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.

ووصف الميتوكوندريا كلاسيكي باسم قوة الخلوية، لأنها هي المقاعد الرئيسية لإنتاج الطاقة في الخلايا المتمايزة. وعلاوة على ذلك، الميتوكوندريا تلعب دورا حاسما في عملية التمثيل الغذائي وتوليد الحرارة، وتعديل الدهون والكالسيوم والتوازن الأكسدة، وتزامن عمليات الخلية إشارات، الخ 1. الميتوكوندريا أيضا أن تلعب دورا نشطا في تحريض الخلايا الموت 2، وكذلك في خلية تنظيم دورة 3. هذه وظائف متعددة تثير الأسئلة الأساسية التالية: أ) كيف الميتوكوندريا أداء كل هذه المهام في وقت واحد وب) وهناك حمامات الميتوكوندريا محددة أو subzones التي تخصصت لوظائف مختلفة؟ في هذا السياق، من المهم أن نلاحظ أن الميتوكوندريا متعددة الوظائف الحيوية في شكلها وحجمها، وهيكل داخل الخلايا الفردية، وأنه شكل حالة استقرار الميتوكوندريا يمكن أن تختلف بين أنواع الخلايا. عقود من البحث من مختلف مختبروتشير الخطابات أن تغيير شكل الميتوكوندريا وحجم وهيكل، التي تسمى مجتمعة ديناميات الميتوكوندريا، أمر بالغ الأهمية للحفاظ على وظائف مختلفة الميتوكوندريا 4،5،6. هذه النتائج تثير إمكانية أن الميتوكوندريا قد يحقق لها وظائف متعددة بحكم ديناميكية الهيكلية. جارية لفهم العلاقة بين الهيكل وظيفة الميتوكوندريا جهود واسعة النطاق. يتم الحفاظ على دينامية هيكل الميتوكوندريا في المقام الأول من خلال قدرتها على الخضوع الانشطار والانصهار الأحداث مع بعضها البعض. انشطار الميتوكوندريا كبيرة تحولها إلى عناصر الميتوكوندريا أصغر، في حين التحام بين مؤسستي الميتوكوندريا أصغر يدمج منهم إلى عنصر الميتوكوندريا أكبر 7. وعلاوة على ذلك، قد يحدث الانصهار عابرة اثنين من الميتوكوندريا للسماح للخلط محتوياتها. تخضع الأحداث الانشطار والانصهار في الأغشية الميتوكوندريا الداخلية والخارجية بعناية من قبل المواصفاتيحدد IFIC من البروتينات. ويتكون الجهاز الانشطار الأساسية من البروتين ذات الصلة dynamin 1 (Drp1)، الذي يتم تجنيده من العصارة الخلوية إلى الميتوكوندريا التي كتبها تفاعلها مع بعض الحسنة بروتينات الميتوكوندريا الحسنة (على سبيل المثال، Fis1 أو Mff1)، في حين أن وظيفة Drp1 يمكن أيضا أن ينظم من قبل بروتينات أخرى على سطح الميتوكوندريا 4. على الرغم من أن Drp1 تعمل على الغشاء الخارجي، والقدرات الانشطار لها تؤثر على الغشاء الداخلي كذلك. تزامن للانشطار أغشية الميتوكوندريا الخارجي والداخلي ليست مفهومة جيدا. من ناحية أخرى، يخضع انصهار الغشاء الداخلي في صلب الأنشطة التي يقوم بها Opa1، في حين يحكم mitofusins انصهار الخارجي غشاء 5. التوازن في مواجهة الانشطار والانصهار أحداث الميتوكوندريا تملي شكل الميتوكوندريا حالة استقرار في خلية. على سبيل المثال، فإن القمع الانشطار الميتوكوندريا يؤدي إلى الانصهار الكامل وبدون معارضة، في حين أن الإفراط في النشاط الميتوكوندرياسيكون ل الانشطار يؤدي إلى تفتت الميتوكوندريا 3. دراسة العلاقة بين الهيكل وظيفة الميتوكوندريا ينطوي في المقام الأول نهجين مجانية: أ) تحليل الظواهر الخلوية والعضوي بعد التلاعب الجيني للبروتينات الانشطار / الانصهار الميتوكوندريا وب) التقييمات المباشرة للهيكل الميتوكوندريا وظيفة. ومن الجدير بالذكر أن التحاليل الجينية قد لا تكشف دائما وظيفة مباشرة للجزيء في متناول اليد (في هذه الحالة، الميتوكوندريا البروتينات الانشطار / الانصهار)، كما قد تنشأ الظواهر بسبب آثارها الجانبية. ولذلك، فإنه من الأهمية بمكان لتطوير واستخدام أدوات لدراسة بنية الميتوكوندريا وظيفة مباشرة. أي تقييم لهيكل الميتوكوندريا يتضمن أدوات الفحص المجهري مختلف. استخدام المجهر مضان من الخلايا الحية قد تقدم كثيرا من الدراسات ديناميات الميتوكوندريا، منذ ديناميكية الميتوكوندريا يمكن رصدها من حيث الكم وquantitatively باستخدام أدوات وتقنيات 8 مضان المجهر المناسبة. وقد تم تطوير الأدوات التي تعتمد على الفحص المجهري مضان لدراسة بنية الميتوكوندريا وظيفة في أنسجة البطن الحية والثابتة ذبابة الفاكهة، توضيح أهمية حيوية الميتوكوندريا في الجسم الحي 9. يتم وصف هذه الأساليب وذات الصلة هنا، وذلك بهدف دراسة بنية الميتوكوندريا وظيفة في المبيض ذبابة الفاكهة. يتكون المبيض ذبابة الفاكهة من سلالة الجرثومية والجسدية الأنساب، التي تنشأ من الخلايا الجذعية البالغة في كل منها الموجودة في germarium 10،11. ستة عشر الخلايا الجرثومية المخلوي (GCS) الحصول على تغليف بواسطة خلايا بصيلات الجسدية (FCS) لتشكيل غرف البيض الفردية التي تنشأ من germarium (الشكل 1). واحدة من 16 GCS الحصول ملتزمة تصبح بويضة، و15 GCS المتبقية تتطور إلى خلايا الممرضة التي تدعم نمو غرفة بويضة، وتسهيل نضوج البويضة قبل وضعه. الغالبية العظمى من السفح الخضوع 9 جولات من الانقسامات الإنقسامية قبل خروجها من دورة الخلية الإنقسامية للتمييز عضال في طبقة الخلايا الظهارية نمط تتكون من خلايا الأمامي جريب (AFCs)، وخلايا الخلفي بصيلات بالفلور، وخلايا الجسم الرئيسية (MBCS) . يتم توصيل الدوائر البيض على التوالي من قبل خلايا ساق، والتي تتمايز الخلايا التي تستمد أيضا من السفح في وقت مبكر في عملية التنمية. شكل الميتوكوندريا التي تنظمها الميتوكوندريا Drp1 البروتين الانشطار وتشارك بنشاط في عملية التمايز خلال التطور الطبيعي للذبابة الفاكهة المبيض طبقة FC 9،12. ووصف الأساليب المستخدمة في هذه الدراسات للتعرف على إشراك Drp1 في ذبابة الفاكهة تطوير طبقة الخلايا المسام هنا.

<table border="0" cellpadding="0" cellspacing="0" width="722">
	<tbody>
		<tr height="38">
			<td height="38" style="height:38px;width:247px;">Grace&#39;s Media (Insect Dissecting Medium)</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:136px;">30611031-2</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:247px;">41 Paraformaldehyde AQ</td>
			<td style="width:148px;">Electronic Microscopy Sciences</td>
			<td style="width:136px;">50-259-99</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:247px;">Mitotracker Green (overall mitochondrial stain)</td>
			<td style="width:148px;">Life Technologies</td>
			<td style="width:136px;">m7514</td>
			<td>Reconstitute and Aliquot</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:247px;">Tetramethylrhodamine ethyl ester perchlorate</td>
			<td style="width:148px;">Sigma Aldrich</td>
			<td style="width:136px;">87917-25MG</td>
			<td>Reconstitute and Aliquot</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">MitoSox (Mito-Ros stain)</td>
			<td style="width:148px;">Life Technologies</td>
			<td style="width:136px;">m36008</td>
			<td>Reconstitute and Aliquot</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">PolyLysine</td>
			<td style="width:148px;">MP Biomedicals</td>
			<td style="width:136px;">ICN15017625</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Fly Vials</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:136px;">AS-515</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Fly Conicals</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:136px;">AS-355</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Fly Vial Flugs</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:136px;">AS273</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Fly Conical Flugs</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:136px;">AS 277</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:247px;">Jazzmix Drosophila food (Drosophila food)</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:136px;">AS153</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Bovine Serum Albumin</td>
			<td style="width:148px;">Sigma Aldrich</td>
			<td style="width:136px;">A9647-50G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Cyclin E Antibody (d-300)</td>
			<td style="width:148px;">Santa Cruz</td>
			<td style="width:136px;">sc- 33748</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:247px;">ATPB antibody [3D5] - Mitochondrial Marker</td>
			<td style="width:148px;">AbCam</td>
			<td style="width:136px;">ab14730</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:247px;">Cy3 AffiniPure Goat Anti-Mouse IgG (H+L)</td>
			<td style="width:148px;">Jackson ImmunoResearch</td>
			<td style="width:136px;">115-165-146</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:247px;">Cy5 AffiniPure Goat Anti-Rabbit IgG (H+L)</td>
			<td style="width:148px;">Jackson ImmunoResearch</td>
			<td style="width:136px;">111-175-144</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:247px;">Hoechst</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:136px;">H3570</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:247px;">VectaShield</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:136px;">H100</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:247px;">Azer Scientific EverMark Select Microscope Slides</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:136px;">22-026-252</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:247px;">Microscope Cover Glass</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:136px;">12-542-B</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:247px;">Name</td>
			<td style="width:148px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="38">
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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.

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

دراسة بنية الميتوكوندريا ووظيفتها في مبايض ذبابة الفاكهة | الأحياء | فيديو

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.

دراسة الميتوكوندريا هيكل وظيفة في<em&gt; ذبابة الفاكهة</em&gt; المبايض

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

studying-mitochondrial-structure-and-function-in-drosophila-ovaries

Nanyang Technological University

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Studying Mitochondrial Structure and Function in Drosophila Ovaries

Cellular Respiration

Cell Structure and Function

SCIENTISTS IN ACTION

23.9K Views.  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. 

Video: Studying Mitochondrial Structure and Function in Drosophila Ovaries

Dissection of Drosophila Ovaries

تشريح المبايض ذبابة الفاكهة

Studying Aggression in Drosophila (fruit flies)

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources. This complex social behavior is influenced by genetic, hormonal and environmental factors. In many organisms, aggression is critical to survival but controlling and suppressing aggression in distinct contexts also has become increasingly important. In recent years, invertebrates have become increasingly useful as model systems for investigating the genetic and systems biological basis of complex social behavior. This is in part due to the diverse repertoire of behaviors exhibited by these organisms. In the accompanying video, we outline a method for analyzing aggression in Drosophila whose design encompasses important eco-ethological constraints. Details include steps for: making a fighting chamber; isolating and painting flies; adding flies to the fight chamber; and video taping fights. This approach is currently being used to identify candidate genes important in aggression and in elaborating the neuronal circuitry that underlies the output of aggression and other social behaviors.

Studying Aggression in Drosophila fruit flies

دراسة العدوان في ذبابة الفاكهة (ذبابة الفاكهة)

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources.  This complex social behavior is influenced by ...

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.

تقنيات Microinjection لدراسة الانقسام الميتوزي في ذبابة الفاكهة السوداء البطن الأجنة المخلوي

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

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

رصد وظيفة القلب في اليرقات ذبابة الفاكهة السوداء البطن للدراسات فسيولوجية

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

على أهمية تركيز البروتين الصحيح لحركية وتقارب في تحديد هيكل وظيفة التحليل

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

أعربت المشبك التصحيح وتقنيات الإرواء لدراسة في قنوات ايون القيطم البويضات

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

تشريح وتلون ذبابة الفاكهة اليرقات المبايض

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

A Protocol for Computer-Based Protein Structure and Function Prediction

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently &gt;20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.

Guidelines for computer based structural and functional characterization of protein using the I-TASSER pipeline is described. Starting from query protein sequence, 3D models are generated using multiple threading alignments and iterative structural assembly simulations. Functional inferences are thereafter drawn based on matches to proteins with known structure and functions.

بروتوكول لبنية البروتين القائم على الحاسوب والتنبؤات وظيفة

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

إعادة إعماره الوظيفية وآخر قناة قياسات تنقية Wildtype والمسخ CFTR البروتين

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

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

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

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