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 ...

The overall goal of this Time-Lapse Video Microscopy is to track the selective Parkin-mediated removal of damaged mitochondria during the mitophagy process. This method can help answer key questions in the molecular metabolism field, such as how mitophagy's affect enduring pathological conditions. The main advantage of this technique is that it provides a very powerful tool for following the dynamics of labeled proteins during their molecular processes.This method can provide insight into the mitochondrial life cycle. It can also be applied to other systems, such as primary cell cultures, embryology studies, and predicting an priority. The immortalized mouse embryonic fibroblasts used in this study are grown in complete DMEM medium on a ten centimeter tissue culture plate at 37 degrees Celsius in a humidified atmosphere containing five percent carbon dioxide.When the cells are at 80 percent confluency, discard the medium by sterile suction and add ten milliliters of sterile PBS. Remove the PBS, and add one milliliter of 0.25 percent Trypsin EDTA. Incubate the plate at 37 degrees Celsius for two to three minutes until the cells are detached.Add four milliliters of complete DMEM medium, and re suspend the cells. Take out ten microliters of the cell suspension for counting with a hemocytometer. Seed one times ten to the sixth cells on to a ten centimeter tissue culture dish, and incubate for 24 hours.On the following day, wash the cells once with PBS and detach the cells by trypsinization, as demonstrated earlier. Add four milliliters of complete DMEM medium, re-suspend the cells, and transfer the cell suspension to a 15 milliliter tube. Spin the cells at 250 gs and four degrees Celsius for five minutes.Discard the supernatant and re suspend the pellet in one milliliter of sterile PBS. Spin the cells again. Discard the supernatant after the second spin.Add 100 microliters of the solution mix for electroporation to the cell pellet and gently re suspend the pellet by pipetting. Then add to the cell suspension two micrograms of EYFP Parkin expression plasmid and one microgram of DsRed2 Mito expression plasmid. Use the disposable Pasteur pipette to transfer the solution to a sterile cuvette.Electroporate the cells using a preset program. Immediately after the electroporation, add 500 microliters of fresh, pre warmed complete DMEM medium to the cells and seed the cells on a six centimeter. live-imaging grade tissue culture plate.Incubate the cells in a humidified atmosphere containing five percent carbon dioxide at 37 degrees Celsius for 24 hours. Prior to starting this procedure, set the temperature of the microscope's chamber at 37 degrees Celsius. Next, prepare a specific live imaging medium by mixing the following, Phenol-free DMEM supplemented with ten percent FBS, two millimolar per liter of L-Glutamine, 100 units per milliliter of penicillin, and 100 milligrams per milliliter of streptomycin.Pre warm the medium at 37 degrees Celsius in a water bath. Discard the medium of the electroparated cells by sterile suction and add one mililiter of pre warmed live imaging medium. Incubate the plate for 30 minutes at 37 degrees Celsius.After confirming that the microscope's chamber is at a stable temperature of 37 degrees Celsius. Influx five percent carbon dioxide into the chamber. Carefully, and without major movements or oscillations, place the plate with the electroporated cells, into the microscope's chamber.Using the software interface, set the microscope to detect the fluorescent signals from the fusion proteins encoded by the EYFP Parkin and DsRed2 Mito expression vectors. Open the time lapse video microscopy software. In the upper menu, select FITC for EYFP Parkin and Rhodamine for DsRed2 Mito.Select a magnification of 20X. Using the software interface, look for a single cell expressing both cotransfected vectors and register the position. Do this for a minimum of 10 cells for every experimental condition.Select the menu, Apps and click on, Multi Dimensional Acquisition. In the Multi Dimensional Acquisition windows, select the parameters needed, such as the number of acquisitions, the interval of time between each acquisition, and the position of the recorded cell. Click on, Acquire to start the basal acquisition of both fluorescent signals.Collect images every five minutes for a total interval of fifteen minutes. While image acquisition is occurring, prepare pre warmed live imaging medium containing Carbonyl Cyanide 4 trifluouromethoxy phenylhydrazone, or FCCP, at a concentration twice the final working concentration. Interrupt the acquisition process, and gently add one milliliter of the pre warmed live imaging medium with FCCP into the plate inside the microscope's chamber.After adding FCCP, it is important to re focus the microscope before restarting the image acquisition. Restart the acquisition process as previously described, and collect images for a total period of three hours using the recorded position. When image acquisition is complete, save all the acquired images for later analysis.Shown here are time lapse images of EYFP-Parkin and DsRED2-Mito fluorescent signals in fibroblasts prior to FCCP administration. The white boxes represent the magnified areas of the respective panels on the right. During the basal time points, EYFP-Parkin, visualized in green, is homogeneously diffused throughout the cell.The mitochondria network, visualized in red, appears to be well interconnected, as indicated by the white arrows. Following FCCP administration, mitochondria fractionation due to membrane depolarization is observed at five minutes. However, EYFP-Parkin is still homogeneously diffused throughout the cytoplasm.At 55 minutes post FCCP administration, EYFP-Parkin is recruited to the damaged mitochondrial membranes to trigger the mitophagy process. The movement of EYFP-Parkin to the mitochondrial membrane following FCCP administration is shown in this representative time lapse movie. Once mastered, this technique can be done in two hours if it is performed properly.After its development, this technique paved the way for researchers in the field of autophagy experiment mitochondrial recycling. Allowing cells to study the mitophagy process as a mitochondrial quality control in the cells.

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Research

JoVE Journal

Biology

7.1K vistas.  Thomas Jefferson University, SKCC. The overall goal of this Time-Lapse Video Microscopy is to track the selective Parkin-mediated removal of damaged mitochondria during the mitophagy process. This method can help answer key questions in the molecular metabolism field, such as how mitophagy's affect enduring pathological conditions. The main advantage of this technique is that it provides a very powerful tool for following the dynamics of labeled proteins during their molecular processes.This method can provide insight i...