研究线粒体结构与功能<em>果蝇</em>卵巢
Summary
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.
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Grace's Media (Insect Dissecting Medium) | Fisher Scientific | 30611031-2 | |
| 41 Paraformaldehyde AQ | Electronic Microscopy Sciences | 50-259-99 | |
| Mitotracker Green (overall mitochondrial stain) | Life Technologies | m7514 | Reconstitute and Aliquot |
| Tetramethylrhodamine ethyl ester perchlorate | Sigma Aldrich | 87917-25MG | Reconstitute and Aliquot |
| MitoSox (Mito-Ros stain) | Life Technologies | m36008 | Reconstitute and Aliquot |
| PolyLysine | MP Biomedicals | ICN15017625 | |
| Fly Vials | Fisher Scientific | AS-515 | |
| Fly Conicals | Fisher Scientific | AS-355 | |
| Fly Vial Flugs | Fisher Scientific | AS273 | |
| Fly Conical Flugs | Fisher Scientific | AS 277 | |
| Jazzmix Drosophila food (Drosophila food) | Fisher Scientific | AS153 | |
| Bovine Serum Albumin | Sigma Aldrich | A9647-50G | |
| Cyclin E Antibody (d-300) | Santa Cruz | sc- 33748 | |
| ATPB antibody [3D5] – Mitochondrial Marker | AbCam | ab14730 | |
| Cy3 AffiniPure Goat Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 115-165-146 | |
| Cy5 AffiniPure Goat Anti-Rabbit IgG (H+L) | Jackson ImmunoResearch | 111-175-144 | |
| Hoechst | Fisher Scientific | H3570 | |
| VectaShield | Fisher Scientific | H100 | |
| Azer Scientific EverMark Select Microscope Slides | Fisher Scientific | 22-026-252 | |
| Microscope Cover Glass | Fisher Scientific | 12-542-B | |
| Name | Company | Catalog Number | Comments |
| Mat Tek Corp Glass Bottom Mircrowell Dish | Fisher Scientific | P35G-0-14-C | |
| Active Dried Yeast | Fisher Scientific | ICN10140001 | |
| Confocal Microscope | Carl Zeiss | LSM 700 | |
| Dumont #5 Forceps | Fine Science Technologies | 11251-20 | |
| Moria Nickel Plated Pin Holder | Fine Science Technologies | 26016-12 | |
| Minutien Pins | Fine Science Technologies | 26002-15 | |
| MYFP ( w[*]; P{w[+mC]=sqh-EYFP-Mito}3 ) | Bloomington Stock Center | 7194 | |
| Fly Pad | Fly stuff | 59-118 | |
| Blowgun | Fly stuff | 54-104 | |
| Blowgun needle | Flystuff | 54-119 | |
| Dissecting Microscope | Carl Zeiss | Stemi 2000 | |
| Analyses software | Carl Zeiss | Zen | |
| Analyses software | Open source | Image J | |
| Research Macro Zoom Microscope | Olympus | MVX10 | |
| QICAM Fast 1394 Cooled Digital Camera, 12-bit, Mono | QImaging | QIC-F-M-12-C | |
| QCapture Pro 5.1 | QImaging |
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