image analysis of  hep-3b cells after drug treatment

예쁜꼬마선충(C. elegans)과 간암 세포주(Liver Cancer Cell line)의 유사분열(Mitophagy) 정량화

Mitochondria are essential for all aerobic animals, including humans. They convert the chemical energy of biomolecules to adenosine triphosphate (ATP) via oxidative phosphorylation1, synthesize heme2, degrade fatty acids through &#946; oxidation3, regulate calcium4 and iron5 homeostasis, control cell death by apoptosis6, and generate reactive oxygen species (ROS), which play a vital role in redox homeostasis7. Two complementary and opposite processes maintain the integrity and proper function of the mitochondria: the synthesis of new mitochondrial components (biogenesis) and the selective removal of damaged ones through mitochondrial autophagy (i.e., mitophagy)8.
Several mitophagy pathways are mediated by enzymes, such as PINK1/Parkin, and receptors, including FUNDC1, FKBP8, and BNIP/NIX9,10. Notably, the selective degradation of mitochondrial components can occur independently of the autophagosome machinery (i.e., through mitochondrial-derived vesicles)11. However, the endpoints of the different selective mitophagy pathways are similar&#160;(i.e.,mitochondrial degradation by lysosomal enzymes)12,13. For this reason, various methods for identifying and measuring mitophagy rely on the colocalization of mitochondrial and lysosomal markers14,15,16,17 and decreased levels of mitochondrial proteins/mitochondrial DNA18.
Below is a concise description of the existing experimental methodologies for measuring mitophagy in animal cells using fluorescence microscopy, emphasizing the mitophagy endpoint phase.
Mitophagy biosensors 
Mitochondrial degradation occurs within the acidic environment of the lysosome19. Therefore, mitochondrial components, including proteins, experience a shift from a neutral to an acidic pH at the endpoint of the mitophagy process. This pattern underpins the mechanism of action of several mitophagy biosensors, including mito-Rosella18 and tandem mCherry-GFP-FIS114. These sensors contain a pH-sensitive green fluorescence protein (GFP) and a pH-insensitive red fluorescence protein (RFP). Therefore, at the endpoint of mitophagy, the green-to-red fluorescence ratio drops significantly due to the quenching of the GFP fluorophore. The major limitations of these sensors are (1) possible F&#246;rster resonance energy transfer (FRET) between the fluorophores; (2) the differential maturation rate of GFP and RFP; (3) dissociation between the GFP and RFP due to proteolytic cleavage of the polypeptide that connects them; (4) fluorescence-emission overlap; and (5) differential fluorophore brightness and quenching15,16.
A sensor that overcomes some of these limitations is the Keima mitochondrial sensor17. The mt-Keima sensor (derived from the coral protein Keima) displays a single emission peak (620 nm). However, its excitation peaks are pH-sensitive. As a result, there is a transition from a green excitation (440 nm) to a red one (586 nm) when shifting from a high pH to an acidic pH16,17. A more recent mitophagy sensor, Mito-SRAI, has advanced the field by allowing for measurements in fixed biological samples20. However, despite the many advantages of genetic sensors, such as the ability to express them in specific tissues/cells and target them to distinct mitochondrial compartments, they also have limitations. One limitation is that the genetic sensors need to be expressed in cells or animals, which can be time-consuming and resource-intensive.
Additionally, the expression of the sensors within mitochondria themselves may influence the mitochondrial function. For example, expressing mitochondrial GFP (mtGFP) in the C. elegans worm body wall muscles expands the mitochondrial network21. This phenotype depends on the function of the stress-activated transcription factor ATFS-1, which plays an essential role in the activation of unfolded protein response in mitochondria (UPRmt)21. Therefore, although genetically encoded mitochondria/mitophagy biosensors are extremely useful for monitoring mitochondria homeostasis in vivo, they may affect the very process they are designed to measure.
Mitochondria/lysosome-specific antibodies and dyes 
Another strategy for testing mitochondrial/lysosome colocalization is to use antibodies against mitochondrial/lysosomal proteins, such as the mitochondrial outer membrane protein TOM20 and lysosomal-associated membrane protein 1 (LAMP1)22. In most cases, secondary antibodies that are conjugated to a fluorophore are used to detect the fluorescence signal via microscopy. Another strategy is to combine genetic constructs with mitochondrial/lysosomal dyes, such as expressing a LAMP1::GFP fusion construct in cells while staining them with a red mitochondrial dye (e.g., Mitotracker Red)16. These methodologies, while effective, require specific antibodies and often involve working with fixed specimens or generating cells/transgenic animals expressing fluorescently labeled mitochondria/lysosomes.
Here, we outline the utilization of a commercial lysosome/mitochondria/nuclear staining kit for assessing the mitophagy-activating properties of synthetic diamine O,O (octane-1,8-diyl)bis(hydroxylamine), hereafter referred to as VL-85023, in C. elegans worms and the human cancer cell line Hep-3B (Figure 1). The staining kit contains a mixture of lysosomal/mitochondrial/nuclear-targeted dyes that specifically stain these organelles23. We previously used this kit to demonstrate the mitophagy activity of 1,8 diaminooctane (hereafter referred to as VL-004) in C. elegans23. Importantly, we validated the staining kit results with the mito-Rosella biosensor and qPCR measurements of the mitochondrial:nuclear DNA content23. This staining kit offers the following advantages. First, there is no need to generate transgenic animals or cells expressing a mitochondrial biosensor. Therefore, we can study unmodified wild-type animals or cells and, thus, save much time, money, and labor. Moreover, as mentioned, expressing mitochondrial biosensors can change the mitochondrial function. Second, the kit is cost-effective, easy to use, and fast. Third, although we demonstrate the method in C. elegans and human cells, it could be modified for other cell types and organisms.
With that said, like any method, the staining kit protocol has drawbacks. For example, the incubation of the worms with the reagent is carried out in the absence of food (we have seen that even dead bacteria significantly decrease the staining efficiency). Although the incubation time is relatively short, it is possible that even in this time frame, homeostatic responses may be altered, including mitophagy. In addition, the binding of the dyes to the ER/mitochondrial/nuclear proteins and other biomolecules may affect the activities of these organelles. Moreover, unlike mitophagy measurement with genetic sensors, we work with worms and cells that have undergone chemical fixation. Therefore, it is impossible to continue monitoring the same worms/cells at different times. Hence, we recommend combining different methodologies to validate the function of mitophagy in a particular physiological process. Below, we present new data demonstrating that VL-850 induces robust mitophagy in C. elegans worms and Hep-3B cells. Therefore, these data further support the hypothesis that VL-850 extends the lifespan of C. elegans and protects C. elegans from oxidative damage through the induction of healthy mitophagy. We have used the proton ionophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), which is a potent mitophagy inducer24, as a positive control.

저자 스포트라이트: Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes(세포기관 특이적 염료를 사용한 예쁜꼬마선충 및 포유류 세포의 유사 분열 검출)  

세포 기관 특이적 염료를 사용한 Caenorhabditis elegans 및 포유류 세포에서 Mitophagy 검출

Degradation of damaged mitochondria by mitophagy is essential for the function of the mitochondrial network. Unfortunately, this process declines with age. We develop and use drugs that activate mitophagy, thereby promoting healthy aging to understand the function of mitophagy in aging and age-related diseases, including Alzheimer's.One of the biggest challenges is to measure the mitophagy process without affecting it. Recent studies have shown that GFP expression in the mitochondria of muscle cells induces stress that is MT UPR. Therefore, using dyes in non-transgenic animals or cells is essential and complements genetic technologies.Our protocol has three main advantages. First, there is no need to generate transgenic animals or cells expressing mitophagy biosensors. Second, no need for expensive infrastructure and expertise such as in case of electron microscopy.And third, the organelle dye cocktail is commercially available and the protocol is simple and fast. The new mitophagy-activating compound VL-850 induces robust mitophagy, and in this way protects from oxidative stress and promote lifespan. Now the big question is to determine the underlying mechanism of action.We explored the mechanism at multiple levels, from the cellular to the organismal using C.elegans and mammalian cells.

Watch this Scientific Journal Video about Image Analysis of  Hep-3B Cells after Drug Treatment at JoVE.com

Image Analysis of  Hep-3B Cells after Drug Treatment  

약물 치료 후 Hep-3B 세포의 이미지 분석

약물 처리된 Hep-3B 세포를 이미징한 후 colocalization 플러그인을 사용하여 이미지 J의 컨포칼 이미지를 엽니다. 이미지 J 서버에서 ND 파일을 열고 대화 상자에서 채널 분할 옵션을 선택합니다. 녹색 및 빨간색 이미지로 작업합니다.이미지를 클릭하고 복제를 선택하거나 키보드 단축키 Shift 플러스를 사용하여 배경을 줄이 D.To 고, 다른 이미지 복제본을 생성하고, 롤링 반경이 100인 배경을 빼고, 배경 만들기 옵션을 선택하여 주어진 이미지 배경으로 이미지를 생성함으로써 원본 이미지를 그대로 유지하기 위해 이러한 이미지의 복제본을 생성합니다. 그런 다음 프로세스로 이동합니다. 이미지 계산기를 클릭하고 두 번째 복제된 이미지에서 첫 번째 복제된 이미지를 뺍니다.결과 이미지를 colocalization 분석에 활용합니다. colocalization 플러그인을 사용하려면 이미지를 클릭하고 유형을 선택한 다음 8비트를 선택하여 녹색 및 빨간색 채널 이미지를 8비트로 변환합니다. 그런 다음 플러그인을 클릭하고 colocalization을 선택합니다.미토콘드리아와 리소좀 신호의 colocalization을 측정하려면 비율을 75%로 설정하고 임계값 빨간색 채널을 80.0으로, 임계값 녹색 채널을 50.0으로 설정합니다. colocalized puncta를 포함하고 RGB 이미지에 3개의 8비트 이미지를 결합한 8비트 이진 이미지가 생성됩니다. 이 이미지를 8비트 이미지로 변환합니다.셀의 관심 영역을 얻으려면 colocalized 점의 이미지를 선택하여 셀 영역을 강조 표시하는 이미지를 생성합니다. 전체 셀 영역을 선택하려면 결과 colocalized 포인트 이미지를 선택하고 이미지를 클릭하고 임계값을 조정한 다음 임계값을 설정하여 모든 셀 영역이 강조 표시되도록 합니다. 셀의 영역을 분석하려면 분석을 선택하고 입자 분석을 클릭한 다음 입자 분석의 기본 설정인 0에서 무한대까지 이미지의 모든 입자를 측정합니다.colocalized 미토콘드리아와 리소좀의 영역을 분석하려면 colocalized 8비트 이미지를 선택합니다. 그런 다음 분석을 선택합니다. 입자 분석을 클릭하고 0.1625제곱마이크로미터에서 4제곱마이크로미터 사이의 반점의 합을 측정합니다.colocalized 미토콘드리아와 리소좀의 면적을 전체 세포 면적으로 나누어 colocalized puncta를 측정합니다. Hep-3B 세포의 공초점 이미지는 미토콘드리아와 리소좀의 공동 국소화를 나타냈습니다. VL 850 및 FCCP는 Hep-3B 세포에서 유의한 미토파지를 유도하였다.

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JoVE Journal

Biology

Image Analysis of  Hep-3B Cells after Drug Treatment

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, ...

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Protocol for Measuring Mitophagy in Caenorhabditis elegans

Protocol for Measuring Mitophagy in Caenorhabditis elegans  

예쁜꼬마선충(Caenorhabditis elegans)에서 유사분열 측정을 위한 프로토콜