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 ...

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Dissection of Drosophila Ovaries

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