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.

Passos críticos dentro do Protocolo Fotodegradação: Prevenção de fotodegradação indevida de amostras fluorescentes é absolutamente necessário para a realização de microscopia confocal eficiente. Portanto, o tempo usado para localizar amostras através da ocular ou para definir os parâmetros de aquisição de imagem através do modo de varredura ao vivo deve ser minimizado para minimizar a fotodegradação. O dano tecidual: Uma ...

As mitocôndrias são classicamente descritos como a potência celular, uma vez que eles são os principais lugares de produção de energia em células diferenciadas. Além disso, as mitocôndrias desempenham um papel crítico no metabolismo, a geração de calor, a modificação lipídica, o cálcio e homeostase redox, a orquestração de processos de sinalização de células, etc 1. As mitocôndrias desempenham também um papel activo na indução de morte celular 2, bem como na regulação do ciclo celular 3. Essa multifuncionalidade levanta as seguintes questões fundamentais: a) como mitocôndrias desempenhar todas estas funções em simultâneo e b) existem piscinas mitocondriais específicos ou subzonas que são especializados para funções distintas? Neste contexto, é importante notar que as mitocôndrias são multifuncionais dinâmica na sua forma, tamanho e estrutura dentro das células individuais e que a forma de estado estacionário de mitocôndrias pode variar entre os tipos de células. Décadas de pesquisa de vários laboratóriooratórios sugerem que a alteração da forma mitocondrial, tamanho e estrutura, chamados coletivamente de dinâmica mitocondrial, é crucial para a manutenção de várias funções mitocondriais 4,5,6. Estes resultados levantam a possibilidade de que mitocôndrias podem cumprir a sua multi-funcionalidade em virtude do seu dinamismo estrutural. grandes esforços estão em curso para entender a relação estrutura-função mitocondrial. A dinâmica da estrutura mitocondrial é mantida principalmente pela sua capacidade de sofrer eventos de fissão e de fusão com o outro. Fissão de grandes mitocôndrias converte-os em elementos mitocondriais menores, enquanto a fusão entre duas mitocôndrias menores funde-os em um elemento mitocondrial maior 7. Além disso, a fusão de duas mitocôndrias transiente pode ocorrer para permitir a mistura do seu conteúdo. Os eventos de fissão e de fusão das membranas mitocondriais interior e exterior são cuidadosamente regulada pela especificaçãoific conjuntos de proteínas. A maquinaria do núcleo fissão é composto de proteína relacionada com dinamina 1 (Drp1), que é recrutados a partir do citosol para a mitocôndria pela sua interacção com certos bona proteínas mitocondriais fide (por exemplo, Fis1 ou Mff1), enquanto função Drp1 também pode ser regulado por outras proteínas na superfície mitocondrial 4. Embora Drp1 opera na membrana externa, as suas capacidades de fissão impacto da membrana interior assim. A orquestração da cisão das membranas mitocondriais exteriores e interiores não é bem compreendido. Por outro lado, a fusão da membrana interna é regulada no núcleo pelas actividades de OPA1, enquanto mitofusins governar a fusão da membrana externa 5. O saldo das contrariando fissão e fusão eventos de mitocôndrias ditar a forma mitocondrial steady-state em uma célula. Por exemplo, a repressão da fissão mitocondrial resultaria em fusão completa e sem oposição, enquanto que a sobre-actividade das mitocôndriasl fissão resultaria em fragmentação da mitocôndria 3. O estudo da relação estrutura-função mitocondrial envolve principalmente duas abordagens complementares: a) análise dos fenótipos celulares e organismais após a manipulação genética de proteínas fissão / fusão mitocondrial e b) as avaliações diretas de estrutura e função mitocondrial. É digno de nota que as análises genéticas podem nem sempre revelar a funcionalidade directa da molécula no lado (neste caso, as proteínas de fissão / fusão mitocondriais), como os fenótipos podem surgir devido a efeitos secundários. Portanto, é de extrema importância para desenvolver e usar ferramentas para estudar a estrutura e função mitocondrial diretamente. Qualquer avaliação da estrutura mitocondrial envolve várias ferramentas de microscopia. Uso de microscopia de fluorescência de células vivas tem avançado muito nos estudos de dinâmica mitocondrial, uma vez que o dinamismo mitocondrial pode ser monitorado de forma qualitativa e Quantitätvamente utilizando as ferramentas e técnicas apropriadas 8 de microscopia de fluorescência. Ferramentas com base em microscopia de fluorescência foram desenvolvidos para estudar a estrutura e a função mitocondrial em tecidos vivos e fixos de Drosophila melanogaster, elucidar a importância de dinamismo mitocondrial in vivo 9. Estes e os métodos relacionados são descritos aqui, com o objectivo de estudar a estrutura e a função mitocondrial no ovário de Drosophila. O ovário Drosophila consiste em germinativas e somáticas linhagens, que surgem a partir de suas respectivas células-tronco adultas que residem no germário 10,11. Dezesseis células germinativas sincicial (GCs) se encapsulado por células do folículo somáticas (FCS) para formar câmaras de ovos individuais que emergem fora do germário (Figura 1). Um dos 16 GCs se comprometeu a tornar-se um oócito, e os restantes 15 GCs desenvolvem em células enfermeira que suportam o crescimento da câmara de oócito, Facilitando a maturação do ovo, antes de ser colocado. A maioria dos CFs submetidos a 9 ciclos de divisões mitóticas antes de sair do ciclo celular mitótico para diferenciar terminalmente em uma camada de células epiteliais modelado que consiste de células anterior dos folículos (AFCs), células foliculares posterior (PFC), e as células do corpo principal (CBMs) . As câmaras de ovo consecutivos são ligados por células haste, que são células que são também derivados das CFs no início do desenvolvimento diferenciadas. Forma mitocondrial regulado pelo Drp1 proteína fissão mitocondrial está ativamente envolvido no processo de diferenciação durante o desenvolvimento normal da camada FC ovário Drosophila 9,12. Os métodos utilizados nestes estudos para identificar o envolvimento de Drp1 em Drosophila desenvolvimento da camada de células do folículo são descritos aqui.

<table border="0" cellpadding="0" cellspacing="0" width="722">
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			<td style="width:136px;">87917-25MG</td>
			<td>Reconstitute and Aliquot</td>
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			<td height="20" style="height:20px;width:247px;">MitoSox (Mito-Ros stain)</td>
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			<td>Reconstitute and Aliquot</td>
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			<td height="20" style="height:20px;width:247px;">Fly Conical Flugs</td>
			<td style="width:148px;">Fisher Scientific</td>
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			<td height="38" style="height:38px;width:247px;">Cy3 AffiniPure Goat Anti-Mouse IgG (H+L)</td>
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			<td height="38" style="height:38px;width:247px;">Azer Scientific EverMark Select Microscope Slides</td>
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			<td height="30" style="height:30px;width:247px;">Microscope Cover Glass</td>
			<td style="width:148px;">Fisher Scientific</td>
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			<td height="30" style="height:30px;width:247px;">Name</td>
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			<td>Comments</td>
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			<td height="38" style="height:38px;width:247px;">Mat Tek Corp&#160;Glass Bottom Mircrowell Dish</td>
			<td style="width:148px;">Fisher Scientific</td>
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			<td style="width:136px;">ICN10140001</td>
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			<td height="20" style="height:20px;width:247px;">Confocal Microscope</td>
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			<td style="width:136px;">LSM 700</td>
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			<td height="47" style="height:47px;width:247px;">Dumont #5 Forceps</td>
			<td style="width:148px;">Fine Science Technologies</td>
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			<td height="51" style="height:51px;width:247px;">Moria Nickel Plated Pin Holder</td>
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			<td height="44" style="height:44px;width:247px;">Minutien Pins</td>
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			<td style="width:136px;">26002-15</td>
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			<td height="46" style="height:46px;width:247px;">MYFP ( w[*]; P{w[+mC]=sqh-EYFP-Mito}3 )</td>
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			<td height="20" style="height:20px;width:247px;">Fly Pad</td>
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			<td height="20" style="height:20px;width:247px;">Blowgun</td>
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			<td style="width:136px;">54-104</td>
			<td></td>
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			<td height="20" style="height:20px;width:247px;">Blowgun needle</td>
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			<td style="width:136px;">54-119</td>
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			<td height="20" style="height:20px;width:247px;">Dissecting Microscope</td>
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			<td height="20" style="height:20px;width:247px;">Analyses software</td>
			<td style="width:148px;">Carl Zeiss</td>
			<td style="width:136px;">Zen&#160;</td>
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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.

Os métodos descritos podem ser utilizados para estudar a estrutura e a função mitocondrial em ovários de Drosophila vivas e fixadas (Figura 2B). São fornecidos alguns exemplos de resultados previstos obtidos com os métodos descritos. A dissecção do ovário Drosophila: Quando dissecados ainda mais, o abdómen cortadas (Figura 3B) a partir do conjunto de Dros...

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

Estudando estrutura e função mitocondrial em ovários de Drosophila | Biologia | Vídeo

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.

Estudar mitocondrial estrutura e função em<em&gt; Drosophila</em&gt; ovários

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 visualizações.  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. 

Vídeo: Studying Mitochondrial Structure and Function in Drosophila Ovaries

Dissection of Drosophila Ovaries

Dissecção da Drosophila Ovários

Biologia

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

Agressão a estudar em Drosophila (mosca da fruta)

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.

Técnicas de microinjeção para estudar na Mitose Drosophila melanogaster Embrião Sincicial

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.

Função de monitorização cardíaca em larval Drosophila melanogaster Para estudos fisiológicos

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.

A importância da concentração de proteína correta para Cinética e Determinação Affinity na estrutura-função Análise

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.

Grampo patch e Técnicas de Perfusão para estudar canais iônicos Expresso em Xenopus Oócitos

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.

Dissecção e coloração de Drosophila Larval Ovários

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.

Um protocolo para Computer-Based Estrutura de Proteínas e de Previsão de Função

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.

Reconstituição funcional e atividade do canal Medidas de purificada tipo selvagem e mutante CFTR Protein

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.

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