速度更快，高分辨率，MTPA-GFP基于线粒体融合含量的收购多个单元格的动力学数据，在并行使用共聚焦显微镜

Summary

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。

Abstract

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 apoptosis^1,2,3,13. 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 networks^4,10,13. 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 assay^1,5. 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 TMRE^9,15. 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 activity^8,15,16,17.

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.

Protocol

1。图像板的制备文化INS1细胞在RPMI媒体含有10％的标准胎牛血清血清，1％青霉素，链霉素，2毫米L-谷氨酰胺，50微米2 -巯基乙醇， 碳酸氢钠 5毫米，2毫米的肝素钠，2毫米丙酮酸，和11毫米葡萄糖80％汇合。 Trypsinize INS1细胞培养与0.05％胰蛋白酶和板到聚-D-赖氨酸涂布盖玻片底成像板（30-40％汇合）。 允许板达到60-80％汇合（〜2天），并添加有针对性的线粒体基质（COXVIII）的PA-GFP腺病毒为24小时（MOI = 5）。交换媒介，使细胞生长成像前2天。 对成像的日子，添加7-15纳米TMRE成像板，平衡至少45分钟。 在此期间对孵化器的导通时间（舞台顶部孵化器已用在这里），并允许在显微镜平衡至37℃，约1小时。打开的5％的CO 2。 </李> 2。蔡司LSM的710共聚焦显微镜成像参数显微镜具有平衡后，根据收购“选项卡上，单击”秀手工工具“，打开成像设立面板，并选择”通道模式“切换跟踪每一个”框架“。 在光路径面板，选择LSM和渠道模式。从标本图标向上的工作，选择“后方”，MBS的690 +和MBS 488。在面板的底部，选择“T型光电倍增管”，使亮场或DIC通道的可视化。 完成调整光路参数设置范围490-540纳米到580-700纳米的TMRE染料的绿色荧光染料。 在采集模式，使用512×512像素的扫描场，平均4倍，并选择一个缩放因子（你可能要保持校准的目的相一致）。 3。成像参数优化使用计划复消色差透镜100X（1.4 NA）的物镜，FOC我们对你的细胞，卤素灯，荧光灯，光毒性，以保护线粒体。 PA-GFP的表达，使用最大开放针孔扫描发现的细胞，是明亮的绿色屏幕。 找到需要激活的PA-GFP和优化成像参数，确保信号不饱和探测器的双光子激光功率最低。检查PA-GFP信号与几个Z系列TMRE信号的共定位。损失的TMRE信号的指示线粒体去极化或光毒性，细胞表现出这些特点，不应该被用于分析。 找到一个PA-GFP表达细胞，并指定一个缩放因子。 设置Z系列范围内收集6片（此范围内，将需要满足所有10个细胞，除非特定的Z-焦点设置在每个位置）。 保存为每个单元格（单元1，单元2等）的成像方法。 4。调整的“自动”ð部分计划和指定的10个细胞，你将遵循为1小时线粒体融合含量的多时间窗口的左侧面板上，选择“节能”面板，文件将被保存在指定位置。 在“收购”面板，载入保存扫描配置1细胞的采集方法和检查Z堆栈框。也可以选择“标记的Z-z的栈中间”。 在“块”面板中，选择“在每个位置的单块”。点击“添加模块”每次要测量的时间间隔。 在“计时”面板，选择“只在第一的位置块前的等待时间间隔”“等待的时间间隔”中键入“0”。块2-4将有“15分钟”这一节。 在位置面板中，选择“移动焦点加载位置之间的位置”和“负载扫描配置时，”禄“或”下一步禄“点击在”编辑位置列表“选择”全部清除“，然后选择”多地点机动的阶段“。 >ER“死神”面板，单击“漂白”框中，指定在“配置”配置文件下拉菜单，在主要软件的窗口中指定。然后，在“漂白”窗口，保存相应的光敏方法。在该地区面板，选择这个特定的细胞的投资回报率，并选择“添加当前区域的投资回报率清单”窗口添加了多时的投资回报率。务必选择相同的位置下拉菜单中的“投资回报率”。 正常工作的时机，每个单元有一个15分钟的时间间隔，这两种方法都需要被保存。在块列表中，第一个将“真正的”光敏配置，其余的将有一个“模拟”的配置，不使用双光子激光。首块也将是唯一的块，不会有延迟（BKIntv = 0）。因此，该方法开始一个由光敏扫描基线扫描，。其余的区块有900秒BkIntv的，和在每个时间点有两个扫描一样在时间0秒，保持时间的一致性。 对于每个10细胞随着时间的推移，执行此序列： 查找PAGFP表达细胞保存其成像方法位置面板标记舞台上的地位，指定成像方法主要的投资回报率面板选择感兴趣的区域，是光活化漂白剂面板保存特定的投资回报率也加载在投资回报率框中： 擦除的投资回报率和重置扫描放大到1 寻找下一个单元格，并设置变焦重复的过程，在未来的9细胞检查每个阶段的位置和所有块有适当的成像方法（扫描配置）。还要确保在每个位置的第一个块对应到相应的光敏方法，而其余包含一个“模拟”的方法。最后，选择“运行”。 5。分析PA-GFP的信号强度<l我>对于细胞在photoactivatable-GFP信号用红色TMRE信号的共定位，减去背景在PAGFP图像（在这里我们使用metamorph）。 然后将数据导出到Excel计算Z-堆叠在每个时间点的平均强度。丢弃有没有信号的光学部分。 原光活化信号的百分比计算，测量信号在每个Z-Stack的稀释。 每个数据点扫描和记录了两次，导致重复的Z-Stack的每个时间点的信息。检查Z-Stack的值一致。如果不是，这是一个问题（如焦点，细胞运动）的指标。 6。代表结果激活和非激活PA-GFP的线粒体之间的融合事件发生时，在线粒体基质PAGFP混合矩阵的非标记和稀释成为了一个更大的区域，降低了信号强度（ 图1A）。在INS1细胞，发生在信号强度显着下降，每15分钟，直到已达到平衡线粒体融合（约1小时）。请注意，在图1B细胞呈现近完整的PA-GFP的和TMRE信号的共存。在这些化验TMRE的浓度非常低（15 nm）的使用，以帮助目标的光敏PAGFP，并同时监测细胞的健康。与去极化线粒体丰富的细胞将有PAGFP和TMRE不完整的共存和不应该加以分析，因为这表明无论是光毒性，或在一个垂死状态的细胞。 线粒体融合的平衡时间通常为1小时INS1细胞，当〜线粒体体积的15％被激活。有时，即使小面积被照亮，大多数线粒体成为由于高度网络化的进一步融合，在这种情况下是难以察觉的光活化。其他类型的细胞可能会出现不同的平衡时间，并应在较短的时间间隔，并在一个较长时期的测试表征线粒体的动态。为了抑制线粒体融合，细胞可以放置内lipotoxic环境。先前已被证明有0.4毫米棕榈片段线粒体，抑制线粒体融合9。这种效果可以看出，在图2，其中线粒体是支离破碎，但信号强度的mtPAGFP不会改变尽可能在正常条件下（ 图1）。因此，稀释的PA-GFP信号可能是由于线粒体融合，而不是核裂变。在其他细胞类型，我们建议使用其他方式诱导线粒体碎片，如沉默OPA1这是必要的线粒体融合14。 图1。 </stroNG>典型稀释的PA-GFP在线粒体融合实验后光敏信号。的PA-GFP逐渐变得黯淡由于线粒体融合的事件，导致蛋白质的稀释，增加面积，可以看到这些投影图像6光学部分量化每15分钟。与PA-GFP TMRE表明，线粒体活跃，而不是去极化。 点击这里查看大图 。 图2。线粒体融合棕榈抑制0.4毫米减少了稀释的PA-GFP 6光学部分，显示短不变的信号强度随着时间的推移，线粒体A.预测B.共存的PA-GFP与TMRE显示，线粒体不德极化。 点击这里查看大图 。

Discussion

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。

Materials

Name of the reagent	Company	Catalogue number
COXIII-adenoviral PA-GFP	Dr. Lippincott-Schwartz
TMRE	Invitrogen	T669
Zeiss LSM 710 confocal	Zeiss