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.

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The authors have no competing financial interests.

プロトコル内の重要なステップ 光退色：蛍光サンプルの過度の光退色を防止することは、効率的な共焦点顕微鏡を実行することに絶対に必要です。したがって、接眼レンズを介してサンプルを検索したり、ライブ走査モードを介して画像取得パラメータを設定するために使用される時間は、光退色を最小にするために最小化されるべきです。 <p class="jove_...

彼らは分化した細胞でのエネルギー産生の主な席があるので、ミトコンドリアは、古典的に、携帯大国として記載されています。また、ミトコンドリア等は1、代謝、発熱、脂質修飾、カルシウム及び酸化還元ホメオスタシスにおける細胞シグナル伝達プロセスのオーケストレーションに重要な役割を演じます。ミトコンドリアは、細胞死2、ならびに細胞周期調節3での誘導に積極的な役割を果たしています。このような多機能性は、以下の基本的な問題を提起する：a）どのようにミトコンドリアが同時にこれらすべての機能を実行し、異なる機能のために特化されている特定のミトコンドリアプールやサブゾーンb）はありますか？この文脈では、多官能性ミトコンドリアが、個々の細胞内での形状、大きさ、および構造に動的であること、およびミトコンドリアの定常状態の形状は、細胞型間で変化し得ることに留意することが重要です。様々な実験室からの研究の十年オラトリオは、ミトコンドリアの形状、大きさ、構造、総称ミトコンドリアのダイナミクスの変化は、様々なミトコンドリアの機能4,5,6を維持するために重要であることを示唆しています。これらの知見は、ミトコンドリアがそれらの構造的ダイナミズムのおかげで、それらの多機能化を達成することができる可能性を提起します。 大規模な努力がミトコンドリアの構造と機能の関係を理解するために進行中です。ミトコンドリアの構造のダイナミズムは、主として、互いに分裂および融合事象を受ける能力によって維持されます。 2つの小さなミトコンドリアの融合が大きくミトコンドリア素子7にそれらをマージしながら、大規模なミトコンドリアの分裂は、より小さなミトコンドリアの要素に変換します。また、2ミトコンドリアの一過性の融合は、その内容の混合を可能にするために発生することがあります。内側と外側のミトコンドリア膜の分裂と融合イベントは慎重仕様によって支配されていますタンパク質のIFICセット。コア核分裂機械はDRP1機能もによって調節することができる一方で、特定の善意のミトコンドリアタンパク質（ 例えば、Fis1またはMff1）との相互作用によってミトコンドリアに細胞質ゾルから募集されたダイナミン関連タンパク質1（DRP1）、から構成されていますミトコンドリアの表面4上の他のタンパク質。 DRP1は、外膜で動作しますが、その核分裂能力は、同様に内膜に影響を与えます。外側と内側のミトコンドリア膜の分裂のオーケストレーションが十分に理解されていません。 mitofusinsは、外膜5の融合を支配しながら、一方で、内膜の融合は、OPA1の活動によりコアに支配されています。ミトコンドリアの対抗核分裂と核融合イベントのバランスは、細胞内の定常状態のミトコンドリア形状を決定します。例えば、ミトコンドリア分裂の抑制は、ミトコンドリアの過活動しながら、完全かつ無競争の融合をもたらすであろうリットルの核分裂はミトコンドリア3の断片化をもたらすであろう。 ミトコンドリアの構造と機能の関係の研究は、主に次の2つの相補的なアプローチを含む：a）は、ミトコンドリア分裂/融合タンパク質の遺伝子操作した後、細胞および生物表現型の分析およびb）ミトコンドリアの構造と機能の直接評価。表現型が原因で二次的な影響を生じる可能性があるとして、遺伝的分析はいつも、（この場合は、ミトコンドリアの分裂/融合タンパク質）手元にある分子の直接の機能を明らかにしない可能性があることに注目すべきです。したがって、直接ミトコンドリアの構造および機能を研究するためのツールを開発し、使用するために極めて重要です。ミトコンドリアの構造のいずれかの評価は、様々な顕微鏡ツールを必要とします。ミトコンドリアの活力を定性的およびquantitatモニターすることができるので、生細胞の蛍光顕微鏡法の使用は、ミトコンドリアの動力学の研究を大幅に進んでいますively適切な蛍光顕微鏡のツールとテクニック8を使用して。蛍光顕微鏡ベースのツールは、 インビボ 9 ミトコンドリアダイナミズムの意義を解明する、ライブ固定キイロショウジョウバエ組織においてミトコンドリアの構造および機能を研究するために開発されてきました。これらおよび関連する方法は、 ショウジョウバエ卵巣でミトコンドリアの構造と機能を研究することを目的に、ここで説明します。 ショウジョウバエの卵巣は、胚腺10,11内に存在し、それぞれの成体幹細胞から生じる生殖系列および体細胞系統、で構成されています。十六合胞体胚細胞（のGC）は、胚腺（ 図1）から出てくる個々の卵室を形成するために体包細胞（FCS）によってカプセル化されます。 16のGCの一つは、卵母細胞になることを約束します、そして残りの15 GCは卵母細胞チャンバの成長を支援ナース細胞に発展しますそれが載置される前に、卵の成熟を促進します。これらは末端前方包細胞（AFCS）、後方包細胞（PFC類）、及び本体細胞からなるパターン化された上皮細胞層に分化する分裂細胞周期を出る前に、FCの大半は、有糸分裂の9ラウンドを受ける（MBCS） 。連続卵室が早期開発中のFCに由来する細胞を分化さ茎細胞によって接続されています。ミトコンドリア分裂タンパク質DRP1によって規制ミトコンドリアの形状は、積極的にショウジョウバエ卵巣FC層9,12の正常な発達の間、分化の過程に関与しています。 ショウジョウバエ卵胞細胞層の発達にDRP1の関与を同定するために、これらの研究で使用した方法は、ここに記載されています。

<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.

記載の方法は、生きて固定ショウジョウバエ卵巣（ 図2B）にミトコンドリアの構造および機能を研究するために使用することができます。記載された方法を用いて得られた期待結果のいくつかの例が提供されます。 ショウジョウバエ卵巣の解剖 ：さらに解剖すると、全体のショウジョ?...

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

ショウジョウバエの卵巣におけるミトコンドリアの構造と機能の研究 |生物学 |ビデオ

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.

ミトコンドリアの構造と機能の研究に<em&gt;ショウジョウバエ</em&gt;卵巣

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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23.9K ビュー.  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. 

ビデオ: Studying Mitochondrial Structure and Function in Drosophila Ovaries

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.

EDU染色によりショウジョウバエアダルト卵巣内のミトコンドリアDNA複製のその場の標識で

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

遺伝学

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.

代謝分析のためのミトコンドリア富化画分の調製で<em&gt;ショウジョウバエ</em

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

発生生物学

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.

ショウジョウバエのティッシュの最小限の量を使用してのグリセリンの繊維でミトコンドリアの酸素消費量の測定

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

生化学

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.

郭清およびショウジョウバエ蛹卵巣の汚損

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

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, Drosophila research lacks popularity in developing countries, possibly due to the misinformed idea that establishing a lab and performing relevant experiments with such tiny insects is difficult and requires expensive, specialized apparatuses. Here, we describe how to build an affordable flylab to quantitatively analyze a myriad of behavioral parameters in D. melanogaster, by 3D-printing many of the necessary pieces of equipment. We provide protocols to build in-house vial racks, courtship arenas, apparatuses for locomotor assays, etc., to be used for general fly maintenance and to perform behavioral experiments using adult flies and larvae. We also provide protocols on how to use more sophisticated systems, such as a high resolution oxygraph, to measure mitochondrial oxygen consumption in larval samples, and show its association with behavioral changes in the larvae upon the xenotopic expression of the mitochondrial alternative oxidase (AOX). AOX increases larval activity and mitochondrial leak respiration, and accelerates development at low temperatures, which is consistent with a thermogenic role for the enzyme. We hope these protocols will inspire researchers, especially from developing countries, to use Drosophila to easily combine behavior and mitochondrial metabolism data, which may lead to information on genes and/or environmental conditions that may also regulate human physiology and disease states.

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

We provide protocols for anyone with a "maker culture" mind to start building a flylab for quantitative analysis of a myriad of behavioral parameters in Drosophila melanogaster, by 3D-printing many of the necessary pieces of equipment. We also describe a high resolution respirometry protocol using larvae to combine behavioral and mitochondrial metabolism data.

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, ...

行動学

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

大人からOenocytesの解剖キイロショウジョウバエ</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

生物学

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.

ショウジョウバエの卵子形成期における分極細胞内輸送と基底膜タンパク質の分泌の共焦点および超解像イメージング

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

ショウジョウバエのミトコンドリア生理学と生体エネルギー学の解析

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.

著者スポットライト:Drosophila melanogasterを使用したミトコンドリア機能と化学的毒性の探索 

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.

ショウ ジョウバエのmelanogaster PINK1B9-Null変異体のミトコンドリア機能の解析(高分解能スピロメトリー)

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

ミトコンドリアの構造と機能の研究に

要約

さらに動画を探す

この動画の章

EDU染色によりショウジョウバエアダルト卵巣内のミトコンドリアDNA複製のその場の標識で

代謝分析のためのミトコンドリア富化画分の調製で

ショウジョウバエのティッシュの最小限の量を使用してのグリセリンの繊維でミトコンドリアの酸素消費量の測定

郭清およびショウジョウバエ蛹卵巣の汚損

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

大人からOenocytesの解剖キイロショウジョウバエ

ショウジョウバエの卵子形成期における分極細胞内輸送と基底膜タンパク質の分泌の共焦点および超解像イメージング

ショウジョウバエのmelanogaster PINK1^B9-Null変異体のミトコンドリア機能の解析(高分解能スピロメトリー)

ショウジョウバエのmelanogaster PINK1^B9-Null変異体のミトコンドリア機能の解析(高分解能スピロメトリー)

ショウジョウバエのmelanogaster PINK1^B9-Null変異体のミトコンドリア機能の解析(高分解能スピロメトリー)

ミトコンドリアの構造と機能の研究に

要約

さらに動画を探す

この動画の章

EDU染色によりショウジョウバエアダルト卵巣内のミトコンドリアDNA複製のその場の標識で

代謝分析のためのミトコンドリア富化画分の調製で

ショウジョウバエのティッシュの最小限の量を使用してのグリセリンの繊維でミトコンドリアの酸素消費量の測定

郭清およびショウジョウバエ蛹卵巣の汚損

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

大人からOenocytesの解剖キイロショウジョウバエ

ショウジョウバエの卵子形成期における分極細胞内輸送と基底膜タンパク質の分泌の共焦点および超解像イメージング

ショウ ジョウバエのmelanogaster PINK1B9-Null変異体のミトコンドリア機能の解析(高分解能スピロメトリー)

ショウ ジョウバエのmelanogaster PINK1B9-Null変異体のミトコンドリア機能の解析(高分解能スピロメトリー)

ショウ ジョウバエのmelanogaster PINK1B9-Null変異体のミトコンドリア機能の解析(高分解能スピロメトリー)

ショウジョウバエのmelanogaster PINK1^B9-Null変異体のミトコンドリア機能の解析(高分解能スピロメトリー)

ショウジョウバエのmelanogaster PINK1^B9-Null変異体のミトコンドリア機能の解析(高分解能スピロメトリー)

ショウジョウバエのmelanogaster PINK1^B9-Null変異体のミトコンドリア機能の解析(高分解能スピロメトリー)