Name: sensitive measurement of mitophagy by flow cytometry using the ph-dependent fluorescent reporter mt-keima
Uploaded: 2018-08-12T14:00:00
Description: Watch this Scientific Journal Video about Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima at JoVE.com

This work was supported by a grant from National Research Foundation of Korea (2016R1D1A1B03931949) (to J. U.), and by the National Research foundation of Korea (NRF) grant funded by the Korea government(MSIT) (No. 2016R1A2B2008887, No. 2016R1A5A2007009) (to J.Y.)

1. Generation of HeLa cells expressing mito-Keima (mt-Keima)
<ol>
	<li>Preparation of mt-Keima lentivirus
	<ol>
		<li>Coat a 100-mm culture dish by adding 2 mL of 0.001% poly-L-lysine/phosphate-buffered saline (PBS) and allow it to stand for 5 min at room temperature.</li>
		<li>Remove the poly-L-lysine solution using a glass pipette connected to a vacuum and wash the culture dish by adding 2 mL of 1x PBS.</li>
		<li>Plate 1.5 x 106 HEK293T cells on the coated culture dish with 10 mL of DMEM...

<ol>
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Here, we present a rapid and sensitive method for using flow cytometry to measure cellular mitophagy activity in cells expressing mt-Keima. Cells undergoing a high level of mitophagy exhibit an increased ratio of PE-CF594 (561 nm)/BV605 (405 nm) excitation. Thus, mitophagy activity can be expressed as the percentage of cells exhibiting a high 561/405 ratio. We calculated the percentage of cells in the &#34;high&#34; gate region on a dot plot of PE-CF594 (561 nm) versus BV605 (405 nm), and the results showed that treatmen...

Mitochondria are organelles that are essential for cell proliferation and physiology. Mitochondria are responsible for generating more than 80% of the ATP supply via oxidative phosphorylation, and they also provide various metabolic intermediates for biosynthesis and metabolism1,2. In addition to their roles in energy supply and metabolism, mitochondria play central roles in many other important processes, including reactive oxygen species (ROS) generation, the regulation of cell death, and intracellular Ca2+ dynamics3. Alterations in mitochondrial function have been associated with human diseases, including cancer, diabetes mellitus and various neurodegenerative diseases4,5. Increased mitochondrial dysfunction and mitochondrial DNA mutations are also thought to contribute to normal aging processes6,7,8. Therefore, quality control is a crucial issue in mitochondria. Mitochondria are highly dynamic organelles that can continuously change their shape between elongated networks and short, fragmented form. Mitochondria dynamics plays an important role in maintaining the function of mitochondria as well as their degradation through mitophagy9.
Mitophagy is a mechanism that involves the selective degradation of whole mitochondria using the autophagy machinery. Mitophagy is the principle mechanism underlying mitochondrial turnover and the removal of damaged or dysfunctional mitochondria. In this process, mitochondria are first surrounded by a membrane, resulting in the formation of autophagosomes, which then fuse with lysosomes for hydrolytic degradation9. Previous genetic studies in Drosophila have suggested that two Parkinson&#39;s disease-related genes, PTEN-induced putative kinase 1 (PINK1) (PARK6) and Parkin (PARK2), function in the same pathway10,11. Subsequence studies have shown that the PINK1-Parkin pathway is responsible for eliminating damaged mitochondria and defects in this pathway result in dysfunctional mitophagy and may contribute to human diseases12,13. Defects in mitophagy processes have recently been found in various human diseases, including cancer, heart disease, liver diseases and neurodegenerative disease 14. In addition, recent studies have also shown that mitophagy is critical to many physiological processes, such as differentiation, development, and the immune response15,16,17,18,19, suggesting that mitophagy may play a more active role in controlling cell functions.
Despite recent confirmation that mitophagy plays an important role in both quality control in mitochondria and human disease, the molecular mechanisms underlying mitophagy remain poorly understood. Although mitophagy-related mechanisms have been systematically studied in yeast, studies aimed at exploring mitophagy-related mechanisms in mammalian cells have mainly focused on the PINK1-Parkin pathway. Previous studies have established that the PINK1-Parkin pathway is primarily responsible for the selective removal of damaged mitochondria via mitophagy12,20,21. However, recent studies have reported that in some models, mitophagy can be activated even in the absence of functional PINK122,23,24. These results suggest that in addition to the PINK1-Parkin pathway, there an additional unidentified pathway that can activate mitophagy.
The absence of a convenient method to assess mitophagy activity is a major obstacle to exploring the pathways that regulate mitophagy and its role in pathophysiological events. Electron microscopy is a powerful tool to directly visualize autophagosome-engulfed mitochondria. However, electron microscopy has limitations in quantifying mitophagy activity. Although strategies that use microtubule-associated protein-1 light chain 3 (LC3)-conjugated fluorescent probes, such as GFP-LC3, are currently the most widely used approaches25, the transient nature of the LC3-based signal and its high rate of false-positive signals limit its sensitivity for detecting mitophagy in cells26. A combination of western blotting to measure mitochondrial protein level, the quantification of mitochondrial DNA, and fluorescence microscopy analysis to colocalize mitochondria with either autophagosomes or lysosomes would be a good approach for assessing mitophagy. However, quantification limitations and a lack of convenience of existing methodologies call for new approaches. The introduction of a new pH-dependent fluorescent protein, the mitochondrial target Keima (mt-Keima), greatly improved the ability to detect mitophagy27. By fusing the mitochondrial-targeting sequence of cytochrome c oxidase subunit VIII (COX VIII) into Keima, mt-Keima is directed to the mitochondrial matrix. The large shift in the excitation peak of Keima from 440 nm to 586 nm (corresponding to pH 7 and pH 4, respectively) can be utilized to assess mitophagy with good sensitively in both in vitro and in vivo experiments28,29. More importantly, the resistance of mt-Keima to lysosomal degradation causes the integration of the mt-Keima signal in lysosomes, allowing for the easy quantitative measurement of mitophagy activity. The fluorescence change that occurs in mt-Keima can be analyzed using either confocal microscopy or flow cytometry28,29. However, a flow cytometry-based method for measuring mitophagy activity using mt-Keima has not been provided in detail to date.
Here, we describe a detailed protocol for a flow cytometry-based technique for measuring mitophagy activity in cells using mt-Keima. Although we have shown here that flow cytometry analysis successfully detected CCCP-induced mitophagy in HeLa cells expressing Parkin, this technique can be applied to a wide variety of cell types.

<table border="0" cellpadding="0" cellspacing="0" style="width:635px;" width="635">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">REAGENTS</td>
			<td style="width:133px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">poly-L-lysine</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:133px;">GIBCO</td>
			<td style="width:119px;">16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">penicillin/streptomycin</td>
			<td style="width:133px;">wellgene</td>
			<td style="width:119px;">LS202-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:133px;">Hyclone</td>
			<td style="width:119px;">SH30013.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEK293T</td>
			<td style="width:133px;">ATCC</td>
			<td style="width:119px;">CRL-3216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:133px;">GIBCO</td>
			<td style="width:119px;">12800-082</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">OPTI-MEM&#160;</td>
			<td style="width:133px;">GIBCO</td>
			<td style="width:119px;">31985-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Turbofect</td>
			<td style="width:133px;">Thermos scientific</td>
			<td style="width:119px;">R0531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.45 &#956;m syringe filter</td>
			<td style="width:133px;">sartorius</td>
			<td align="right" style="width:119px;">16555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HeLa</td>
			<td style="width:133px;">ATCC</td>
			<td style="width:119px;">CCL-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polybrene</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">H9268</td>
			<td>8 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">puromycin</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8833</td>
			<td>2 mg/ml&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Carbonyl cyanide m-chlorophenyl hydrazine (CCCP)</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">C2759</td>
			<td>10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">trypsin-EDTA</td>
			<td style="width:133px;">wellgene</td>
			<td style="width:119px;">LS015-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EQUIPMENTS</td>
			<td style="width:133px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD LSRFortessa</td>
			<td style="width:133px;">BD Bioscience</td>
			<td style="width:119px;">LSRFortessa</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FACSDIVA</td>
			<td style="width:133px;">BD Bioscience</td>
			<td style="width:119px;">FACSDIVA (v8.0.1)</td>
			<td></td>
		</tr>
	</tbody>
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sensitive measurement of mitophagy by flow cytometry using the ph-dependent fluorescent reporter mt-keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

An example of the flow cytometry analysis of CCCP-induced mitophagy in HeLa-Parkin cells is shown in Figure 4. Using the flow cytometry analysis method described above, we can detect a dramatic increase in mitophagic cells in the &#34;high&#34; gate. The percentage of cells in the &#34;high&#34; gate was increased more than 10-fold compared with untreated control cells (Figure 4A). This increase in mitophagy activity was complete...

Watch this Scientific Journal Video about Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima at JoVE.com

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

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Biology

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