线粒体融合是衡量跟踪矩阵针对性photoconverted整个网络随着时间的推移线粒体的GFP平衡的。到目前为止,只有一个细胞可能受到的时间在一个小时长的动力学分析。我们提出了一个方法,同时测量多个细胞,从而加快了数据采集过程。
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.
这种方法允许约10细胞成像的时间,如果收购发生光敏后每隔15分钟。细胞的确切数目将取决于如何迅速,是能够找到和标记mtPAGFP表达细胞在培养皿中,如何快速,可以设置所有软件参数。使自动化运行顺利,甚至细胞层应使用指定的Z-Stack的利润率,因为将适用于所有细胞。
这一初步的光敏面积的大小,将治理的平衡时间。为了能够测量线粒体融合,这是重要的,以photoactivate只有10-20%的网络,例如,可随着时间的推移监测网络的其余部分的信号传播。如果太多的网络是光活化,这是完全融合可能会出现太快,而不会被捕获的事件。
必须采取非常谨慎调整激光功率的双光子激光以及TMRE浓度,以避免光毒性,从而导致线粒体去极化。确保该mtPAGFP信号TMRE信号的共定位,可以帮助评估光毒性和一般细胞的健康8,15。 epifluoerescent光照明应避免。而细胞表达mtPAGFP,针孔,应该最大限度地开放,而低功率488nm激发扫描。调整的双光子激光的权力photoactivate足够的PA-GFP来衡量1小时以上的信号,但不oversaturate细胞可能会非常棘手8。然而,时间应在这个优化步骤花了,因为一次自动启动程序,它是乏味停止,选择更多的细胞,并恢复。
对于质量控制,图像采集的微分干涉对比(DIC)(或透射光)重点监察细胞可以很乐于助人,也是一个很好的方法来检测扫描过程中形成的泡沫在浸油,这有时会发生从舞台的动作。
虽然使用此mtPAGFP方法收集数据上的光活化线粒体那些没有标记的线粒体基质蛋白的单向流动,可以想象的是,利用这种技术来研究其他进程。例如,特定的荧光可以连接到膜蛋白,观察其特定的运动融合事件,已显示为ABC,我从15水溶性基质蛋白的混合不同的时间尺度上发生融合。
The authors have nothing to disclose.
我们感谢塔夫茨大学神经科学研究中心,P30的NS047243(杰克逊),线粒体亲和性研究协作(mtARC)埃文斯在波士顿大学医学校区,链接医药公司的跨学科的生物医学研究中心的支持,并支持这项工作的蔡司。
Name of the reagent | Company | Catalogue number |
COXIII-adenoviral PA-GFP | Dr. Lippincott-Schwartz | |
TMRE | Invitrogen | T669 |
Zeiss LSM 710 confocal | Zeiss |