We acknowledge Leena Patel and Diamond Woodard for helping in the Drosophila medium preparation and Dr. Igor Chesnokov for providing access to the camera-attached stereomicroscope.

1.制备果蝇 （在图2A中描绘所需的工具） <ol><li>任何所描述的实验中，收集果蝇 （保持在室温，或25℃）内羽化的5天，将它们放置在盛有5小瓶-毫升果蝇 7食品（参见材料数据表 ），以不超过25苍蝇在每个瓶子;维护女:2男:1的比例。 </li><li>洒造粒酵母少量刺激果蝇产蛋量。 4天 - 2内进行实验操作。 </li></ol> 2.解剖果蝇卵巢的（图2A描绘所需的工具） <ol><li>暖昆虫解剖培养基（见材料表 ），以室温，25℃。填充八孔玻璃解剖盘的三个孔，以在每个培养基的200微升良好。 </li><li>麻醉果蝇与CO <sub。通过放置喷枪的针药瓶塞下&gt; 2。把他们放在一个飞盘。使用解剖显微镜，理清5女，并将其放置在解剖盘的第一口井。现场表演时，显微镜，一次处理一个果蝇 。 </li><li>同时期待通过解剖显微镜的目镜，从断绝使用两对镊子腹部胸部。使用镊子，仔细的腹部转移到菜的第二口井。 </li><li>用一对镊子的保持在后端腹部，并慢慢地与另一对钳子的推卵巢出来（与其他腹部内容一起）。如果这种尝试失败了，小心插入钳入前端释放卵巢取出腹腔外骨骼。 </li><li>使用镊子，持有不透明后端个人卵巢（ 即蛋黄填充，后期鸡蛋），并小心将其移动到菜的第三井为挑逗处理它实时显微镜（步骤3）或用于固定到执行免疫染色（步骤7）。 </li><li>通过从后轻轻扫过挑逗针对每个卵巢的前端，同时用一对镊子的保持它用的后端小心挑逗从卵巢周围保护套。 注意:要取笑期间尽量减少损失，弯曲针尖端和通过增加显微镜（ 图2A）的放大率放大各卵巢。挑逗应该足以打破鞘有效的，但它也应仔细进行保存卵巢管的完整性。 </li></ol> 3.准备用于活组织显微镜 注意:在图2A中描绘所需的工具。 <ol><li> 果蝇解剖之前，准备多聚赖氨酸涂覆室。为此，将多聚赖氨酸（0.1毫克/毫升）的两个20微升滴上covergl驴，以防止液滴的合并在彼此合理距离玻璃底培养皿（35 mm）上的（14毫米，0号公报）。风干平板在37ºC1小时并标记在板的与可擦除标记在底部的白色多聚赖氨酸膜的边缘。 注:多聚赖氨酸包被的腔室可以被储存在4℃下一周。如果使用预先涂覆室中，在开始果蝇解剖前使其至室温。 </li><li>上设置共焦显微镜扫描参数，以确保可将样品立即解剖和安装完成后，如下成像。 </li><li>放置一解剖并逗卵巢（以下的步骤2和染色，如果需要的话，在步骤5）上的标记的多聚赖氨酸包被的区域，传播卵巢与挑逗针头至卵巢管分离。将昆虫解剖介质对卵巢的前10μL下降，确保覆盖Ť他整个多聚赖氨酸包被的区域。盖培养皿。 </li><li>立即执行在室温下安装的样品的共焦显微镜。 注意:只有一个果蝇应解剖，进行处理，并在时间成像。此外，显微镜不应在37℃，这会模仿果蝇组织热休克环境中执行。 </li></ol> 4.荧光损失在漂白（翻转）检测，以评估线粒体基质连续性 注:在熔融线粒体结构线粒体基质连续性以下通过中间步骤的进展中的线粒体内膜和外膜的完全熔化后建立的。线粒体裂变可以遵循同样的步骤，但在相反的方向（ 图3A）。 FLIP是一个时间推移基于显微镜半定量方法，该方法可用于评估在线粒体基质连续性体外线粒体的最终熔融状态（ 图3A中的步骤3和4）在活果蝇卵巢9。对FLIP测定法作为表示在被定期（ 图3A中FLIP ROI）的光漂白线粒体基质的荧光分子的线粒体的感兴趣区域（ROI）的小区域进行。其结果是，是与对FLIP的ROI（实验ROI在图3A）连续任何周围线粒体区域将失去的信号，由于分子中的连续线粒体基质的交换。这里展示的FLIP实验转基因果蝇表达mitoYFP，其中包含标记YFP人类细胞色素氧化酶亚基第八的线粒体靶向序列，将其定位到线粒体基质中自由扩散的形式进行。类似的实验，也可以与线粒体pUASP-线粒体-GFP转基因进行的，如先前报道&lt;SUP&gt; 9。类似FLIP协议可以与靶向线粒体膜间空间中的探针可以用于能够检测从外部的融合所得的连续性而不是内线粒体膜（步骤2在图3A）。 <ol><li>在共聚焦显微镜打开图像采集软件，并在"获取"标签（ 表1）设置适当的扫描参数。检查"时间序列"，"漂白"，和"区域"框以打开单个标签。出台相应的采集参数值在每个选项卡（ 表1）。 注:针孔应保持开路，因为此实验的目的是监测从单个细胞的全线粒体人口整体信号。 </li><li>使用目镜快速定位在安装现场组织感兴趣的领域。 注:选择是很好铺在玻璃bottome的卵巢管D盘，由于共焦显微镜不能浮动卵巢管进行的。 </li><li>点击"活"来获取感兴趣的选定字段的实时图像。点击"停止"，停止现场扫描。 </li><li>如果需要的话，调节采集参数，使得所检测到的荧光信号是低于饱和水平（由缺乏红色像素表示当"范围指示器"选项时），与所定义的背景为通过调整所述偏移值设定。 </li><li>绘制使用从"区域"标签贝塞尔绘图工具来划分由现场扫描获取的图像上的光漂白区的小的投资回报。 注:ROI的大小应在20 -在细胞内的总荧光线粒体信号的50％。 </li><li>通过点击进行图像采集"开始实验"。 </li><li>量化使用专有或开源软件的荧光强度（见材料表）。记录从投资回报率的平均信号，其中重复漂白是针对（FLIP）;其中，漂白尚未在相同小区进行的感兴趣区（试验）;从在同一视场的另一个未漂白细胞的投资回报率，为试验期间（漂白）评估整体漂白;并且在背景区域（背景）的投资回报率。减去在其他感兴趣区域的平均信号获得的平均背景信号。正常化与用于各个小区中的初始漂白前信号的荧光信号。 </li><li>绘制使用任何标准绘图软件的标准化数据。 </li></ol> 5.现场染色荧光染料线粒体 注:稳态线粒体结构和潜在的可使用特异性结合到线粒体的活细胞和组织的染料进行评估。活果蝇卵巢可体外染色荧光线粒体渍形象化线粒体，以评估线粒体活性氧（丝裂ROS）的生产，并评估每单位质量线粒体电位。这可以通过共染色与线粒体电位染料四甲乙酯（TMRE）和代表线粒体质量相容的活线粒体染色来完成（见材料表的特定染料）。 <ol><li>稀释在温暖的昆虫污渍解剖介质的最终工作浓度的股票:线粒体染色，250纳米; TMRE，50nM的;和线粒体-ROS染色，5微米。 </li><li>解剖和戏弄卵巢以下步骤2中后，将卵巢成任何特定的染色溶液的200微升在解剖盘的好。孵育他们10分钟并通过合适的盒用铝箔包裹，以保护它免受光所覆盖的培养皿中。用钳子小心地移动它们到连续的3口井containin洗染色卵巢无污点摹网上平台。 </li><li>对于共染色与TMRE和兼容整体线粒体染色，按照上述协议，立即先用TMRE，然后用染色的整体线粒体染色（不之间的任何洗涤步骤）。 </li><li>安装在一个多聚赖氨酸涂覆的玻璃底培养皿下列步骤3卵巢和用于与适当的扫描参数（ 表1）共聚焦显微镜制备，下列步骤4.2-4.4。 注:从掺入染料的信号，再尝试安装该染色样品中安装介质中时并没有持续。 </li><li>检查Z-切片框打开的选项卡。转动调焦环对样品的底部，而它正在实时扫描，然后单击"设置第一"来定义底层Z-部分。做相同的同时移动焦点车轮朝另一个方向以限定的最上面部分。 </li><li>通过点击进行图像采集"开始实验"。</li><li>量化来自感兴趣区为背景信号和各个单元的背景校正的荧光强度（如在步骤4.7）和使用任何绘图软件绘制数据。 </li></ol> 6.代DRP1空马赛克 注意:此处所用的克隆策略介绍一个GFP阳性，表型的野生型背景下，对于DRP1无效突变9基因型杂的背景绿色荧光蛋白（GFP）阴性DRP1空克隆。热休克诱导翻转，翻转识别目标（FLP-FRT）介导的位点特异性有丝分裂重组创建功能无效drpKG03815等位基因的纯合子克隆。 果蝇携带突变DRP1的基因型为drpKG03815 FRT40A / CYO，而基因型携带热休克诱导FLP（hsFLP）和UbiGFP克隆标记是hsflp;泛素NLS-GFP（UbiGFP）FRT 40A / CYO。的本身的基因型上述基因型之间的交叉lected后代hsFLP / +; drpKG03815 FRT40A / UbiGFPFRT40A。 <ol><li>通过将移动到新鲜食品的新瓶，每2〜3天同步果蝇的处女集合。日常监控pupariating瓶，以便收集新兴处女的女性。 </li><li>从DRP1突变基因型每天晚上收集红眼，卷曲翼处女的女性，一旦在上午，一次，并将其放置在一个单独的小瓶内。同时，收集果蝇雄性暗红色的眼睛和内羽化5天携带hsflp和UbiGFP直翅膀。 </li><li> 2男女比例:通过增加男性的处女女性与女性建立一个跨1。 </li><li>洒造粒酵母少量刺激产蛋量。 注:小心地移动至果蝇到的媒体新鲜小瓶，每2至3天，以增加后代的数量，并减少小瓶拥挤。 </li><li>一个esthetize，排序，并收集直翅，红眼睛的女性后代中羽化5天。 </li><li>对热休克，将收集的果蝇到空的小瓶造粒酵母少量和一个小的，柔软的擦拭（热休克期间吸收水分）。放置与果蝇小瓶在水浴中在38ºC1小时，以产生主要毛囊细胞克隆，并在37ºC1小时，一天两次（允许2热冲击之间的至少5小时），用于连续2天，同时生成的种系和卵泡细胞克隆。 注:确保将小瓶中的水至插头的水平完全淹没。 </li><li>热休克可能使果蝇不动。允许热休克果蝇 ，为在室温下1小时恢复当他们再次成为移动。 </li><li>以相同的比例增加男性回女性在十字架上，并将其移动到与mediu新鲜瓶米撒上颗粒酵母少量。保持果蝇至少5天。 </li><li>解剖卵巢根据需要为一个活的或固定的实验。 注意:从亲果蝇表达UbiGFP分离卵巢应该被用作阴性对照，以确认由热休克GFP阴性的克隆的有效诱导。 </li></ol> 7.联合免疫染色细胞周期素E和线粒体 注:要检测果蝇细胞周期素E（dCyclinE），我们已经使用了专门针对dCyclinE 9募集市售获得的抗体（见材料表 ）。作为线粒体标记物，我们使用针对的ATP-B的抗体（线粒体ATP合酶复合物的亚基）9。 <ol><li>暖4％低聚甲醛（PFA），以室温，25℃。 警告！多聚甲醛是有毒的。 注:开场后安瓿，PFA应储存在4℃和7天内使用。这是因为，PFA的存储可允许氧化甲醇，这将在定影期间极大地改变线粒体膜，即使痕量存在。 </li><li>解剖后，立即将其放入新鲜的PFA 200μL在玻璃解剖盘的井（无揶揄）修复解剖卵巢。请在通风橱中的菜15分钟。通过用钳子小心移动它们到连续的3个孔，1×磷酸盐缓冲盐水的各自含有200微升（PBS）洗涤固定的卵巢。 注:实验可以在这里停止，并且可将样本在4ºC留1天。 </li><li>在PBS更彻底逗卵巢（类似于步骤2.6），以小心地取出防护纤维鞘可能妨碍了抗体抗原访问。 </li><li>通过将它们与500微升新鲜的0离心管通透戏弄卵巢。5％的PBS-的Triton-X100（PBS-TX）和孵育它为30分钟，同时在25转摆动。 注意:在摇动，放置管平行于摇摆运动;将它们垂直地可以允许组织粘附到管的盖，从而导致组织损失。 </li><li>以除去PBS-TX放置离心管在机架以允许组织在管的底部沉淀下来。目视检查他们，挖掘管，如果需要的话，给人们带来任何浮动的组织下到谷底。从顶部小心地吸出溶液，并确保在管底部的组织保持原状。 </li><li>通过添加2％牛血清白蛋白的200μL的（BSA）溶解在0.5％的PBS-TX方框卵巢，并在室温下孵育它在摇臂1小时。通过以下步骤7.5移除阻断剂。 </li><li>添加第一抗体在新鲜封闭剂的200微升:抗兔dCyclinE抗体（1:100）和抗小鼠的ATP-B抗体（1:100）。孵育在摇臂的组织，在室温下2小时。 </li><li>用PBS-TX洗卵巢3次，每次15分钟，随后的步骤7.5。 </li><li>添加200μL的在新鲜的PBS-TX的适当的二级抗体:抗小鼠CY3（1:1000）和抗 - 兔CY5（1:500）。孵育该摇杆在室温下1小时。 </li><li>用PBS-TX洗卵巢3次，每次15分钟，随后的步骤7.5。 </li><li>染色的DNA，加入赫斯特（1:1000稀释）最终PBS-TX洗。 </li><li>最后，离开组织在1X PBS 500微升。 </li><li>使用1毫升微量，请从离心管的免疫染色卵巢到含有PBS的200微升玻璃解剖盘的新鲜好。 </li><li>基于甘油安装介质一滴添加到载玻片并且由一个加到安装平台的免疫染色卵巢之一。 注:确保卵巢确实转移到安装介质。未能一切都做得那么Ø会导致组织损失。 </li><li>同时期待通过解剖显微镜，轻轻掐使用戏弄针保持与镊子卵巢的不透明部分不透明成熟的卵子商会透明卵巢管（年龄阶段）。从安装介质中取出成熟的卵子商会。 </li><li>将在幻灯片上，轻压一个玻璃罩（22毫米，1号），以确保安装介质传播均匀下方。 注:按上玻璃罩也确保沿玻璃罩的组织的适当对准。未能这样最佳地做可以允许较小的阶段漂浮在所述安装平台，从而防止那些组织的最佳显微镜。 </li><li>空气干燥15分钟，将样品和密封玻璃罩的边缘仔细指甲油。 </li><li>执行共焦显微镜根据实验的需要（ 表1）。 </li></ol>

<ol>
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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=At+the+crossroads+of+differentiation+and+proliferation:+precise+control+of+cell-cycle+changes+by+multiple+signaling+pathways+in+Drosophila+follicle+cells.">At the crossroads of differentiation and proliferation: precise control of cell-cycle changes by multiple signaling pathways in Drosophila follicle cells.</a> Bioessays. 33 (2), 124-134 (2011).</li><li>Sahai-Hernandez, P., Castanieto, A., Nystul, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+models+of+epithelial+stem+cells+and+their+niches.">Drosophila models of epithelial stem cells and their niches.</a> Wiley Interdiscip Rev Dev Biol. 1 (3), 447-457 (2012).</li><li>Parker, D. J., 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=A+new+mitochondrial+pool+of+cyclin+E,+regulated+by+Drp1,+is+linked+to+cell-density-dependent+cell+proliferation.">A new mitochondrial pool of cyclin E, regulated by Drp1, is linked to cell-density-dependent cell proliferation.</a> J Cell Sci. 128 (22), 4171-4182 (2015).</li><li>Mitra, K., Wunder, C., Roysam, B., Lin, G., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hyperfused+mitochondrial+state+achieved+at+G1-S+regulates+cyclin+E+buildup+and+entry+into+S+phase.">A hyperfused mitochondrial state achieved at G1-S regulates cyclin E buildup and entry into S phase.</a> Proc Natl Acad Sci U S A. 106 (29), 11960-11965 (2009).</li><li>Shidara, Y., Hollenbeck, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defects+in+mitochondrial+axonal+transport+and+membrane+potential+without+increased+reactive+oxygen+species+production+in+a+Drosophila+model+of+Friedreich+ataxia.">Defects in mitochondrial axonal transport and membrane potential without increased reactive oxygen species production in a Drosophila model of Friedreich ataxia.</a> J Neurosci. 30 (34), 11369-11378 (2010).</li><li>Zielonka, J., Kalyanaraman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroethidine-+and+MitoSOX-derived+red+fluorescence+is+not+a+reliable+indicator+of+intracellular+superoxide+formation:+another+inconvenient+truth.">Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth.</a> Free Radic Biol Med. 48 (8), 983-1001 (2010).</li><li>Haack, T., Bergstralh, D. T., St Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Damage+to+the+Drosophila+follicle+cell+epithelium+produces+&amp;#34;false+clones&amp;#34;+with+apparent+polarity+phenotypes.">Damage to the Drosophila follicle cell epithelium produces &amp;#34;false clones&amp;#34; with apparent polarity phenotypes.</a> Biol Open. 2 (12), 1313-1320 (2013).</li><li>Murphy, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+mitochondria+produce+reactive+oxygen+species.">How mitochondria produce reactive oxygen species.</a> Biochem J. 417 (1), 1-13 (2009).</li><li>Mandal, S., Guptan, P., Owusu-Ansah, E., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+regulation+of+cell+cycle+progression+during+development+as+revealed+by+the+tenured+mutation+in+Drosophila.">Mitochondrial regulation of cell cycle progression during development as revealed by the tenured mutation in Drosophila.</a> Dev Cell. 9 (6), 843-854 (2005).</li><li>Tipping, M., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+context-dependent+tumorigenesis.">Drosophila as a model for context-dependent tumorigenesis.</a> J Cell Physiol. 229 (1), 27-33 (2014).</li><li>Herranz, H., Eichenlaub, T., Cohen, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+in+Drosophila:+Imaginal+Discs+as+a+Model+for+Epithelial+Tumor+Formation.">Cancer in Drosophila: Imaginal Discs as a Model for Epithelial Tumor Formation.</a> Curr Top Dev Biol. 116, 181-199 (2016).</li><li>Pandey, U. B., Nichols, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+disease+models+in+Drosophila+melanogaster+and+the+role+of+the+fly+in+therapeutic+drug+discovery.">Human disease models in Drosophila melanogaster and the role of the fly in therapeutic drug discovery.</a> Pharmacol Rev. 63 (2), 411-436 (2011).</li></ol>

The authors have no competing financial interests.

该议定书中的关键步骤 漂白:防止荧光样品的过度的光漂白是绝对必要的进行高效率的共焦显微镜。因此，用于通过目镜来定位样品或通过实时扫描模式设置图像采集参数的时间应尽量减少，以减少光漂白。 的组织损伤 :由于线粒体被认为是细胞健康的传感器，这是非常重要的，以确保使用所描述的方法获得的数据是生理学相关，并不?...

线粒体经典描述为蜂窝重地，因为它们是能源生产的在分化的细胞的主座。此外，线粒体起到代谢，发热，脂质修饰，钙和氧化还原平衡的关键作用，细胞信号传导过程的流程等 1。线粒体也发挥在细胞周期调控3细胞死亡2诱导的积极作用，以及。这种多功能性提出了以下基本问题:a）如何线粒体同时执行所有这些功能和b）是否有专门的是针对不同的功能，具体的线粒体池或子区域？在此情况下，必须注意的是，多官能线粒体在它们的形状，大小和单个细胞内结构是动态的，并且线粒体的稳态形状可以细胞类型之间变化是重要的。从不同的实验室数十年的研究礼拜堂表明线粒体形状，大小和结构，统称线粒体动力学的改变，是为了保持各种线粒体功能4,5,6至关重要。这些发现提出线粒体可能凭借其结构的活力完成其多功能的可能性。 广泛正在努力了解线粒体结构与功能的关系。线粒体结构的动力主要是由他们的经历裂变和聚变事件与对方保持能力。大线粒体裂变将它们转换成更小的线粒体的元素，而两个较小的线粒体之间的融合将它们合并成一个更大的线粒体元件7。此外，可以发生两种线粒体的瞬态融合，以允许其内容的混合。内和外线粒体膜的裂变和聚变事件仔细规格支配IFIC设置蛋白质。芯裂变机械由dynamin上相关蛋白1（DRP1），这是从细胞质通过其与某些善意线粒体蛋白质（ 例如，FIS1或Mff1）相互作用招募到线粒体，而DRP1功能也可以通过调节的线粒体表面4上的其它蛋白质。虽然DRP1外膜上运行时，其裂变能力产生影响的内膜为好。外和内线粒体膜的裂变的编排还不是很清楚。另一方面，内膜的融合在由OPA1的活动芯部管辖，而mitofusins管辖外膜5的融合。线粒体的反作用裂变和融合事件的平衡决定在细胞稳态线粒体形状。例如，线粒体裂变的压迫会导致完全和无对抗的融合，而线粒体的过度活动升裂变将导致线粒体3的碎片。 线粒体结构 - 功能关系的研究主要涉及两个互补的方法:一）线粒体裂变/融合蛋白的基因操作后的细胞和有机体的表型分析和b）线粒体的结构和功能的直接评估。值得注意的是，遗传分析可能不总是揭示分子的手头的直接功能（在此情况下，线粒体裂变/融合蛋白）中，作为表型可能是由于次级效应引起的。因此，它是极为重要的发展和使用的工具来直接研究线粒体的结构和功能。线粒体结构的任何评估都涉及各种显微镜的工具。活细胞荧光显微镜的使用具有极大地促进线粒体动力学的研究，因为线粒体活力可以监测定性和quantitatively使用适当的荧光显微镜的工具和技术8。荧光显微镜基础的工具已经发展到线粒体的研究结构和功能的现场和固定果蝇组织，阐明体内线粒体9活力的重要意义。这些和相关的方法在这里所描述，在果蝇卵巢研究线粒体的结构和功能的目的。 果蝇卵巢由种系和体细胞谱系，从驻留在germarium 10,11各自的成体干细胞，其产生的。十六合胞生殖细胞（GCS）通过体毛囊细胞（FC）中得到包封以形成浮现出germarium（ 图1）的个人卵腔室。之一的16个选区的获得致力于成为卵母细胞，和其余15选区发展成支持卵腔的生长护士细胞，促进卵成熟它铺设之前。多数的FC经过9轮有丝分裂的它们退出有丝分裂的细胞周期到最终分化成由前卵泡电池（AFC），后囊细胞（全氟化碳），和主体单元的图案的上皮细胞层之前（的MBC） 。连续蛋室由茎细胞，其分化，它们也从FC的在发展的早期的细胞连接。由线粒体分裂蛋白DRP1调节线粒体形状果蝇卵巢FC层9,12的正常发展过程中，积极参与分化过程。在这些研究中使用，以确定DRP1的果蝇卵泡细胞层发展参与方法说明如下。

<table><tbody><tr><td>Grace&#039;s Media (Insect Dissecting Medium)</td><td>Fisher Scientific</td><td>30611031-2</td><td></td></tr><tr><td>41 Paraformaldehyde AQ</td><td>Electronic Microscopy Sciences</td><td>50-259-99</td><td></td></tr><tr><td>Mitotracker Green (overall mitochondrial stain)</td><td>Life Technologies</td><td>m7514</td><td>Reconstitute and Aliquot</td></tr><tr><td>Tetramethylrhodamine ethyl ester perchlorate</td><td>Sigma Aldrich</td><td>87917-25MG</td><td>Reconstitute and Aliquot</td></tr><tr><td>MitoSox (Mito-Ros stain)</td><td>Life Technologies</td><td>m36008</td><td>Reconstitute and Aliquot</td></tr><tr><td>PolyLysine</td><td>MP Biomedicals</td><td>ICN15017625</td><td></td></tr><tr><td>Fly Vials</td><td>Fisher Scientific</td><td>AS-515</td><td></td></tr><tr><td>Fly Conicals</td><td>Fisher Scientific</td><td>AS-355</td><td></td></tr><tr><td>Fly Vial Flugs</td><td>Fisher Scientific</td><td>AS273</td><td></td></tr><tr><td>Fly Conical Flugs</td><td>Fisher Scientific</td><td>AS 277</td><td></td></tr><tr><td>Jazzmix Drosophila food (Drosophila food)</td><td>Fisher Scientific</td><td>AS153</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A9647-50G</td><td></td></tr><tr><td>Cyclin E Antibody (d-300)</td><td>Santa Cruz</td><td>sc- 33748</td><td></td></tr><tr><td>ATPB antibody [3D5] - Mitochondrial Marker</td><td>AbCam</td><td>ab14730</td><td></td></tr><tr><td>Cy3 AffiniPure Goat Anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>115-165-146</td><td></td></tr><tr><td>Cy5 AffiniPure Goat Anti-Rabbit IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>111-175-144</td><td></td></tr><tr><td>Hoechst</td><td>Fisher Scientific</td><td>H3570</td><td></td></tr><tr><td>VectaShield</td><td>Fisher Scientific</td><td>H100</td><td></td></tr><tr><td>Azer Scientific EverMark Select Microscope Slides</td><td>Fisher Scientific</td><td>22-026-252</td><td></td></tr><tr><td>Microscope Cover Glass</td><td>Fisher Scientific</td><td>12-542-B</td><td></td></tr><tr><td>Mat Tek Corp&amp;nbsp;Glass Bottom Mircrowell Dish</td><td>Fisher Scientific</td><td>P35G-0-14-C</td><td></td></tr><tr><td>Active Dried Yeast</td><td>Fisher Scientific</td><td>ICN10140001</td><td></td></tr><tr><td>Confocal Microscope</td><td>Carl Zeiss</td><td>LSM 700</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Technologies</td><td>11251-20</td><td></td></tr><tr><td>Moria Nickel Plated Pin Holder</td><td>Fine Science Technologies</td><td>26016-12</td><td></td></tr><tr><td>Minutien Pins</td><td>Fine Science Technologies</td><td>26002-15</td><td></td></tr><tr><td>MYFP ( w[*]; P{w[+mC]=sqh-EYFP-Mito}3 )</td><td>Bloomington Stock Center</td><td>7194</td><td></td></tr><tr><td>Fly Pad</td><td>Fly stuff</td><td>59-118</td><td></td></tr><tr><td>Blowgun</td><td>Fly stuff</td><td>54-104</td><td></td></tr><tr><td>Blowgun needle</td><td>Flystuff</td><td>54-119</td><td></td></tr><tr><td>Dissecting Microscope</td><td>Carl Zeiss</td><td>Stemi 2000</td><td></td></tr><tr><td>Analyses software</td><td>Carl Zeiss</td><td>Zen&amp;nbsp;</td><td></td></tr><tr><td>Analyses software</td><td>Open source</td><td>Image J</td><td></td></tr><tr><td>Research Macro Zoom Microscope</td><td>Olympus</td><td>MVX10</td><td></td></tr><tr><td>QICAM Fast 1394 Cooled Digital Camera, 12-bit, Mono&amp;nbsp;</td><td>QImaging</td><td>QIC-F-M-12-C</td><td></td></tr><tr><td>QCapture Pro 5.1</td><td>QImaging</td><td></td><td></td></tr></tbody></table>

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.

所描述的方法可用于研究线粒体的结构和功能在现场和固定果蝇卵巢（ 图2B）。提供与所描述的方法获得的预期结果的一些例子。 果蝇卵巢的解剖 :当进一步解剖，从整个果蝇 （ 图3A）被切断的腹部（ 图3B）应该释放腹腔内容，包括来自每个单独的果蝇 2?...

Watch this Scientific Journal Video about Studying Mitochondrial Structure and Function in Drosophila Ovaries at JoVE.com

果蝇卵巢线粒体结构和功能的研究- 中国优秀硕士学位论文全文数据库生物学 |视频

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 ...

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Studying Mitochondrial Structure and Function in Drosophila Ovaries

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Cell Structure and Function

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23.9K 次观看.  University of Alabama at Birmingham. 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. 

视频: Studying Mitochondrial Structure and Function in Drosophila Ovaries

Dissection of Drosophila Ovaries

剖析果蝇卵巢

科研

生物学

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.

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 ...

遗传学

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions. 
Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

在代谢分析准备线粒体富集级的<em&gt;果蝇</em

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often ...

发育生物学

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

用最小组织量测定果蝇透纤维中线粒体耗氧量

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

生物化学

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

果蝇蛹卵巢的解剖与染色

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

果蝇卵生过程中极化细胞内运输和基底膜蛋白分泌的共聚焦和超分辨率成像

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment ...

分析果蝇的线粒体生理学和生物能量学

Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1B9-Null Mutant Using High-resolution Respirometry

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy metabolism with impairment of mitochondrial functions. Mitochondria are organelles that control both energy metabolism and cellular processes involved in cell survival and death. For this reason, approaches to evaluate mitochondrial function can offer important insights into cellular conditions in pathological and physiological processes. In this regard, high-resolution respirometry (HRR) protocols allow evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes. Furthermore, studying mitochondrial physiology and bioenergetics requires genetically and experimentally tractable models such as Drosophila melanogaster.
This model presents several advantages, such as its similarity to human physiology, its rapid life cycle, easy maintenance, cost-effectiveness, high throughput capabilities, and a minimized number of ethical concerns. These attributes collectively establish it as an invaluable tool for dissecting complex cellular processes. The present work explains how to analyze mitochondrial function using the Drosophila melanogaster PINK1B9-null mutant. The pink1 gene is responsible for encoding PTEN-induced putative kinase 1, through a process recognized as mitophagy, which is crucial for the removal of dysfunctional mitochondria from the mitochondrial network. Mutations in this gene have been associated with an autosomal recessive early-onset familial form of PD. This model can be used to study mitochondrial dysfunction involved in the pathophysiology of PD.

作者聚焦：使用黑腹果蝇探索线粒体功能和化学毒性 

Here we present a high-resolution respirometry protocol to analyze bioenergetics in PINK1B9-null mutant fruit flies. The method uses the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

使用高分辨率呼吸测量法分析 果蝇黑腹 果蝇 PINK1B9-Null 突变体的线粒体功能

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy ...

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

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

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

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

研究线粒体结构与功能

摘要

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此视频中的章节

剖析果蝇卵巢

在果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

在代谢分析准备线粒体富集级的

用最小组织量测定果蝇透纤维中线粒体耗氧量

果蝇蛹卵巢的解剖与染色

果蝇卵生过程中极化细胞内运输和基底膜蛋白分泌的共聚焦和超分辨率成像

使用高分辨率呼吸测量法分析果蝇黑腹果蝇 PINK1^B9-Null 突变体的线粒体功能

使用高分辨率呼吸测量法分析果蝇黑腹果蝇 PINK1^B9-Null 突变体的线粒体功能

使用高分辨率呼吸测量法分析果蝇黑腹果蝇 PINK1^B9-Null 突变体的线粒体功能

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

研究线粒体结构与功能

摘要

探索更多视频

此视频中的章节

剖析果蝇卵巢

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

在代谢分析准备线粒体富集级的

用最小组织量测定果蝇透纤维中线粒体耗氧量

果蝇蛹卵巢的解剖与染色

果蝇卵生过程中极化细胞内运输和基底膜蛋白分泌的共聚焦和超分辨率成像

使用高分辨率呼吸测量法分析 果蝇黑腹 果蝇 PINK1B9-Null 突变体的线粒体功能

使用高分辨率呼吸测量法分析 果蝇黑腹 果蝇 PINK1B9-Null 突变体的线粒体功能

使用高分辨率呼吸测量法分析 果蝇黑腹 果蝇 PINK1B9-Null 突变体的线粒体功能

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

在果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

使用高分辨率呼吸测量法分析果蝇黑腹果蝇 PINK1^B9-Null 突变体的线粒体功能

使用高分辨率呼吸测量法分析果蝇黑腹果蝇 PINK1^B9-Null 突变体的线粒体功能

使用高分辨率呼吸测量法分析果蝇黑腹果蝇 PINK1^B9-Null 突变体的线粒体功能