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  • 摘要
  • 摘要
  • 引言
  • 研究方案
  • 结果
  • 讨论
  • 披露声明
  • 致谢
  • 材料
  • 参考文献
  • 转载和许可

摘要

Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

摘要

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach.

引言

Macro autophagy is an intracellular process that involves the catabolic degradation of both damaged and dysfunctional cellular components, such as organelles and proteins for the purpose of either recycling or energy production. To initiate this metabolic process, the cell engulfs the damaged cellular components into a double-membrane structure, known as an autophagosome, which fuses with a lysosome and its content is degraded and recycled 1,2. There are two major types of autophagy, the non-selective and selective. The non-selective autophagy process occurs when the cell is under nutrient deprivation conditions and needs to scavenge for both essential nutrients and energy. However, selective autophagy occurs to mediate the removal of both dysfunctional/damaged organelles and proteins that otherwise could be toxic. One of the most studied selective autophagy process is the removal of mitochondria, termed mitophagy 1,3-5.

Mitochondria are the central organelles for cell metabolism and the primary source of adenosine triphosphate (ATP) via oxidative phosphorylation through the electron transport chain, fatty acid oxidation, and tricarboxylic acid (TCA) cycle. Moreover, mitochondria regulate reactive oxygen species (ROS) production and release proteins that participate in cell death pathways 6-8.

PTEN-induced putative kinase 1 (PINK1) and Parkin RBR E3 ubiquitin ligase (Parkin) are the key proteins implicated in the mitophagy process. Parkin can protect against cell death by keeping the cell healthy through mitochondrial quality control9. Upon the loss of mitochondrial membrane potential, cytosolic Parkin is recruited to the mitochondria by PINK1. This recruitment triggers the sequential events of mitophagy 10. There is a broad range of evidence that mitophagy is a fundamental mitochondria quality control process and abnormalities in this process drive disease 7. For instance, autosomal recessive Parkinson's disease has been associated with mutations in the genes that encode for Parkin and PINK1 (PARK2 and PINK1, respectively) 11. The quality control of mitochondrial health is essential for the removal of mitochondria that contribute to the accumulation of ROS12. Excessive presence of intracellular ROS can lead to damage of both nuclear and mitochondrial DNA (DNA and mt DNA, respectively).

Herein, we show a time-lapse video microscopy approach to follow the aggregation of Parkin after the induction of Parkin-mediated mitophagy in immortalized mouse embryonic fibroblasts via in vitro administration of carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP), an uncoupling agent. FCCP disrupts ATP synthesis by short circuiting protons across the outer mitochondria membrane and hence uncoupling oxidative phosphorylation from the electron transport chain 13. Triggering the depolarization of the mitochondrial membrane leads to the disruption of mitochondria and selective Parkin-dependent removal. Therefore, transfecting the cells of interest with an expression vector encoding Parkin fused with a fluorescent marker (enhanced yellow fluorescent protein, EYFP) can be used as a fluorescent tag to follow the recruitment of Parkin during the mitophagic process. In order to visualize the mitochondria, we co-transfected pDsRed2-Mito, which encodes red fluorescent protein (DsRed2) that contains a mitochondrial targeting sequence of cytochrome c oxidase subunit VIII (Mito). pDsRed2-Mito is designed for fluorescent labeling of mitochondria14. The time required for Parkin translocation into the mitochondrial membrane can be measured and gives an indirect measure of cellular health. For example, we can say that if a cell line knocked-out for a particular gene of interest shows either a faster or slower recruitment of Parkin after the induction of mitophagy by FCCP, that gene product would be a key player in order to keep the metabolic rates of the cell at the physiological status and prevent the development of diseases. Therefore, the time-lapse video microscopy provides a very powerful tool for both basic and clinical research applications in following the dynamic of labeled proteins during their molecular processes and understanding how these processes are affected during a pathological condition.

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研究方案

1.成纤维细胞的电穿孔用两种表达载体EYFP - 帕金和pDsRed2-美图

  1. 生长在10厘米组织培养板使用的DMEM（Dulbecco氏改进的Eagle培养基）补充有10％胎牛血清，2毫摩尔/升L-谷氨酰胺，100U / ml青霉素介质永生化小鼠胚胎成纤维细胞，和100毫克/毫升链霉素在含有5％的CO 2，在37℃潮湿气氛。
    1. 在80％的细胞汇合，通过无菌吸弃完整的DMEM培养基并加入10毫升的无菌1×磷酸盐缓冲液（PBS）（80克NaCl，2.0g的氯化钾，14.4克Na 2 HPO 4 2.4克KH的2 PO 4和1升蒸馏水2 O，PH值:7.4）。
    2. 无菌吸弃PBS，并加入1毫升胰蛋白酶EDTA 0.25％。在37℃下孵育该板直至细胞被分离（2 - 3分钟）。加入4 ml完全DMEM培养基，重悬细胞，并取出10 _6;细胞悬浮液的血球计数的湖
      注:血细胞计数被设计为使得在一组的16角正方形的细胞数量相当于细胞×10 4个 / ml的数。
  2. 种子1×10 6细胞到10厘米组织培养皿前24小时到电穿孔过程。
  3. 无菌吸弃完整的DMEM中，加入10毫升无菌PBS的。无菌吸弃PBS，并加入1毫升胰蛋白酶EDTA 0.25％。在37℃下孵育该板直至细胞被分离（2 - 3分钟）。加入4 ml完全DMEM培养基和悬浮细胞在一个15毫升管。
  4. 使用冷冻离心机（4℃）纺细胞向下，在250×g离心5分钟。丢弃无菌吸上清，悬浮颗粒在1毫升无菌PBS。旋细胞向下，在250×g离心5分钟，4℃。
  5. 丢弃无菌吸上清，加100微升Ø˚F解决方案组合为电（82微升电解决方案V +18μl的补充解决方案1）的细胞沉淀和移液器轻轻悬浮颗粒（欲了解更多详情，请参阅用户指南）。在细胞悬液中加入2微克EYFP - 帕金（激发/发射527分之514）和1微克pDsRed2-美图（激发/发射六百二十〇分之五百六十五）的。
  6. 转移溶液至用一次性巴斯德灭菌反应杯（这两个工具都随试剂盒提供），使用预先设定的程序的NIH / 3T3的U-030（单脉冲，电压200伏，电容960μF，脉冲时间20毫秒电穿孔，脉冲数:1）。
  7. 电穿孔后，立即在37℃下在含有5％的CO 2的潮湿气氛中加入500μl新鲜预热完全DMEM培养基和种子上将6cm组织培养板活成像同类细胞孵育它们24小时。

2.时间推移​​视频显微镜

    设置在显微镜的腔室在37℃至使用前的温度。
  1. 在这一点上，准备一个特定的实时成像介质进行与实验协议。准备实时成像介质如下;混合DMEM苯酚 - 自由补充有10％胎牛血清，2毫摩尔/升L-谷氨酰胺，100U / ml青霉素，和100毫克/毫升链霉素。在37℃的水浴中预暖实时成像介质。
  2. 丢弃电穿孔的细胞无菌抽吸培养基并加入预热实时成像介质1毫升，使用P1000吸管，并在37℃下孵育该板30分钟。
  3. 在板潜伏期，流入5％的CO 2在显微镜的腔室已经在37℃的稳定温度。
  4. 放置板的电穿孔细胞进入显微镜的房间，避免重大变动或振荡。
  5. 使用软件接口，以便同时检测荧光设置显微镜从由共转染载体编码的融合蛋白刘哲民信号，EYFP-帕金（励磁范围495〜510 nm，发射范围520至550nm，绿色）和pDsRed2-三刀（励磁最大558纳米和最大发射583纳米，红）。
    1. 打开时间推移视频显微镜软件。在上面的菜单中选择EYFP - 帕金和罗丹明对pDsRed2-三刀FITC。在上面的菜单中选择放大倍率（20X）。
  6. 使用软件界面，寻找既表达共转染载体的单细胞和注册的位置。直到最少10个细胞的收集对于每个实验条件（实验组）重复此步骤。
    1. 选择菜单"应用程序"，然后单击多维采集。在多维采集窗口选择所需的参数，如收购的数量，每个采集和记录单元的位置之间的时间间隔。点击"采集"开始采集过程。
  7. 启动基底采集既EYFP-帕金和采集图像每5分钟的15分钟的总时间间隔的DsRed2-三刀荧光信号。
  8. 制备预热实时成像介质用浓度FCCP的比最终工作浓度高两倍（0.1 - 10μM，细胞类型依赖性的）。
  9. 中断采集过程，并添加轻轻地加入1ml用FCCP预热实时成像介质的成用吸管P1000显微镜的室内板。
  10. 如前所述，为3小时的总时间，使用所记录的位置重新开始获取过程。保存所有采集到的图像，以分析它们，当收购是在创建过程mitophagic的视频。

3.分析

  1. 收集的个人计算机上的所有在".TIFF"格式所获取的图像
  2. 与图形软打开图片洁具及定义时间间隔由线粒体引起的去极化与FCCP以显示进入线粒体膜帕金招募的第一个形象出现（图像采集每5分钟）。重复此分析在实验组中的每个小区。
  3. 标签上与实验组名的电子表格的列的第一个单元。对于每个实验组，测量为帕金招募最少10个细胞的的时间间隔。作出帕金招募所计算的时间间隔的平均值，并定义为每个实验组的标准偏差（平均值±SD，N ​​= 10）。
    1. 整理成单个计算时间间隔列。每列包含N =实验组10次测量。
    2. 选择从公式生成器菜单功能"平均"。选择列中的数据。选择从公式生成器菜单功能"标准差"。塞莱克拉列中的数据。
  4. 使用统计方法，比较以确定在实验组之间mitophagy的动态一个显著差施加适当的统计实验组。 （例如，两组=学生t-检验 ，三个或更多个基团= ANOVA加一个事后检验 ）

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结果

这里，我们显示的时间推移的视频显微镜如何是一个强大的技术，可用于遵循荧光标记的蛋白质的分子事件在单个小区。代表结果还表明，该技术是如何允许采集高质量的图像。当分子过程的图象，而得到的，我们有机会来分析他们以不同的方式。这里，我们分析的感应mitophagy过程，这是至关重要的分子过程，维持细胞稳态选择性地除去受损的线粒体的起始之间的时间间?...

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讨论

时间推移显微镜可以被定义为，能在一次观测中时间延长活细胞成像到细胞动力学的过长时间的观察的技术。这种方法是从简单的共焦或活细胞显微镜分辨，因为它允许观察者能够实时识别单个荧光标记的蛋白质，并按照其一个单一的活细胞内的动态。事实上，共焦显微镜可以使用荧光抗体容易识别免疫标记的蛋白质，但它不允许观察细胞，并在其实际环境中的分子事件。

?...

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披露声明

The authors have nothing to disclose.

致谢

This work was supported in part by NIH grants (1R01CA137494, R01CA132115, R01CA086072 to R.G.P.), the Kimmel Cancer Center NIH Cancer Center Core grant P30CA056036 (R.G.P.), a grant from the Breast Cancer Research Foundation, generous grants from the Dr. Ralph and Marian C. Falk Medical Research Trust (R.G.P.) and a grant from the Pennsylvania Department of Health (R.G.P.). In part this work was supported by an American Italian Cancer Foundation postdoctoral fellowship (G.D.) and Bioimaging Shared Resource of the Sidney Kimmel Cancer Center (NCI 5 P30 CA-56036).The Department specifically disclaims responsibility for an analysis, interpretations or conclusions. There are no conflicts of interest associated with this manuscript.

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材料

NameCompanyCatalog NumberComments
DMEMCorning Life Science10-013-CVPre-warm at 37 °C before use
Phenol-free DMEMCorning Life Science17-205-CVPre-warm at 37 °C before use
Fetal Bovine SerumSigma-AldrichF2442Pre-warm at 37 °C before use
L-GlutamineGibco25030Pre-warm at 37 °C before use
Penicillin/streptomycinCorning Life Science30-002-CIPre-warm at 37 °C before use
EYFP-PARKIN expression vectorAddgene23955
pDsRed2-Mito expression vectorClontech632421
Nucleofector 2B deviceLONZAAAD-1001S
Nucleofector for kit R NIH/3T3LONZAVCA-1001
ZEISS AXIOVERT 200M inverted microscopeCARL ZEISS
Carbonyl Cyanide 4-(trifluoromethoxy)-Phenylhydrazone (FCCP)Sigma-AldrichC2920
MetaMorphMolecular DevicesExperimental Builder
ImageJNational Institute of HealthExperimental Builder

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