扩散在整个主体18。最常见的做法是，药物加入到飞食品或蔗糖/药物饱和滤纸。例如，微管组装，秋水仙碱的抑制剂，被卵巢切除19之前供给到果蝇2-3天。因此，为了评估药物递送方法，选择适当的浓度是很重要的。 为了确保在果蝇卵巢线粒体DNA复制的成功的成像，有几个关键的步骤必须认真执行。最重要的是，夹层和的EdU掺入过程中保留了卵巢的生存能力和健康是必要的（步骤1和2）。需要被加热至室温与FBS施耐德的果蝇中使用前。每个人都应该尽量减少蛋室和解剖工具或枪头之间的直接接触。卵巢应在解决方案沉浸任何时候，以避免脱水。处理不当的组织可以很容易导致晕倒或无荧光信号。在组织安装步骤，卵巢管应该从彼此分离并摊开在显微镜载玻片。确保蛋室不堆叠在彼此的顶部。我们注意到，鸡蛋室超出14期小显示掺入的EdU。此外，由于大尺寸，后期鸡蛋商会往往会造成邻近的年轻蛋商会是失焦。因此，建议在后期蛋室被丢弃。 在这里，我们提供的标签在果蝇卵巢成年线粒体DNA复制了详细的方案。此方法允许线粒体DNA复制的简单量化在各种遗传学和药理学扰动并且将成为潜在的解剖发育线粒体生物合成和线粒体DNA遗传机制非常有用。

除核基因组中，每个的真核细胞还包含数千个在线粒体基质小环状DNA的拷贝。而线粒体DNA（mtDNA的）编码的电子传递链的必需亚基，大多数线粒体蛋白质，包括所有的因素对复制和线粒体DNA的转录是由核基因组编码。相反，下面的继承孟德尔定律的核基因组，动物线粒体基因组是通过母系遗传独占。因此，理解的mtDNA是如何增殖和从一代传递到下一个通过女性的卵母细胞是至关重要的。然而，有过线粒体DNA复制是如何在阴生殖调节继续争论。此外，广为接受的mtDNA瓶颈理论线粒体传输表明线粒体DNA中的原始生殖细胞的种群二次采样到一个相对较小的数都灵3G发展1 2。这也意味着，线粒体DNA的复制是时间和卵子发生过程监管空间。因此，种系发育过程中原位检测线粒体DNA复制的将促进线粒体遗传的机制的理解。 果蝇卵子提供了一个听话的基因系统来研究线粒体DNA复制和传播。在每两个果蝇卵巢，有卵腔室16-20独立串称为卵巢管3，其是产蛋的功能单元（参见图1A）。每个卵巢管包含卵子发生，其中，前尖端是由一个称为germarium结构的渐进线性组织。该germarium被进一步分成包含在不同发育阶段的生殖细胞的四个区域。在区域1，生殖系干细胞通过不对称分裂产生称为为c子细胞ystoblasts。区域2a包含已经完成了他们最后的分工cystoblasts。 Cystoblasts经过四轮分工生产16电池组。 16细胞仍然被称为环运河桥梁细胞质相互连接。仅其中的一个单元承诺分化为卵母细胞，而其他15发展为多倍体护士细胞。必不可少的胞质结构，被称为fusome，建议用环管的形成，以促进，确定囊肿极性和护士细胞-卵母细胞的相互作用4,5。作为孢囊向区域2b移动，囊肿结构跨越germarium的整个宽度，并变得更加与毛囊细胞相关联。该germarium结构结束与包含第一个初露头角的卵室区3。随后，将蛋室在germarium，它通过卵巢管在14形态不同阶段进行的后端装配。卵母细胞的生长依赖于滋养细胞，WHICH转运蛋白，mRNA和经由环管进入卵母细胞内膜结构（ 例如 ，高尔基）。期间果蝇卵子发生，人们发现，每个16细胞囊肿内的线粒体的一个级分与fusome相关联，通过环管移动，并且被输送到单个卵母细胞在称为巴尔比亚尼体6大的质量。这种现象提出了有助于通过女性卵巢线粒体遗传的质量控制。 线粒体DNA合成的检测依赖于标记的DNA前体掺入到细胞DNA中。传统上，胸苷5-溴-2&#39;-脱氧尿苷的核苷类似物（BrdU）标记用于标记在组织培养细胞7,8- mtDNA的复制。然而，基于抗体的BrdU标记显示出若干局限性，特别是对于整个贴装组织染色。 BrdU标记的一个主要缺点是，它需要的DNA变性，以暴露所述的BrdU表位，因此，它可以被抗BrdU抗体进行检测。检体已苛刻的变性条件如化学品（ 如，盐酸或甲醇和乙酸的混合物），热或用DNase消化，这可能会破坏试样的结构和下列染色步骤9复杂化下处理， 10。 这里，另一种胸苷类似物5-乙炔基-2&#39;-脱氧尿苷（EDU）被用来标记在果蝇成年卵巢复制线粒体DNA。这种方法是快速，高度敏感。的EdU DNA复制过程中容易地结合到细胞DNA中。下面检测是基于一个"点击"反应中，铜（Ⅰ）催化的末端炔基和荧光叠氮化物10之间的共价反应。由于反应不需要检体的变性，它允许良好的结构保存。此外，染料的大小zide只有1/500的一种抗体分子10，使整体安装组织的快速，高效的渗透。我们已经用这种方法果蝇卵子发生过程中检测线粒体DNA的复制，发现在果蝇卵巢11 germarium区一个惊人的空间格局，这导致我们提出了一个相关的复制线粒体DNA选择性的继承机制。我们在座的果蝇卵巢线粒体DNA复制的标签的EdU了详细的方案。为了证明该协议的应用，我们还测试了线粒体DNA突变体果蝇的线粒体DNA复制（ 公吨:COI T300I）11，以及与线粒体解偶联剂，其消散线粒体膜电位和潜在破坏线粒体DNA复制的多样的治疗。

<table><tbody><tr><td>Schneider&amp;rsquo;s Drosophila medium</td><td>Invitrogen</td><td>21720-024</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>10100-147</td><td></td></tr><tr><td>Pair of Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>Aphidicolin</td><td>Sigma</td><td>A0781</td><td>Aliquot after dissolving in DMSO. Avoid repetitive thawing and freezing. Protect from light. May be used within 6 weeks after dissolving.&amp;nbsp;</td></tr><tr><td>FCCP</td><td>Sigma</td><td>C2920</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Paraformaldehyde, 16% EM grade</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Formaldehyde is toxic; it should be handled in a fume hood with skin and eye protection.</td></tr><tr><td>PBS</td><td>KD Medical</td><td>RGF-3190</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A7030</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T9284</td><td></td></tr><tr><td>Click-iT EdU Alexa Fluor 488 Imaging Kit</td><td>Invitrogen</td><td>C10337</td><td>EdU, CuSO&lt;sub&gt;4&lt;/sub&gt;, Alexa Fluor 488 azide, EdU reaction buffer and Edu buffer additive are included</td></tr><tr><td>Mouse ATP synthase subunit &amp;alpha; antibody, (15H4C4)</td><td>MitoSciences</td><td>Ab14748</td><td>1:1,000 dilution</td></tr><tr><td>Mouse Hts antibody (clone RC)</td><td>Developmental Studies Hybridoma Bank (DSHB)</td><td>hts RC</td><td>1:1,000 dilution</td></tr><tr><td>Goat anti-mouse Alexa Fluor 568 secondary antibody</td><td>Invitrogen</td><td>A-11004</td><td>1:200 dilution</td></tr><tr><td>Vectashield mounting medium with DAPI</td><td>Vector Laboratories</td><td>H-1500</td><td></td></tr><tr><td>Glass coverslips, #1.5 22 mm x 22 mm</td><td>Fisher Scientific</td><td>12-541-B</td><td></td></tr><tr><td>Microscope slide</td><td>Fisher Scientific</td><td>22-038-103</td><td></td></tr><tr><td>Nail polish</td><td>Elf</td><td></td><td>Many of the pigments used in nail polishes are fluorescent and leach into specimens. Only clear nail polish should be used.</td></tr></tbody></table>

in situ labeling of mitochondrial dna replication in drosophila adult ovaries by edu staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

上述协议允许与线粒体（ 图1B-C）相关联的点状结构，这表明果蝇卵子中的线粒体DNA复制的可视化。所述的EdU泪点与由染色ATP合酶α亚基（ 图2）标记的线粒体本地化。所观察到的信号是在用溴化乙锭11，对线粒体DNA复制14抑制剂治疗的卵巢缺席，验证这些泪点确实标签复制mtDNA.Aphidicolin用于抑制核DNA染色，而不会影响线粒体DNA复制（ 图2）。无阿非迪霉素治疗，紧张的EdU信号标记细胞核，并几乎没有观察到mtDNA泪点（ 图3）。然而，在阿非迪霉素的存在下，核掺入显着降低，并观察到与线粒体有关的许多泪点。 图1B）线粒体DNA的复制水平高。然而，值得注意的是，线粒体DNA复制显示在germarium的空间格局。如通过的EdU泪点的数字表示，有线粒体DNA复制在germarium的区域1中等水平，但几乎没有的EdU掺入区域2A（ 图1C）。作为囊肿在germarium向下移动到区域2B，线粒体DNA复制恢复，并在区域2B的后部囊肿的EdU泪点的数目比在区域2A（ 图1C）高得多。具体而言，密集的EdU掺入集中在环运河和fusome结构，由胡力邵泰（HTS）蛋白染色。线粒体DNA保持在较高的水平在germarium的区域3复制（ 图1C） 为了证明特异性的协会集成电路的基因或与线粒体增殖治疗， 果蝇卵巢可经受基因操作或药物治疗。我们治疗卵巢用不同浓度FCCP，一个典型的线粒体protonophore，消散线粒体膜电位。作为对照，DMSO对线粒体DNA的复制（ 图4A）没有影响。高剂量FCCP（10μM）几乎完全耗尽整个germarium（ 图4D）线粒体DNA的复制。尽管如此，FCCP（2或5微米）的低浓度对线粒体DNA复制在区域1中的区域2B和3（ 图4B-C）的影响较小，但抑制复制，表明区域2B和3是线粒体破坏更敏感，或它们保持相对较慢的复制动力学。上述结果表明，线粒体DNA复制与线粒体活性相关。特别是，germarium不同地区的反应不同线粒体小鬼airment。 <img alt="图1" src="/files/ftp_upload/54516/54516fig1.jpg" /> 果蝇卵子发生过程 图1. 线粒体DNA的复制。（A） 果蝇卵巢管的图和germarium的放大图。在卵巢管示出由左到右，前至后的蛋室中，连续的发育阶段。在germarium，所述fusome（红），生殖系干细胞（蓝色），线粒体（绿），未来的卵母细胞（虚线，由定位，fusome结构和线粒体簇识别），显影囊肿（桃）和四个发育区域示。 （B）埃杜标记野生型卵巢管和抗体对HTS-RC，为环运河和fusome标记代表Z堆叠投影。在阿非迪霉素的存在下，所述的EdU被掺入线粒体DNA（箭头）和细胞核（箭头）。比例尺，10微米。（ 三 ） 乙方在盒装区域概述germaria的放大图。四个发育区域表示。比例尺，5微米11。 <a href="https://www.jove.com/files/ftp_upload/54516/54516fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54516/54516fig2.jpg" /> 图2. 在通过的EdU掺入用阿非迪霉素治疗可视果蝇 germarium线粒体DNA复制（A） - （D）示出的EdU掺入野生型germarium的代表性共聚焦部（绿色，b）中，线粒体，标记被ATP合酶α亚基染色（红色，C），以及核，标记用DAPI染色（蓝色，D）的预孵育DNA聚合酶α抑制剂阿非迪霉素。 （A＆＃39 ;-D&#39;）中流露出的EdU掺入（B方框区域的放大图像（A）"），线粒体（C&#39;）和核（D&#39;）。在阿非迪霉素的存在下，核掺入（箭头）被降低和许多泪点中线粒体（箭头）内本地化。比例尺，10μm以下。这个数字已经从11修改。 <a href="https://www.jove.com/files/ftp_upload/54516/54516fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54516/54516fig3.jpg" /> 图3.线粒体DNA复制是在未经阿非迪霉素治疗果蝇 germarium几乎检测不到（A） - （D）示出的EdU掺入（绿色）的野生型germarium的代表性共聚焦部分，线粒体，标记由ATP合酶α亚基染色（红色），以及核，标有在不存在的DNA聚合酶α抑制剂阿非迪霉素的DAPI染色（蓝色）。 （A&#39;-D&#39;），（A）中方框区域的放大图像显示掺入的EdU（B&#39;），线粒体（C&#39;）和核（D&#39;）。阿非迪霉素无，勉强被检测到强烈的EdU信号标签核（箭头），和线粒体DNA泪点。比例尺，10μm以下。这个数字已经从11修改。 <a href="https://www.jove.com/files/ftp_upload/54516/54516fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54516/54516fig4.jpg" /> 图4.线粒体解偶联剂损害线粒体DNA的复制。说明野生型germarium代表Z堆栈项目DMSO处理（A）或在2微米（B）中，5微米（C），10μM的EdU掺入期间（D）的浓度的线粒体解偶联剂FCCP。注意，在与2微米FCCP（B）的处理过的区域2B中的受损的EdU标记，并在这两个区域2B和3与5微米FCCP处理（在盒子中概述）。四个发育区域被示出。箭头，核DNA;箭头，线粒体DNA。比例尺，10微米。这个数字已经从11修改。 <a href="https://www.jove.com/files/ftp_upload/54516/54516fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining at JoVE.com

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

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In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

9.1K 次观看.  National Institutes of Health. Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2´-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

视频: In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

通过DNA含量测定的哺乳动物复制时序的全基因组的测定

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

科研

遗传学

Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy cells1-3. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication4. We developed a sensitive technique to label newly synthesized mtDNA in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of 5-ethynyl-2'-deoxyuridine (EdU)5-7 with a tyramide signal amplification (TSA)8 protocol to visualize mtDNA replication within subcellular compartments of neurons. EdU is superior to other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), because the initial click reaction to label EdU5-7 does not require the harsh acid treatments or enzyme digests that are required for exposing the BrdU epitope. The milder labeling of EdU allows for direct comparison of its incorporation with other cellular markers9-10. The ability to visualize and quantify mtDNA biogenesis provides an essential tool for investigating the mechanisms used to regulate mitochondrial biogenesis and would provide insight into the pathogenesis associated with drug toxicity, aging, cancer and neurodegenerative diseases. Our technique is applicable to sensory neurons as well as other cell types. The use of this technique to measure mtDNA biogenesis has significant implications in furthering the understanding of both normal cellular physiology as well as impaired disease states.

We developed a sensitive technique to label newly synthesized mitochondrial DNA (mtDNA) in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of EdU together with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons.

在单个细胞中的线粒体DNA复制的可视化EDU信号放大

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy ...

神经科学

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.

种系线虫与胸苷模拟教育的细胞周期分析

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...

发育生物学

mtDNA 合成和分布的高通量定量

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

作者聚焦:基于图像的高通量线粒体 DNA 合成和分布定量  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

基于图像的高通量线粒体DNA合成和分布定量

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...

生物化学

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these mechanisms is crucial for developing neural regenerative therapies. Drosophila melanogaster is a leading model for studies of neural development but historically has not been exploited to investigate adult brain regeneration. This is primarily because the adult brain exhibits very low mitotic activity. Nonetheless, penetrating traumatic brain injury (PTBI) to the adult Drosophila central brain triggers the generation of new neurons and new glia. The powerful genetic tools available in Drosophila combined with the simple but rigorous injury protocol described here now make adult Drosophila brain a robust model for neural regeneration research. Provided here are detailed instructions for (1) penetrating injuries to the adult central brain and (2) dissection, immunohistochemistry, and imaging post-injury. These protocols yield highly reproducible results and will facilitate additional studies to dissect mechanisms underlying neural regeneration.

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

This article provides detailed protocols for inflicting Penetrating Traumatic Brain Injury (PTBI) to adult Drosophila and examining the resulting neurogenesis.

刺激和分析 果蝇 中枢脑中的成人神经发生

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

实验方法来研究线粒体定位和核细胞周期激酶CDK1功能

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

生物学

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 ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

可视化和跟踪活果蝇黑色素细胞卵室的内源性 mRNA

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...

在果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

摘要

探索更多视频

此视频中的章节

通过DNA含量测定的哺乳动物复制时序的全基因组的测定

在单个细胞中的线粒体DNA复制的可视化EDU信号放大

种系线虫与胸苷模拟教育的细胞周期分析

基于图像的高通量线粒体DNA合成和分布定量

基于图像的高通量线粒体DNA合成和分布定量

基于图像的高通量线粒体DNA合成和分布定量

刺激和分析果蝇中枢脑中的成人神经发生

实验方法来研究线粒体定位和核细胞周期激酶CDK1功能

研究线粒体结构与功能

可视化和跟踪活果蝇黑色素细胞卵室的内源性 mRNA

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

摘要

探索更多视频

此视频中的章节

通过DNA含量测定的哺乳动物复制时序的全基因组的测定

在单个细胞中的线粒体DNA复制的可视化EDU信号放大

种系线虫与胸苷模拟教育的细胞周期分析

基于图像的高通量线粒体DNA合成和分布定量

基于图像的高通量线粒体DNA合成和分布定量

基于图像的高通量线粒体DNA合成和分布定量

刺激和分析 果蝇 中枢脑中的成人神经发生

实验方法来研究线粒体定位和核细胞周期激酶CDK1功能

研究线粒体结构与功能

可视化和跟踪活果蝇黑色素细胞卵室的内源性 mRNA

在果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

刺激和分析果蝇中枢脑中的成人神经发生