Mitochondrial 융합은 시간이 지남에 mitochondrial 네트워크 photoconverted 매트릭스 타겟 GFP의 평균 추적에 의해 측정되었다. 지금까지 하나의 세포는 한 번에 한 시간 동안 운동 분석을 받다 수 있습니다. 우리는 동시에이를 통해 데이터 수집 과정을 속도, 여러 개의 세포를 측정하는 방법을 제시한다.
Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment.
Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis1,2,3,13. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer’s disease, Parkinson’s disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks4,10,13. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function18. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust14. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis9. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary.
Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay7, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay1,5. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process 4,5. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment.
A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP)6,11. Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal6. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE9,15. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE.
The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity8,15,16,17.
In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15nM TMRE8 in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.
인수 photoactivation 후 15 분마다 발생하는 경우이 방법은 한 번에 약 10 세포 이미징을 허용합니다. 세포의 정확한 숫자는 하나 mtPAGFP 표현 문화 접시 내에서 세포, 그리고 얼마나 빨리 하나가 모든 소프트웨어 매개 변수를 설정할 수를 찾아 표시할 수있게하는 속도에 따라 다릅니다. 지정된 Z-스택 마진은 모든 세포를 신청하기 때문에 자동화가 원활하게 실행하기 위해서 세포의도 레이어를 사용해야합니다.
이 초기 photoactivation 영역의 크기는 평균 시간을 적용합니다. mitochondrial 융합을 측정할 수있게하기 위해서는 네트워크의 나머지 부분에 대한 신호의 확산은 시간이 지남에 따라 모니터링할 수 있도록 네트워크의 유일한 10~20%를 photoactivate하는 것이 중요합니다. 네트워크의 너무 많이가 photoactivated 경우, 그것은 완전한 융합도 빠르게 발생 가능성이 있으며, 이벤트가 캡처되지 않습니다.
익스 트림주의로 이동합니다두 광자 레이저의 레이저 파워뿐만 아니라 mitochondrial 탈분극로 연결 phototoxicity을 피하기 위해 TMRE 농도를 조정합니다. mtPAGFP 신호가 TMRE 신호로 colocalizes 수 있도록하는 것은 phototoxicity과 일반 세포 건강 8,15를 평가하는 데 유용합니다. epifluoerescent 빛이있는 조명은 피해야한다. mtPAGFP을 표현 세포를 검색하는 동안 저전력 488nm의 여기로 스캔하는 동안, 핀홀이 maximally 열려 있어야합니다. PA-GFP는 1 시간 이상 신호를 측정할 수 있지만 세포 중 8 까다롭습 수 oversaturate하는 데 충분하지 photoactivate하는 두 광자 레이저 파워 조절. 일단 자동으로 프로그램이 시작되기 때문에 그러나 시간이 최적화 단계에서 지출되어야합니다 그것은 세포를 선택하여, 그것을 중지하고 재개하는 지루한이다.
품질 관리의 경우 차등 간섭 대비 (DIC)의 인수 세포에 초점을 모니터 이미지 (또는 전송 빛) 매우 될 수 있습니다도움이 또한 스캔하는 동안 침지 기름에 형성된 거품을 감지하는 좋은 방법, 이것은 때로는 무대의 움직임에서 발생합니다.
이 mtPAGFP 방법을 사용하면 분류되지 않은 사람들에게 photoactivated mitochondria에서 mitochondrial 매트릭스 단백질의 단방향 운동에서 데이터를 수집하지만, 그것은 다른 프로세스를 연구하기 위해이 기술을 활용하는 생각할 수있다. 예를 들어, 특정 fluorochromes는 ABC-날 15 가용성 매트릭스 단백질의 혼합에서 다른 시간 척도에서 발생하는의 융합에 표시되었습니다대로 퓨전 이벤트 기간 동안 자신의 구체적인 움직임을 관찰하는 멤브레인 단백질에 첨부하실 수 있습니다.
The authors have nothing to disclose.
우리는 신경 과학 연구를위한 Tufts 센터, P30 NS047243 (잭슨), 보스턴 대학 의과 대학, 링크 의학 공단에서 학제간 바이오 메디컬 연구 에반스 센터에서 지원 Mitochondria 동질 연구 협력 (mtARC), 그리고이 작품을 지원하기위한 자이스 혈구 감사드립니다.
Name of the reagent | Company | Catalogue number |
COXIII-adenoviral PA-GFP | Dr. Lippincott-Schwartz | |
TMRE | Invitrogen | T669 |
Zeiss LSM 710 confocal | Zeiss |