Name: studying mitochondrial structure and function in drosophila ovaries
Uploaded: 2017-01-04T13:00:00
Description: Watch this Scientific Journal Video about Studying Mitochondrial Structure and Function in Drosophila Ovaries at JoVE.com

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

1. Preparation of Drosophila (the tools required are depicted in Figure 2A)
<ol>
	<li>For any of the experiments described, collect Drosophila (maintained at room temperature, or 25 &#186;C) within 5 days of eclosion and place them in a vial filled with 5 - 7 mL of Drosophila food (see Materials Table), with no more than 25 flies in each vial; maintain a female:male ratio of 2:1.</li>
	<li>Sprinkle a small amount of granulated yeast to stimulate Drosophila egg produc...

<ol>
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Chapter. , 21-21 (2010).</li><li>Mitra, K., Rikhy, R., Lilly, M., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DRP1-dependent+mitochondrial+fission+initiates+follicle+cell+differentiation+during+Drosophila+oogenesis.">DRP1-dependent mitochondrial fission initiates follicle cell differentiation during Drosophila oogenesis.</a> J Cell Biol. 197 (4), 487-497 (2012).</li><li>Klusza, S., Deng, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=At+the+crossroads+of+differentiation+and+proliferation:+precise+control+of+cell-cycle+changes+by+multiple+signaling+pathways+in+Drosophila+follicle+cells.">At the crossroads of differentiation and proliferation: precise control of cell-cycle changes by multiple signaling pathways in Drosophila follicle cells.</a> Bioessays. 33 (2), 124-134 (2011).</li><li>Sahai-Hernandez, P., Castanieto, A., Nystul, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+models+of+epithelial+stem+cells+and+their+niches.">Drosophila models of epithelial stem cells and their niches.</a> Wiley Interdiscip Rev Dev Biol. 1 (3), 447-457 (2012).</li><li>Parker, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mitochondrial+pool+of+cyclin+E,+regulated+by+Drp1,+is+linked+to+cell-density-dependent+cell+proliferation.">A new mitochondrial pool of cyclin E, regulated by Drp1, is linked to cell-density-dependent cell proliferation.</a> J Cell Sci. 128 (22), 4171-4182 (2015).</li><li>Mitra, K., Wunder, C., Roysam, B., Lin, G., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hyperfused+mitochondrial+state+achieved+at+G1-S+regulates+cyclin+E+buildup+and+entry+into+S+phase.">A hyperfused mitochondrial state achieved at G1-S regulates cyclin E buildup and entry into S phase.</a> Proc Natl Acad Sci U S A. 106 (29), 11960-11965 (2009).</li><li>Shidara, Y., Hollenbeck, P. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+mitochondria+produce+reactive+oxygen+species.">How mitochondria produce reactive oxygen species.</a> Biochem J. 417 (1), 1-13 (2009).</li><li>Mandal, S., Guptan, P., Owusu-Ansah, E., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+regulation+of+cell+cycle+progression+during+development+as+revealed+by+the+tenured+mutation+in+Drosophila.">Mitochondrial regulation of cell cycle progression during development as revealed by the tenured mutation in Drosophila.</a> Dev Cell. 9 (6), 843-854 (2005).</li><li>Tipping, M., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+context-dependent+tumorigenesis.">Drosophila as a model for context-dependent tumorigenesis.</a> J Cell Physiol. 229 (1), 27-33 (2014).</li><li>Herranz, H., Eichenlaub, T., Cohen, S. 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Critical Steps within the Protocol
Photobleaching: Preventing undue photobleaching of fluorescent samples is absolutely necessary to performing efficient confocal microscopy. Therefore, the time used to locate samples through the eyepiece or to set image acquisition parameters through the live scanning mode should be minimized to minimize photobleaching.
Tissue damage: Since mitochondria are considered to be the sensors of cellular health, it...

Mitochondria are classically described as the cellular powerhouse, since they are the main seats of energy production in differentiated cells. Moreover, mitochondria play a critical role in metabolism, heat generation, lipid modification, calcium and redox homeostasis, the orchestration of cell signaling processes, etc1. Mitochondria also play an active role in the induction of cell death2, as well as in cell cycle regulation3. Such multi-functionality raises the following fundamental questions: a) how do mitochondria perform all these functions simultaneously and b) are there specific mitochondrial pools or subzones that are specialized for distinct functions? In this context, it is important to note that the multifunctional mitochondria are dynamic in their shape, size, and structure within individual cells and that the steady-state shape of mitochondria can vary between cell types. Decades of research from various laboratories suggest that the alteration of mitochondrial shape, size, and structure, collectively called mitochondrial dynamics, is crucial for maintaining various mitochondrial functions4,5,6. These findings raise the possibility that mitochondria may accomplish their multi-functionality by virtue of their structural dynamism.
Extensive efforts are underway to understand the mitochondrial structure-function relationship. The dynamism of mitochondrial structure is primarily maintained by their ability to undergo fission and fusion events with each other. Fission of large mitochondria converts them into smaller mitochondrial elements, while fusion between two smaller mitochondria merges them into a larger mitochondrial element7. Moreover, transient fusion of two mitochondria may occur to allow the mixing of their contents. The fission and fusion events of the inner and outer mitochondrial membranes are carefully governed by specific sets of proteins. The core fission machinery is composed of dynamin-related protein 1 (Drp1), which is recruited from the cytosol to the mitochondria by its interaction with certain bona fide mitochondrial proteins (e.g., Fis1 or Mff1), while Drp1 function can also be regulated by other proteins on the mitochondrial surface4. Although Drp1 operates on the outer membrane, its fission abilities impact the inner membrane as well. The orchestration of the fission of outer and inner mitochondrial membranes is not well understood. On the other hand, fusion of the inner membrane is governed at the core by the activities of Opa1, while mitofusins govern the fusion of the outer-membrane5. The balance of the counteracting fission and fusion events of mitochondria dictate the steady-state mitochondrial shape in a cell. For example, repression of mitochondrial fission would result in complete and unopposed fusion, while the over-activity of mitochondrial fission would result in fragmentation of mitochondria3.
The study of the mitochondrial structure-function relationship primarily involves two complimentary approaches: a) analyses of the cellular and organismal phenotypes after genetic manipulation of mitochondrial fission/fusion proteins and b) direct assessments of mitochondrial structure and function. It is noteworthy that genetic analyses may not always reveal the direct functionality of the molecule at hand (in this case, mitochondrial fission/fusion proteins), as the phenotypes may arise due to secondary effects. Therefore, it is of the utmost importance to develop and use tools to study mitochondrial structure and function directly. Any assessment of mitochondrial structure involves various microscopy tools. Use of fluorescence microscopy of live cells has greatly advanced the studies of mitochondrial dynamics, since mitochondrial dynamism can be monitored both qualitatively and quantitatively using the appropriate fluorescence microscopy tools and techniques8. Fluorescence microscopy-based tools have been developed to study mitochondrial structure and function in live and fixed Drosophila melanogaster tissues, elucidating the significance of mitochondrial dynamism in vivo9. These and related methods are described here, with the goal of studying mitochondrial structure and function in the Drosophila ovary.
The Drosophila ovary consists of germline and somatic lineages, which arise from their respective adult stem cells that reside in the germarium10,11. Sixteen syncytial germ cells (GCs) get encapsulated by somatic follicle cells (FCs) to form individual egg chambers that emerge out of the germarium (Figure 1). One of the 16 GCs get committed to become an oocyte, and the remaining 15 GCs develop into nurse cells that support the growth of the oocyte chamber, facilitating the maturation of the egg before it is laid. The majority of the FCs undergo 9 rounds of mitotic divisions before they exit the mitotic cell cycle to terminally differentiate into a patterned epithelial cell layer consisting of anterior follicle cells (AFCs), posterior follicle cells (PFCs), and main body cells (MBCs). The consecutive egg chambers are connected by stalk cells, which are differentiated cells that are also derived from the FCs early in development. Mitochondrial shape regulated by the mitochondrial fission protein Drp1 is actively involved in the process of differentiation during the normal development of the Drosophila ovarian FC layer9,12. The methods used in these studies to identify the involvement of Drp1 in Drosophila follicle cell layer development are described here.

<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>
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			<td style="width:136px;">30611031-2</td>
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			<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>
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			<td height="38" style="height:38px;width:247px;">Mitotracker Green (overall mitochondrial stain)</td>
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			<td style="width:136px;">m7514</td>
			<td>Reconstitute and Aliquot</td>
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			<td height="38" style="height:38px;width:247px;">Tetramethylrhodamine ethyl ester perchlorate</td>
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			<td height="38" style="height:38px;width:247px;">Jazzmix Drosophila food (Drosophila food)</td>
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			<td height="38" style="height:38px;width:247px;">ATPB antibody [3D5] - Mitochondrial Marker</td>
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			<td style="width:136px;">ab14730</td>
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			<td height="30" style="height:30px;width:247px;">Microscope Cover Glass</td>
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			<td height="30" style="height:30px;width:247px;">Name</td>
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			<td height="20" style="height:20px;width:247px;">Active Dried Yeast</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:136px;">ICN10140001</td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Confocal Microscope</td>
			<td style="width:148px;">Carl Zeiss</td>
			<td style="width:136px;">LSM 700</td>
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			<td height="47" style="height:47px;width:247px;">Dumont #5 Forceps</td>
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			<td style="width:136px;">11251-20</td>
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			<td height="51" style="height:51px;width:247px;">Moria Nickel Plated Pin Holder</td>
			<td style="width:148px;">Fine Science Technologies</td>
			<td style="width:136px;">26016-12</td>
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			<td height="44" style="height:44px;width:247px;">Minutien Pins</td>
			<td style="width:148px;">Fine Science Technologies</td>
			<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>
			<td style="width:148px;">Bloomington Stock Center</td>
			<td style="width:136px;">7194</td>
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			<td height="20" style="height:20px;width:247px;">Fly Pad</td>
			<td style="width:148px;">Fly stuff</td>
			<td style="width:136px;">59-118</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Blowgun</td>
			<td style="width:148px;">Fly stuff</td>
			<td style="width:136px;">54-104</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Blowgun needle</td>
			<td style="width:148px;">Flystuff</td>
			<td style="width:136px;">54-119</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Dissecting Microscope</td>
			<td style="width:148px;">Carl Zeiss</td>
			<td style="width:136px;">Stemi 2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<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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		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Analyses software</td>
			<td style="width:148px;">Open source</td>
			<td style="width:136px;">Image J</td>
			<td></td>
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			<td height="20" style="height:20px;width:247px;">Research Macro Zoom Microscope</td>
			<td style="width:148px;">Olympus</td>
			<td style="width:136px;">MVX10</td>
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			<td height="39" style="height:39px;width:247px;">QICAM Fast 1394 Cooled Digital Camera, 12-bit, Mono&#160;</td>
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			<td style="width:136px;">QIC-F-M-12-C</td>
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			<td height="21" style="height:21px;width:247px;">QCapture Pro 5.1</td>
			<td style="width:148px;">QImaging</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
	</tbody>
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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.

The described methods can be used to study mitochondrial structure and function in live and fixed Drosophila ovaries (Figure 2B). Provided are some examples of anticipated results obtained with the described methods.
Dissection of the Drosophila ovary: When dissected further, the severed abdomens (Figure 3B) from the whole Drosophila (Figure 3A)...

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

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.

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 ...

The overall goal of this procedure, is to demonstrate methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism drosophila melanogaster. This method can help answer key questions in the field of Mitochondrial Biology. Such as the regulatory mechanisms of mitochondrial function.The main advantage of this technique is that it performs detailed analysis of the mitochondrial structure/function relationship. After sorting out five anesthetized female flies according to the text protocol, under the dissection microscope, use two pairs of forceps to sever the thorax from the abdomen. Then, carefully transfer the abdomens to the second well of the dish.Using one pair of forceps to hold the abdomen at the posterior end, use the other pair of forceps to slowly push the ovaries out. Then use the forceps to hold an individual ovary by the opaque posterior end and carefully move it to the third well of the dish for teasing or fixing. Carefully tease the protective sheath from around the ovaries, by using a pair of forceps to hold the posterior end and lightly sweeping a teasing needle from the posterior to the anterior end.To prepare polylysene coated chambers, place two separated 20 microliter drops of polylysene on the cover glass of a glass-bottom petri dish. Air dry the plates at 37 degrees celsius for one hour, and outline the polylysine on the underside of the plate. Set the scanning parameters on the confocal microscope by opening the image acquisition software, and in the acquisition tab, set the appropriate scanning parameters.Check the time series, bleaching and regions boxes to open the individual tabs. Then, enter the appropriate acquisition parameter values into each tab. Next, place one dissected and teased ovary onto a marked polylysine coated region and use a teasing needle to spread the ovary and separate the ovarials.Place a 10 micro liter drop of insect dissecting medium on top of the ovary, making sure to cover the entire polylysine coated area. Then, cover the petri dish. Immediately after mounting the ovary, place the sample on the microscope stage and use the eye piece to quickly locate the field of interest in the live tissue.Click live to acquire a live image of the selected field of interest, then click stop to stop live scanning. If necessary, adjust the acquisition parameters so that the detected fluorescent signal is below the saturation levels with the defined background as set by adjusting the offset values. From the graphic tab, use the bezier drawing tool to draw a small ROI to demarcate the photo bleaching zone on the image acquired by scanning.Then perform image acquisition, by clicking on start experiment. To carry out live-staining with fluorescent mitochondrial dyes, dilute the stock of stains in warm dissecting medium to final working concentrations. After dissection and teasing is demonstrated earlier, place the ovaries into 200 microliters of staining solution in a well of a dissection dish.Cover the dish with aluminum foil wrapped box to protect it from light and incubate the tissue for 10 minutes. Next, wash the stained ovaries by using forceps to move them carefully into three consecutive wells containing medium without stain. For co-staining with TMRE, in the compatible overall mitrochondrial stain, carry-out staining with TMRE first, then without washing the tissue, immediately transfer the sample into the overall mitochondrial stain.After washing and mounting the ovaries as demonstrated earlier in this video, to carry out confocal imaging on the sample, check the z-sectioning box to open the tab. Turn the focus wheel towards the bottom of the sample while it is being live-scanned and click on set-first to define the bottom-most z-section. Then move the focus wheel in the opposite direction to define the top-most section.Perform image acquisition by clicking on start experiment. In the case of live xvivamicroscopy, the data should be obtained within 15 minutes of tissue mounting, otherwise, the experimental results may be affected by alterations of physiology due to insuing death of the tissue. Immediately after dissection under a fume hood place ovaries into 200 microliters of fresh room temperature PFA in the well of a glass dissecting dish.Incubate the tissue for 15 minutes to fix it. After washing the ovaries in PBS according to the text protocol, while still in PBS, tease the ovaries to carefully remove the protective fiber sheath that may hinder antigen access from the antibody. Permeabilize the teased ovaries by placing them in a microfuse tube with 500 microliters of 0.5 percent tritan x 100 in PBS.Next, place the tube in a covered box and set to rock at 25 RPM's for 30 minutes. Remove the PBS/TX according to the text protocol block the tissue by adding 200 microliters of BSA dissolved in 0.5 percent PBS/TX. Incubate the samples on the rocker at room temperature for one hour.After removing the blocking solution, add anti-rabid desycline E and anti-mouse ATPB antibodies. Incubate the tissue on the rocker at room temperature for two hours. Following the incubation, use PBS-TX to wash the ovaries three times for 15 minutes each.Then, add 200 microliters of the appropriate secondary antibodies and fresh PBS-TX and incubate at room temperature for one hour. After washing the tissue two times, add hooks to the third wash. Then with a one milliliter micropipet, remove the immuno-stained ovaries from the microfuse tube into a fresh well of a glass dissecting dish containing 200 microliters of PBS.Add one drop of glycerol based mounting medium to a glass slide and add the immuno-stained ovaries one-by-one to the mounting medium. Under a dissection microscope, while holding the opaque portion of the ovary with forceps, use a teasing needle to gently pluck the transparent ovarials from the opaque mature egg chambers. Then, remove the mature egg chambers from the mounting medium.Place a cover glass on the slide and lightly press to ensure the mounting medium spreads uniformly. Air dry the samples for 15 minutes before using nail polish to seal the edges. Finally, perform confocal microscopy as needed.As shown here, flip targeted to one of the mitochondrial clouds associated with the nerse cells leads to greater than 50 percent loss of the fluorescent signal from the blue and white ROI's surrounding the red flip ROI within 200 seconds while the yellow control ROI's minimally bleached. The drosophilla ovaries exhibited a decrease in signal with a depth of tissue, and it is important to discern the maximal depth of tissue within which the phlorophores such as TMRE and mitostain, can be detected during live imaging. A qualitative analysis from co-staining with TMRE and the overall mitochondrial stain, shows that the drisophilla germarium exhibits higher mitochondrial potential per unit mass in stage 2B.In this figure, semi-qualitative analysis demonstrates the difference in mitochondrial potential per unit mass between AFC's and the MBC's as analyzed from a stage 9 egg chamber. These images reveal that mitosox uptake occurs in the mitotic and post-mitotic drisophilla follicle cells. In this experiment, co-immuno staining identifies elevated decycline signal overlapping with the mitochondrial ATPB signal and the GFP negative DRP1 nole clones in the Germarium and in a stage 9 egg chamber.The described methods can be modified and employed to study mitochondrial structure function in other drisophilla tissues. After watching this video, you should have a good understanding of how to dissect, stain and image fixed and live drisophilla ovaries to study mitochonrial structure and function.

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Dissection of Drosophila Ovaries

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

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.

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

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.

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

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.

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.

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.

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

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight muscles (IFMs) of flies, specifically the dorso-longitudinal muscles (DLM), as the experimental subject to measure the atrophic phenotype brought about by different genetic causes. In this protocol, we describe how to embed fly thorax muscles for semi thin sectioning, how to obtain a good contrast between muscle and the surrounding tissue, and how to process optical microscope images for semiautomatic acquisition of quantifiable data and analysis. We describe three specific applications of the methodological pipeline. First, we show how the method can be applied to quantify muscle degeneration in a myotonic dystrophy fly model; second, measurement of muscle cross-sectional area can help to identify genes that either promote or prevent muscle atrophy and/or muscle degeneration; third, this protocol can be applied to determine whether a candidate compound is able to significantly modify a given atrophic phenotype induced by a disease-causing mutation or by an environmental trigger.

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

We report a method to quantify muscle area, which is an indirect method to determine muscle mass in Drosophila adults. We demonstrate the application of our methodology by analyzing the indirect flight muscles in a Drosophila model of Myotonic Dystrophy disease.

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...