Name: time-lapse video microscopy for assessment of eyfp-parkin aggregation as a marker for cellular mitophagy
Uploaded: 2016-05-04T13:00:00
Description: Watch this Scientific Journal Video about Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy at JoVE.com

This work was supported in part by NIH grants (1R01CA137494, R01CA132115, R01CA086072 to R.G.P.), the Kimmel Cancer Center NIH Cancer Center Core grant P30CA056036 (R.G.P.), a grant from the Breast Cancer Research Foundation, generous grants from the Dr. Ralph and Marian C. Falk Medical Research Trust (R.G.P.) and a grant from the Pennsylvania Department of Health (R.G.P.). In part this work was supported by an American Italian Cancer Foundation postdoctoral fellowship (G.D.) and Bioimaging Shared Resource of the Sidney Kimmel Cancer Center (NCI 5 P30 CA-56036).The Department specifically disclaims responsibility for an analysis, interpretations or conclusions. There are no conflicts of interest associated with this manuscript.

1. Electroporation of Fibroblast with Both Expression Vectors EYFP-Parkin and pDsRed2-Mito
<ol>
	<li>Grow the immortalized mouse embryonic fibroblast cells on 10 centimeter tissue-culture plate using DMEM (Dulbecco&#39;s modified Eagle&#39;s medium) medium supplemented with 10% fetal bovine serum, 2 mmol/l L-glutamine, 100 U/ml&#160;penicillin, and 100 mg/ml streptomycin in humidified atmosphere containing 5% CO2 at 37 &#176;C.
	<ol>
		<li>At 80% cell confluency, discard the complete DMEM medium by sterile s...

<ol>
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Time-lapse microscopy can be defined as the technique that extends live cell imaging from a single observation in time to the observation of cellular dynamics over long periods of time. This methodology is distinguished from a simple confocal or live cell microscopy because it allows the observer to identify in real time a single fluorescent-tagged protein and follow its dynamic inside a single live cell. In fact, the confocal microscopy can easily identifies immuno-labeled proteins using fluorescent antibody, but it doe...

Macro autophagy is an intracellular process that involves the catabolic degradation of both damaged and dysfunctional cellular components, such as organelles and proteins for the purpose of either recycling or energy production. To initiate this metabolic process, the cell engulfs the damaged cellular components into a double-membrane structure, known as an autophagosome, which fuses with a lysosome and its content is degraded and recycled 1,2. There are two major types of autophagy, the non-selective and selective. The non-selective autophagy process occurs when the cell is under nutrient deprivation conditions and needs to scavenge for both essential nutrients and energy. However, selective autophagy occurs to mediate the removal of both dysfunctional/damaged organelles and proteins that otherwise could be toxic. One of the most studied selective autophagy process is the removal of mitochondria, termed mitophagy 1,3-5.
Mitochondria are the central organelles for cell metabolism and the primary source of adenosine triphosphate (ATP) via oxidative phosphorylation through the electron transport chain, fatty acid oxidation, and tricarboxylic acid (TCA) cycle. Moreover, mitochondria regulate reactive oxygen species (ROS) production and release proteins that participate in cell death pathways 6-8.
PTEN-induced putative kinase 1 (PINK1) and Parkin RBR E3 ubiquitin ligase (Parkin) are the key proteins implicated in the mitophagy process. Parkin can protect against cell death by keeping the cell healthy through mitochondrial quality control9. Upon the loss of mitochondrial membrane potential, cytosolic Parkin is recruited to the mitochondria by PINK1. This recruitment triggers the sequential events of mitophagy 10. There is a broad range of evidence that mitophagy is a fundamental mitochondria quality control process and abnormalities in this process drive disease 7. For instance, autosomal recessive Parkinson's disease has been associated with mutations in the genes that encode for Parkin and PINK1 (PARK2 and PINK1, respectively) 11. The quality control of mitochondrial health is essential for the removal of mitochondria that contribute to the accumulation of ROS12. Excessive presence of intracellular ROS can lead to damage of both nuclear and mitochondrial DNA (DNA and mt DNA, respectively).
Herein, we show a time-lapse video microscopy approach to follow the aggregation of Parkin after the induction of Parkin-mediated mitophagy in immortalized mouse embryonic fibroblasts via in vitro administration of carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP), an uncoupling agent. FCCP disrupts ATP synthesis by short circuiting protons across the outer mitochondria membrane and hence uncoupling oxidative phosphorylation from the electron transport chain 13. Triggering the depolarization of the mitochondrial membrane leads to the disruption of mitochondria and selective Parkin-dependent removal. Therefore, transfecting the cells of interest with an expression vector encoding Parkin fused with a fluorescent marker (enhanced yellow fluorescent protein, EYFP) can be used as a fluorescent tag to follow the recruitment of Parkin during the mitophagic process. In order to visualize the mitochondria, we co-transfected pDsRed2-Mito, which encodes red fluorescent protein (DsRed2) that contains a mitochondrial targeting sequence of cytochrome c oxidase subunit VIII (Mito). pDsRed2-Mito is designed for fluorescent labeling of mitochondria14. The time required for Parkin translocation into the mitochondrial membrane can be measured and gives an indirect measure of cellular health. For example, we can say that if a cell line knocked-out for a particular gene of interest shows either a faster or slower recruitment of Parkin after the induction of mitophagy by FCCP, that gene product would be a key player in order to keep the metabolic rates of the cell at the physiological status and prevent the development of diseases. Therefore, the time-lapse video microscopy provides a very powerful tool for both basic and clinical research applications in following the dynamic of labeled proteins during their molecular processes and understanding how these processes are affected during a pathological condition.

<table border="0" cellpadding="0" cellspacing="0" width="1736">
	<tbody>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">DMEM</td>
			<td style="width:357px;">Corning Life Science</td>
			<td style="width:276px;">10-013-CV</td>
			<td style="width:369px;">Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">PHENOL-FREE DMEM</td>
			<td style="width:357px;">Corning Life Science</td>
			<td style="width:276px;">17-205-CV</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">FETAL BOVINE SERUM</td>
			<td style="width:357px;">Sigma-Aldrich</td>
			<td style="width:276px;">F2442</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">L-GLUTAMINE</td>
			<td style="width:357px;">Gibco</td>
			<td style="width:276px;">25030</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">PENICILLIN/ STREPTOMYCIN</td>
			<td style="width:357px;">Corning Life Science</td>
			<td style="width:276px;">30-002-CI</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">EYFP-PARKIN EXPRESSION VECTOR</td>
			<td style="width:357px;">Addgene</td>
			<td style="width:276px;">23955</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">pDsRed2-Mito EXPRESSION VECTOR</td>
			<td style="width:357px;">Clontech</td>
			<td style="width:276px;">632421</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">NUCLEOFECTOR 2B DEVICE</td>
			<td style="width:357px;">LONZA</td>
			<td style="width:276px;">AAD-1001S</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">NUCLEOFECTOR FOR KIT R NIH/3T3</td>
			<td style="width:357px;">LONZA</td>
			<td style="width:276px;">VCA-1001</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">ZEISS AXIOVERT 200M INVERTED MICROSCOPE</td>
			<td style="width:357px;">CARL ZEISS</td>
			<td style="width:276px;"></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Carbonyl Cyanide 4-(trifluoromethoxy)-Phenylhydrazone (FCCP)</td>
			<td style="width:357px;">Sigma-Aldrich</td>
			<td style="width:276px;">C2920</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">MetaMorph</td>
			<td style="width:357px;">Molecular Devices</td>
			<td style="width:276px;">Experimental Builder</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:735px;">ImageJ</td>
			<td style="width:357px;">National Institute of Health</td>
			<td style="width:276px;">Experimental Builder</td>
			<td></td>
		</tr>
	</tbody>
</table>

time-lapse video microscopy for assessment of eyfp-parkin aggregation as a marker for cellular mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach.

Herein, we show how time-lapse video microscopy is a powerful technique that can be used to follow molecular events of fluorescently-tagged proteins in a single cell. The representative results also show how this technique allows for the acquisition of high quality images. When images of the molecular process, are obtained, we have the opportunity to analyze them in different ways. Here, we analyze the interval of time between initiation of the induced mitophagy process, which is the cruc...

Watch this Scientific Journal Video about Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy at JoVE.com

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time ...

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In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...