威康信托基金资助下，该协议。我们感谢为维护飞股票和多年来的准备飞食品莫琳·辛克莱女士。我们还想感谢他的帮助和技术支持，在此协议的发展迈克尔·艾奇逊先生。

1。转基因果蝇和维护<ol><li> 转基因果蝇在此示范使用:-UBQ UBQ-GFP-CDC20（二），RFP-His2B（X）; UBQ-GFP-CDC20（二）UBQ-GFP-CDC20（二）; MAD2 安永 （三*）和UBQ-GFP-CDC20转基因果蝇先前在实验室中产生的，通过一个标准的P-元素介导的转基因方法10,11和UBQ RFP-His2B的是在UMR CNRS的144 /研究所居里从YohannsBelaïche样的礼物法国巴黎。他们介绍到Mad2的通过标准的果蝇基因突变的背景。 MAD2 安永原突变系从布卢明顿股票中心购买。我们将不讨论用于提高在本协议中的转化过程。 注:*表示的染色体数目。 </li><li> 维修:转基因果蝇保持在25°C间塑料瓶containin中Ğ飞食品与顶端额外的干酵母粉。小瓶经常更换每3-4周，根据生长条件（ 图1）。 </li></ol> 2。飞食品制备（实验室规模） <ol><li>苍蝇的食物混合适量，加热并不断搅拌，溶解的组成部分。 </li><li>作为浆分发到每个塑料瓶使用Jencons科技有限公司蠕动泵（直径为2.5厘米×8厘米长），约8-10毫升这一媒介。 </li><li>当食品浆料和冷却至室温，小瓶，然后用棉花泡沫插头插入。这些食物小瓶被放置在一个托盘，然后将其密封在一个塑料袋内，并保存在4℃，以后使用。 </li></ol> 3。小规模的蛋收集<ol><li>约50 2-3日龄成年果蝇对被转移到一个新的飞食品小瓶提供额外干酵母粉在其表面在25°C间裁员ING胚胎。 </li><li>苍蝇，然后转移到一个新的小瓶，每隔一小时30分钟离开的胚胎在小瓶，以确保收集的一些胚胎周围核的分裂周期8-10岁的细胞核逐渐迁移到皮质和组织作为单面单层。通常被丢弃，因为它往往包含被保留在女性的身体条件不适合铺设的中年胚胎的第一个小时集合。 </li></ol> 4。准备盖玻片和幻灯片<ol><li>取出一个50×22毫米的盖玻片，稍湿的水非常小的量使用湿润的细笔刷，放在显微镜幻灯片一侧的四个角落，使盖玻片不移动，因为毛细管表面张力薄液膜所造成的。 </li><li>适用的薄带钢跨中间盖玻片庚胶水，庚应在几秒钟内蒸发盖玻片上留下的胶水。 </li><li>剪下一个小方块（约1.5毫米2）金刚石笔的另一盖玻片，拿起一个放在胶条结束（这是用来打开需要显微注射时针头），保存以供将来的人使用。 </li><li>再举显微镜幻灯片和双面胶带粘片约2厘米长，它和封面纸剥离。 （参见图2A和B）。 </li></ol> 5。 dechorionate胚胎<ol><li>转移到一个新的食品小瓶苍蝇，并用适量的液体湿的细笔刷双面胶带将其转移到幻灯片上的约50鸡蛋，让鸡蛋散开。当液体蒸发的鸡蛋会坚持到磁带上。 </li><li>在解剖显微镜下触摸沿其长轴蛋多次使用到一半的镊子绒毛膜外侧轻轻被打破，开放，但留在楚胚胎离子外壳，以防止快速脱水。继续，直到鸡蛋10-20（在一个单一的实验所需的鸡蛋的数量而定）已被视为这一进程。 </li><li>胚胎，然后拿起并从绒毛膜壳和胶条轻轻地放在一个。鸡蛋可以放在与胚胎平行或反平行于长边胶条，取决于如果他们将被显微注射的长边对齐。 </li><li>胚胎可以立即被覆盖的Voltalef 10S油适量（西格玛700 Holocarbon油和27油的5:1混合，），以防止进一步的即时成像干燥或干燥室中放置3-8分钟他们进一步脱水取决于房间的湿度和实验的目的。 10S油脱水的胚胎应涵盖在适当的时候，以防止过度脱水，如果需要显微注射。 （参见图2C，丁。急症室） </li></ol> 6。成像胚胎<ol><li>时间推移或单个活胚胎图像采集用1 HCX PL亚洲生产力组织的政务司司长40X ​​1.25油浸物镜（60或100倍的目标太取决于实验的目的，可以使用的Leica TCS SP2倒置共聚焦显微镜系统放大倍率使用漂白会发生更多的照片）。 </li><li>当多个荧光成像，其中有重叠的光谱和共同表达在同一胚胎，每个荧光蛋白的发光波长连续收集。激发和发射波长的Leica TCS SP2系统是:绿色荧光蛋白，λ:488 nm和λEM:500-600纳米。利福平，λ:543 nm和λEM:555-700纳米。 </li><li>一个单一的形象普遍获得平均2扫描线与平均2帧扫描。这需要至少6.3秒，扫描512×512带够ixel同时扫描模式，15.44秒的顺序扫描模式和图像产生一个很好的信号信噪比。设立时间推移图像采集的时间框架时需要考虑这些时间。 </li><li>针孔一般设置1-2个AU（轻快单位），和激光功率调整，以可能的最低程度，以避免任何重大的漂白。 </li></ol> 7。通过适当的试剂微量注射胚胎挑起国资委<ol><li>实验前的准备针。我们喜欢拉siliconised玻璃毛细管（GC-100-10，哈佛）针头使用燃烧/棕色微量器（萨特仪器，型号:P-97）的参数为:热量= 600，拉= 90，VEL = 70 ，德尔= 150。 </li><li>回填罚款装载头（20μL的Eppendorf很多:W215818J）使用适当的浓度在注射液的试剂1-2毫升的针。 </li><li>安装在针针议员lder连续离心喷射控制系统相连的压力管道的显微镜。 </li><li>查找胚根据40X客观的和不改变针移动到视野的中心，并逐步降低它的权利焦平面焦平面针的影子。 </li><li>将胚胎轻轻找到破盖玻片预先安装胶条一端的小广场。很轻轻移动针头，直到它击中平方米的小盖玻片的边缘，轻轻地打破它打开。 </li><li>将胚胎放回视图，并选择胚胎注射合适的年龄。我通常在5-7注入，因为这是胚胎细胞核移植前向外皮质核分裂周期。核的分裂周期的各个阶段，可以决定由GFP-CDC20或核组蛋白的RFP-2B信号之前注射9荧光信号的快速扫描。 </li><li>小心移动了安莉芳Ø关闭和调整焦平面胚胎和针的针。移动胚胎进针，注入一滴试剂所需的位置和移动距离胚胎（或后续的显微注射系统的指示）。胚胎可以注射后或从侧面取决于实验的目的。 </li><li>一旦注入胚胎，准备在适当的时候成像。 （参见图2F＆G公司 ） </li></ol> 8。代表结果<img alt="图1" src="/files/ftp_upload/3763/3763fig1.jpg" /> 图1。所需材料:1。细笔刷，B。小方破眼镜的容器，C。 22×50毫米盖玻片，D。显微镜幻灯片，电子。双面胶带，F。孔上的盖子，G在试管中的干酵母粉。酵母粉煤灰维修，H用的颗粒。飞食品瓶， 我 。一个用于dechorionate胚胎1镊子的一半，J一对镊子，K。庚胶液容器（容量瓶）。 <img alt="图2" src="/files/ftp_upload/3763/3763fig2.jpg" /> 图2。图表显示在步骤4，步骤5和针注射在第7步准备在胚胎dechorionation盖玻片和幻灯片的准备。一个 。顶部的图片显示了一个跨的中间地带，庚胶水和小广场的一端粘在一个破碎的盖玻片盖玻片。底图为一个在其四个角的薄水层举行盖玻片显微镜幻灯片。使用幻灯片举行盖玻片的原因是要保持大致相同的焦距，发现当你在幻灯片上双面胶带，从而避免重新聚焦显微镜，而你ARË解剖和转移磁带和盖玻片之间的胚胎，B。用双面透明胶带的长度短的幻灯片应用前剥离其封面纸用于dechorionating胚胎，C。左边的图片显示有显着的前部和后部两端完整绒毛膜炮弹的胚胎;右侧图为在破碎的蛋壳壳的胚胎之前，他们被转移到盖玻片胶条，D，E图显示两个。胶水带dechorionated胚胎转移，安置和调整的方式。胚胎受10S油经过适当的干燥期。F＆G图显示如何打开针位置，使注入的尖端。 从上述描述的实验中获得的单个或延时的图像可直接分析使用莱卡软件或TIF作为一个开放的FO文件中保存rmat可为进一步量化，或使用其他常见的图像分析程序，如Photoshop和图像J，MetaMorph等两个例子来说明这个编辑分析讨论如下: 例1:一个时间推移电影（ 图3），显示着丝粒的GFP-CDC20和生活果蝇合胞胚胎染色体运动的动态招聘。时间推移从原来的序列图像感兴趣区域确定/编辑和使用Photoshop软件自动批量转换。电影组装使用QuickTime软件。 <img alt="图3" src="/files/ftp_upload/3763/3763fig3.jpg" /> 图3。一时间推移电影显示着丝粒的GFP-CDC20和生活果蝇合胞胚胎染色体运动的动态招聘。 （一）时间推移图像合胞从转基因胚胎，共同表达GFP-CDC20（绿色）和RFP-2B组蛋白（红色）融合蛋白，并记录在18°C在细胞核分裂周期7​​-8使用了Leica TCS SP2共聚焦系统。帧被每10秒钟。细胞已经在前期的帧被视为零时间10点， <a href="http://www.jove.com/files/ftp_upload/3763/3763movie3A.mov" target="_blank">点击这里查看图3A的电影</a> 。（二）绿色荧光蛋白CDC20可以随时观察前期和早中期，着丝粒（B3＆4，白色箭头）和坚持在中期和后期着丝粒（B5＆6，白色箭头）。 GFP-CDC20被排除从相间细胞核（白色箭头），早前期进入细胞核。 （B8-14）作为标记共同表达His2BmRFP的使用染色质形态。酒吧为5mm。 例2:国资委的功能可以通过操纵胚胎显微注射荧光标记的抗体，蛋白质或chemica研究L化合物的利益，可能引发国资委。例如注射秋水仙碱，使微管解聚挑起国资委表示在图4 10。 <img alt="图4" src="/files/ftp_upload/3763/3763fig4.jpg" /> 图4。 MAD2是必不可少的秋水仙碱援引国资委功能 10。 GFP-CDC20; MAD2 + / + GFP-CDC20; MAD2 安永 （MAD2 - / -空突变）和"绿色荧光蛋白MAD2; MAD2 安永胚胎内注射秋水仙碱治疗。时间推移聚焦图像被前（01，07和13）或注射后（02-06，前面板; 08-12，中板; 14-18，底部面板）。绿色荧光蛋白CDC20（顶部和中间板）或GFP-MAD2（底部面板）着丝粒信号被用来作为细胞周期进展的标志。顶部面板，箭头表示在02-06被捕的着丝粒。在01箭头标记的区域表明，秋水仙碱治疗前，GFP-DC20被排除后期相间核。中间面板，在缺乏内源性MAD2，GFP的-CDC20信号，继续振荡和细胞核中，开启和关闭的着丝粒，虽然胞质出现有缺陷，将预计在缺乏微管的胚胎，在存在秋水仙碱（在07-12箭头表示）建议在逮捕细胞秋水仙碱治疗失败的国资委功能。女儿细胞核分离失败图片10。底部面板，在14-18的箭头表明被捕的着丝粒积累的GFP-MAD2融合蛋白MAD2突变胚胎中的国资委缺陷型救援功能的GFP-MAD2建议。 13箭头表明在晚间期核中细胞MAD2积累。禁止为5mm。胚胎显微注射〜1％的100mg/ml秋水仙碱在1×PBS原液的蛋量。

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+Techniques+for+Studying+Mitosis+in+the+Drosophila+melanogaster+Syncytial+Embryo.">Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo.</a> J. Vis. Exp. (31), e1382 (2009).</li><li>Figard, L., Sokac, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Cell+Shape+Change+in+Living+Drosophila+Embryos.">Imaging Cell Shape Change in Living Drosophila Embryos.</a> J. Vis. Exp. (49), e2503 (2011).</li></ol>

我们什么都没有透露。

这里描述的协议是一个成像飞合胞胚胎及使用的Leica TCS SP2激光共聚焦扫描显微镜，可以修改，以适应其他显微镜系统，也可以适应其他基因的功能，利用果蝇合胞胚胎研究的一般方法。我们已经用此协议纺锤体组装检查点，蛋白质动力学和蛋白质水解，使用转基因果蝇或多克隆抗体，以可视化的蛋白定位在生活或固定样本4,6,12-14研究的许多方面。 这是最简单的收集鸡蛋的小号码（约10 - 100）时，以保持新鲜飞食品瓶苍蝇。这样可以减少决策和准备额外的苹果汁琼脂平板和鸡蛋收集室在15,16其他出版物中所述的麻烦。盖玻片玻璃破碎的小广场一个在另外一端的盖玻片上的胶条使得它容易得多打开针注射量，并确定通过调整显微注射系统设置，而针浸泡在10S石油。重要的是，为避免泄漏和维持胚胎的可行性，特别是对于那些需要很长一段时间，或提高注射后转化时，实验成功的注射胚胎的干燥程度。然而，有没有需要究竟有多长干燥的金科玉律。这种变化从实验，实验和依赖注入的解决方案和工作环境的湿度量。 利益的特定蛋白质的功能，可以操纵特定的蛋白质抑制剂，通过显微注射单或多克隆抗体对一个特定的蛋白质，信使RNA或野生型或基因突变的背景下荧光标记的肽。这些突变线或GFP标记的转基因株系的许多公开AV从果蝇库存中心，其中大部分是上市ailable果蝇（ <a href="http://flybase.org/" target="_blank">http://flybase.org/~~V</a> ），可在线搜索。

必要的设备和试剂: 共聚焦成像系统:在本协议中所述的成像系统是在Leica TCS SP2激光扫描共聚焦倒置显微镜系统。介绍的方法也适用于可能与其他成像系统的一些小的修改。 解剖显微镜:我们使用的是奥林巴斯SZX7与东风PLAPO 1X-4镜头。 针拔:火焰山/棕色微量车夫（萨特仪器，型号:P-97）。 显微注射系统的Eppendorf显微注射系统的描述，但可用于任何其他适当的系统。 飞食品经销商:Jencons科技有限公司蠕动泵。 试剂: <table border="1"><tbody><tr><td> 成分 </td><td> 重量小号</td><td> 资源 </td><td> 地段编号 </td></tr><tr><td>玉米粉</td><td> 100.0克</td><td>英国的SUMA， </td><td></td></tr><tr><td>红糖</td><td> 50.0克</td><td>林顿的，英国</td><td></td></tr><tr><td>干酵母</td><td> 25.0克</td><td> DCL的酵母有限公司英国</td><td></td></tr><tr><td>洋菜</td><td> 12.5克</td><td> Fisher Scientific则</td><td> 106556 </td></tr><tr><td>山梨酸</td><td> 0.4克</td><td> BDH问题，英国VWR国际有限公司</td><td> 8829310 </td></tr><tr><td>苯甲酸</td><td> 2.9克</td><td> Fisher Scientific则</td><td> 1019599 </td></tr><tr><td>尼泊金 （甲基-4 - 海德鲁 xybenzoate） </td><td> 0.9克</td><td> BDH问题，英国VWR国际有限公司</td><td> K35969015 </td></tr><tr><td colspan="3"> H 2 O上升至1L </td><td></td></tr></tbody></table><p类="jove_content"&gt; 表1。飞食品配料。 holocarbon 700和27油购自Sigma。 干酵母:托马斯·阿利森，英国。 准备庚胶:以约1.5米的双面透明胶带，把成猎鹰15毫升管庚烷5毫升，和旋转为3-5小时或过夜。时间后，分成4等分等分1.5毫升的Eppendorf离心管中，并在台式离心机在旋转速度为5分钟，以消除任何碎片。存储在10毫升容量瓶庚胶液，并继续使用。胶水的强度会有所不同，根据所使用的胶水的浓度和金额。通常情况下，更薄的胶水产生更低的背景噪音时，由共聚焦显微镜扫描。 <ul><li> 盖玻片:BDH问题，英国VWR国际有限公司</li><li> 显微镜幻灯片:BDH问题，英国VWR国际有限公司&lt;/ LI&gt; <li> siliconised玻璃毛细管 GC-100-10，哈佛</li><li> 罚款加载提示:20μL的Eppendorf很多:W215818J </li><li>镊子</li><li>细笔刷</li></ul>

studying mitotic checkpoint by illustrating dynamic kinetochore protein behavior and chromosome motion in living drosophila syncytial embryos

纺锤体装配检验点（SAC）的机制是一个积极的信号，它监视染色体着丝粒和纺锤体微管之间的相互作用，以防止后期发病，直到染色体是否正确连接。细胞利用这种机制，以防止非整倍体或基因组不稳定，因此，癌症和其他人类疾病，如出生缺陷和阿尔茨海默氏症1。 Mad1的，如国资委组件的数量，MAD2，BUB1，BUBR1，BUB3，Mps1，ZW10，罗德和Aurora B激酶已被确定，他们是所有着丝粒的动态蛋白质2。有证据表明，着丝粒是SAC信号启动。 CDC20的是国资委的首要监管目标。 CDC20是必要的APC /℃（， 一个 naphase 带够romotingÇomplex或Çyclosome）激活3之一，也是一个着丝粒的动态蛋白质4-6。当被激活时，国资委抑制活性的APC /防止两个关键基材，细胞周期蛋白B及securin的破坏，从而防止中期向后期过渡7,8。国资委信号究竟是如何启动和组装上的着丝粒和传达到APC / C的抑制其功能仍然难以捉摸。 果蝇是一个非常听话的实验系统;一个更简单和更易于理解的有机体相比，人类只有一个，普通股的基本过程。也许，这是一个最好的生物体活细胞生物成像研究中使用，特别是对有丝分裂活动在时间和空间可视化，为早期胚胎通过13快速核分裂周期同步（8-10分钟每个周期在25°C），并逐步组织在刚刚下皮层9单面单层的细胞核。 在这里，我提出了一种生物成像的方法，使用转基因果蝇表达星GFP（绿色荧光蛋白）或其变种针对性利息的Leica TCS SP2激光共聚焦扫描显微镜系统研究苍蝇国资委的功能，图像显示一些国资委组件的GFP融合蛋白，CDC20和Mad2蛋白作为例子。

Watch this Scientific Journal Video about Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos at JoVE.com

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

着丝粒是地方国资委启动其信号监测的有丝分裂的姐妹染色单体隔离。一种方法是描述与染色体运动的可视化招聘和营业额的着丝粒蛋白及其协调<em&gt;果蝇</em&gt;胚胎使用徕卡激光扫描共聚焦系统。

龙虎斗动粒蛋白的动态行为与染色体运动，在生活学习有丝分裂关卡<em&gt;果蝇</em&gt;合胞胚胎

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

studying-mitotic-checkpoint-illustrating-dynamic-kinetochore-protein

Research

JoVE Journal

Biology

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

11.3K 次观看.  University of Newcastle, United Kingdom. 着丝粒是地方国资委启动其信号监测的有丝分裂的姐妹染色单体隔离。一种方法是描述与染色体运动的可视化招聘和营业额的着丝粒蛋白及其协调果蝇胚胎使用徕卡激光扫描共聚焦系统。

视频: Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

利用荧光原位杂交 (fish) 监测中期和中期 I. 型果蝇卵母细胞的臂部凝聚状态

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

科研

发育生物学

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

研究有丝分裂的显微注射技术果蝇合胞体胚胎

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

生物学

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

的实时成像<em&gt;果蝇</em&gt;幼虫神经母细胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

神经科学

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

用于活、完整组织中细胞分部分析的果蝇拉瓦尔和普帕尔睾试验的制备

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

精子发生的细胞学分析:Live和固定制剂<em&gt;果蝇</em&gt;睾丸

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

成像前中期和减数分裂中期的我固定技术<em&gt;果蝇</em&gt;卵母细胞

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

有丝分裂细胞同步与高分辨共聚焦显微镜联合应用研究细胞周期蛋白在有丝分裂过程中的作用

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, misunderstood, or misinterpreted by the fixed-cell analysis. While the fixed cell imaging and analysis is robust and sufficient to observe cellular steady-state, it can be limited in defining a temporal order of events at the cellular level and is ill-equipped to assess the transient nature of dynamic processes including mitotic progression. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular processes at the single-cell level over time and has the capacity to capture the dynamics of processes that would otherwise be poorly represented in fixed cell imaging. Here we describe an approach to generate cells carrying fluorescently labeled markers of chromatin and microtubules and their use in live cell imaging approaches to monitor metaphase chromosome alignment and mitotic exit. We describe imaging-based techniques to assess the dynamics of spindle formation and mitotic progression, including the identification of cells at various stages in mitosis, identification&#160;and tracking of mitotic defects, and analysis of spindle dynamics and mitotic cell fate following the treatment with mitotic inhibitors.

Here we present a protocol to assess the dynamics of spindle formation and mitotic progression. Our application of time-lapse imaging enables the user to identify cells at various stages of mitosis, track and identify mitotic defects, and analyze spindle dynamics and mitotic cell fate upon exposure to anti-mitotic drugs.

活细胞成像，以评估在斜接主轴扰动后元相定时和细胞命运的动态

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, ...

龙虎斗动粒蛋白的动态行为与染色体运动，在生活学习有丝分裂关卡

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