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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

1. Electroporation of Fibroblast with Both Expression Vectors EYFP-Parkin and pDsRed2-Mito

1. Grow the immortalized mouse embryonic fibroblast cells on 10 centimeter tissue-culture plate using DMEM (Dulbecco's modified Eagle's medium) medium supplemented with 10% fetal bovine serum, 2 mmol/l L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin in humidified atmosphere containing 5% CO₂ at 37 °C.
   1. At 80% cell confluency, discard the complete DMEM medium by sterile suction and add 10 ml of sterile 1x phosphate buffer solution (PBS) (80 g of NaCl, 2.0 g of KCl, 14.4 g of Na₂HPO₄, 2.4 g of KH₂PO₄ and 1 L distilled H₂O, PH: 7.4).
   2. Discard the PBS by sterile suction and add 1 ml of Trypsin-EDTA 0.25%. Incubate the plate at 37 °C until the cells are detached (2 - 3 min). Add 4 ml of complete DMEM medium, resuspend the cells and take out 10 μl of the cell suspension for the hemocytometer counting.
      NOTE: The hemocytometer is designed so that the number of cells in one set of 16 corner squares is equivalent to the number of cells x 10⁴/ml.
2. Seed 1 x 10⁶ cells onto 10 cm tissue culture dishes 24 hours prior to the electroporation process.
3. Discard the complete DMEM medium by sterile suction and add 10 ml of sterile PBS. Discard the PBS by sterile suction and add 1 ml of Trypsin-EDTA 0.25%. Incubate the plate at 37 °C until the cells are detached (2 - 3 min). Add 4 ml of complete DMEM medium and resuspend the cells in a 15 ml tube.
4. Spin the cells down at 250 x g for 5 min using a refrigerated-centrifuge (4 °C). Discard the supernatant by sterile suction and resuspend the pellet in 1 ml of sterile PBS. Spin the cells down at 250 x g for 5 min at 4 °C.
5. Discard the supernatant by sterile suction, and add 100 µl of solution mix for electroporation (82 µl of electroporation Solution V + 18 µl of Supplemental solution 1) to the cell pellet and gently resuspend the pellet by pipetting (For more details refer to user's guide). Add 2 µg of EYFP-Parkin (excitation/emission 514/527) and 1 µg pDsRed2-Mito (excitation/emission 565/620) at the cell suspension.
6. Transfer the solution to the sterile cuvette using a disposable pasteur (both tools are provided with the kit) and electroporate using the pre-set program NIH/3T3 U-030 (Single pulse, voltage 200 V, capacitance 960 µF, pulse time 20 milliseconds, pulse number: 1).
7. Immediately after the electroporation, add 500 µl of fresh pre-warmed complete DMEM medium and seed the cell on a 6 cm tissue-culture plate live-imaging-grade and incubate them in a humidified atmosphere containing 5% CO₂ at 37 °C for 24 hours.

2. Time-Lapse Video Microscopy

1. Set the temperature of the microscope's chamber at 37 °C prior to use.
2. At this point, prepare a specific live imaging medium to proceed with the experimental protocol. Prepare the live imaging medium as follows; mix DMEM phenol-free supplemented with 10% fetal bovine serum, 2 mmol/l L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Pre-warm the live imaging medium at 37 °C in a water bath.
3. Discard the medium of the electroporated cells by sterile suction and add 1 ml of pre-warmed live imaging medium, using a P1000 pipet and incubate the plate for 30 min at 37 °C.
4. During the plate incubation period, influx 5% CO₂ in the microscope's chamber already at a stable temperature of 37 °C.
5. Place the plate with the electroporated cells into the microscope's chamber avoiding major movements or oscillations.
6. Using the software interface, set the microscope in order to detect both fluorescence signals from the fusion proteins encoded by the co-transfected vectors, EYFP-Parkin (Excitation range 495 to 510 nm and emission range 520 to 550 nm, green) and the pDsRed2-Mito (Excitation maximum 558 nm and emission maximum 583 nm, red).
   1. Open the time-lapse video microscopy software. In the upper menu select the FITC for EYFP-Parkin and Rhodamine for pDsRed2-Mito. In the upper menu select the magnification (20X).
7. Using the software interface, look for the single cell expressing both co-transfected vectors and register the position. Repeat this step until a minimum of 10 cells is collected for every experimental condition (Experimental group).
   1. 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 acquisition process.
8. Start the basal acquisition of both EYFP-Parkin and DsRed2-Mito fluorescent signal collecting images every 5 min for a total interval of 15 min.
9. Prepare pre-warmed live imaging medium with a concentration of FCCP twice higher than the final working concentration (0.1 - 10 µM, cell type-dependent).
10. Interrupt the acquisition process and add gently 1 ml of pre-warmed live imaging medium with FCCP into the plate inside the microscope's chamber using a P1000 pipet.
11. Restart the acquisition process as previously described, for a total period of 3 hours, using the recorded position. Save all the acquired images in order to analyze them and create a video of the mitophagic process when the acquisition is over.

3. Analysis

1. Collect all the acquired images in ".tiff " format on a personal computer
2. Open the images with a graphic software and define the temporal interval occurred from the mitochondrial induced-depolarization with FCCP to the first image showing the recruitment of Parkin into the mitochondrial membrane (Images are acquired every 5 min). Repeat this analysis for each cell in the experimental group.
3. Label the first cell of the column on a spreadsheet with the experimental group name. For each experimental group, measure the temporal intervals for Parkin recruitment of a minimum of 10 cells. Make an average of the calculated temporal intervals for Parkin recruitment and define the standard deviation for each experimental group (Mean ± S.D., N = 10).
   1. Organize into columns the single calculated temporal intervals. Every column contains the n = 10 measurements of the experimental group.
   2. Select the function "average" from the Formula Builder menu. Select the data in the column. Select the function "Standard Deviation" from the Formula Builder menu. Select the data in the column.
4. Using a statistical approach, compare the experimental groups applying the appropriate statistic in order to define a significant difference in the dynamic of mitophagy between the experimental groups. (For instance, two groups = Student's t-Test, three or more groups = ANOVA plus a post-hoc test)

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Corning Life Science	10-013-CV	Pre-warm at 37 °C before use
Phenol-free DMEM	Corning Life Science	17-205-CV	Pre-warm at 37 °C before use
Fetal Bovine Serum	Sigma-Aldrich	F2442	Pre-warm at 37 °C before use
L-Glutamine	Gibco	25030	Pre-warm at 37 °C before use
Penicillin/streptomycin	Corning Life Science	30-002-CI	Pre-warm at 37 °C before use
EYFP-PARKIN expression vector	Addgene	23955
pDsRed2-Mito expression vector	Clontech	632421
Nucleofector 2B device	LONZA	AAD-1001S
Nucleofector for kit R NIH/3T3	LONZA	VCA-1001
ZEISS AXIOVERT 200M inverted microscope	CARL ZEISS
Carbonyl Cyanide 4-(trifluoromethoxy)-Phenylhydrazone (FCCP)	Sigma-Aldrich	C2920
MetaMorph	Molecular Devices	Experimental Builder
ImageJ	National Institute of Health	Experimental Builder