长期活细胞成像对紫杉醇反应细胞命运的评估
Summary
活细胞成像提供了丰富的信息, 单个细胞或整个人口, 是无法实现的固定细胞成像单独。这里描述了用抗有丝分裂药物紫杉醇治疗后细胞命运决定的活细胞成像协议。
Abstract
活细胞成像是一种强大的技术, 可以用来直接可视化的生物现象在单个细胞的长期时间。在过去的十年中, 新的和创新的技术大大提高了活细胞成像的实用性。在 37 °C 和 5% CO2单元格培养条件下, 单元格现在可以保持焦点并连续数天进行映像。此外, 可以同时获取多个表示不同实验条件的视场, 从而提供高通量的实验数据。活细胞成像通过允许直接可视化和时间定量的动态细胞事件提供了一个显著的优势比固定细胞成像。活体细胞成像还可以识别在使用基于人口的化验方法时可能遗漏的单个细胞行为的变化。在这里, 我们描述活细胞成像协议, 以评估细胞命运决定后, 抗有丝分裂药物紫杉醇治疗。我们展示的方法, 以可视化的 mitotically 被逮捕的细胞直接死于有丝分裂或滑回之间的界面。我们还描述了如何使用荧光泛的细胞周期指示器 (FUCCI) 系统来评估产生于有丝分裂滑移的周期性细胞的分数, 能够重新进入细胞周期。最后, 我们描述了一种用于识别核包络破裂事件的活细胞成像方法。
Introduction
抗有丝分裂药物长期用于各种类型的实体肿瘤的化疗方案, 并经常显示出很大的功效1,2,3。机械化, 这些药物扰乱正常的有丝分裂进展, 并促进有丝分裂在迅速增殖的癌细胞的抑制。然而, 细胞的命运, 对有丝分裂的反应是高度可变的: 虽然细胞的一小部分经历细胞死亡直接从有丝分裂, 其他退出有丝分裂和返回到间期作为二、四倍体杂交细胞 (一个过程称为有丝分裂滑移)4, 5,6,7,8。这些界面细胞可以执行细胞凋亡, 接受永久性细胞周期骤停, 甚至重新进入细胞周期4,5,6,7,8,9 ,10,11。细胞通过滑入间期而逃避有丝分裂细胞死亡, 只会在药物去除后重新进入细胞周期, 因此可能有助于癌细胞的重新出现。此外, 从有丝分裂中滑出的细胞是二、四倍体杂交的, tetraploidy 被称为促进染色体不稳定, 驱动肿瘤复发12, 13, 14, 15.因此, 确定控制细胞命运的因素对抗有丝分裂药物治疗是至关重要的, 以优化目前的治疗方法。
在本议定书中, 我们描述的方法, 以直接观察和研究细胞的命运, 经过长期的有丝分裂, 以回应抗有丝分裂药物紫杉醇。紫杉醇是临床上行之有效的治疗, 在许多肿瘤类型, 包括乳房, 卵巢和肺部, 已证明是非常有效的16,17,18,19, 20. 紫杉醇是从红豆杉树皮中提取的植物生物碱, 稳定微管, 从而防止其动态性 21, 22.虽然紫杉醇抑制微管动力学不会影响从 g1到 g 2 的细胞周期进展,但该药物确实导致在有丝分裂期间主轴组件检查点的持续激活, 阻碍动粒微管附件 (此处深度查看 23, 24) 25.因此, 在紫杉醇治疗的细胞中防止后期发作, 并导致长期的有丝分裂停止。
本议定书将首先描述在活体细胞成像实验中识别有丝分裂细胞的方法。由于两种明显的细胞生物学变化, 有丝分裂可以在黏附组织培养细胞中可视化。首先, 染色体在核包络击穿前立即变得高度浓缩。虽然通常可以通过标准的相衬显微镜检测到染色体凝结, 但使用标记染色体的荧光标记 (例如荧光标记的组蛋白) 能更清楚地发现。其次, 有丝分裂细胞也可以通过细胞四舍五入所产生的戏剧性形态学变化来识别。
该协议将演示如何使用活细胞成像方法来跟踪发生长期有丝分裂骤停的细胞的命运。在有丝分裂中被捕的细胞经历了三种截然不同的命运。首先, 细胞在有丝分裂过程中会发生细胞死亡。这种现象很容易被光镜所形象化, 因为死亡细胞被观察到收缩、疱疹和/或破裂。第二, 细胞可以从有丝分裂中退出, 回到无染色体分离或胞质分裂的间期, 这一过程称为有丝分裂滑移。染色体的 decondensation 和/或有丝分裂细胞的扁平化容易识别这个过程。从有丝分裂中滑出的细胞也经常显示不规则的, 多裂的细胞核, 并且经常有几个微核的5。第三, 在有丝分裂中被拘捕的细胞可以在长时间延迟后进行有丝分裂, 并进行减数分裂。虽然不常见的较高药物浓度, 这种行为表明, 被逮捕的细胞可能已满足主轴装配检查点, 或主轴装配检查点部分减弱或缺陷。染色体分离和后续胞质分裂使用活细胞成像可以可视化后期发病。
还将描述活细胞成像方法, 以跟踪细胞的命运, 以避免有丝分裂细胞死亡的有丝分裂延迟。发生有丝分裂滑移的细胞在随后的界面中死亡, 触发持久性 G1细胞周期的停止, 或重新进入细胞周期以启动新一轮细胞分裂4。使用 FUCCI (荧光泛细胞周期指示器) 系统来确定细胞分裂后重新进入细胞周期的分数将被描述。FUCCI 允许直接可视化 G1/S 转换, 并且可以与长期活细胞成像同时使用, 这两个体外和在体内26,27。FUCCI 系统利用两个荧光标记的蛋白质, 截断形式的 hCdt1 (染色质许可和 DNA 复制因子 1) 和 hGeminin, 其水平振荡的基础上细胞周期的位置。在 G1阶段, hCdt1 (融合到红色荧光蛋白) 在高级别存在, 它的作用是许可 DNA 进行复制, 但泛素化 E3 泛素连接酶超临界 Skp2 并在 S/G 2/M 阶段降级, 以防止重新复制 DNA26。相比之下, hGeminin (融合到绿色荧光蛋白), 是 hCdt1 的抑制剂, 其水平峰值在 S/G2/M, 但泛素化由 E3 泛素连接酶APC Cdh1 和退化在有丝分裂结束和整个G 126. 因此, FUCCI 提供了细胞周期阶段的直接荧光读数, 因为细胞在 G1,和绿色荧光期间在 S/G2/m 期间呈现红色荧光。FUCCI 系统是一个重要的进步, 超过其他方法 (如 bromodeoxyuridine 染色), 以确定增生细胞, 因为它不需要细胞固定, 并允许单细胞成像不需要额外的药理同步细胞群的治疗方法。尽管在本协议中没有讨论, 还开发了额外的活细胞传感器来可视化细胞周期进展, 包括 G128的旋 B 传感器、DNA 连接酶-RFP 29 和 PCDNA-GFP 30 传感器 对于 s-相位和最近的 FUCCI-4 传感器, 它检测单元周期的所有阶段31。
最后介绍了一种检测核包络破裂的活细胞成像方法。最近的研究表明, 癌细胞的核包不稳定, 容易爆裂, 从而使 nucleoplasm 和细胞质的内容混合。这种现象, 称为核破裂, 可以促进 DNA 损伤和刺激先天免疫应答32,33,34,35,36,37,38,39,40,41,42,43,44. 虽然核破裂的根本原因仍然不完整, 但众所周知, 核结构的变形与核破裂的发生率42的增加有关联。紫杉醇治疗的一个众所周知的作用是在有丝分裂后产生惊人的异常核结构;因此, 用活细胞成像方法来量化核破裂将被描述, 同时也探讨紫杉醇治疗是否增加核破裂事件的频率。核破裂可以通过观察到的核靶荧光蛋白泄漏到细胞质中来检测 (例如与核定位信号融合的 RFP 串联二聚体重复 TDRFP)。这种渗漏是明显可见的眼睛, 这使得简单的定量的破裂事件。
该协议需要一个 widefield 荧光显微镜, 配备了编码阶段和调焦软件。编码阶段允许精确的自动移动到定义的 X Y 坐标, 而自动对焦软件在成像周期的持续时间内维护焦点的单元格。此外, 该协议要求设备保持37°c 的细胞与湿润 5% CO2大气。这可以通过在温度和空气控制的外壳内封闭整个显微镜, 或者使用在当地保持温度和环境的舞台顶部设备来实现。该协议中使用的相对比目标是一个0.30 的数值孔径的计划。然而, 20X 目标也足以在单个焦平面中识别圆形有丝分裂和扁平化的间相细胞。如果执行相对比成像 (如本方法所述), 盖子可以是玻璃或塑料。如果使用差动干涉造影 (DIC) 显微镜, 必须使用玻璃罩防止光的退极化。
Protocol
Representative Results
Discussion
长期活细胞成像的最关键的方面是确保成像细胞的健康。必须让细胞接触到极小的外部环境压力源, 例如温度、湿度和/或 CO2级的不合格条件。同样重要的是, 限制光损伤的荧光激发照明, 因为成像应力是已知的影响细胞行为的 50.限制光损伤可以以多种方式实现, 如在其他48中详细说明.一般而言, 最好使用最短的曝光时间来产生足够明亮的荧光信号, 从而?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
AFB、微型飞行器和 NJG 希望感谢瑞安昆顿对《 TDRFP-NLS 》的手稿和艾利安 Salic 的评论。AFB 和微型飞行器由研究院生物分子药理训练补助金5T32GM008541 资助。NJG 是沙米姆·卡齐和阿什拉夫 Dahod 乳腺癌研究实验室的成员, 并得到 NIH 赠款 GM117150 和 CA-154531、凯琳 Grunebaum 基金会、史密斯家族奖计划、塞尔学者计划和黑色素瘤研究的支持。联盟。
Materials
| phenol red-free, DMEM:F12 media | Hyclone | SH3027202 | |
| 10% fetal bovine serum (FBS) | Gibco | 10438026 | |
| 100 IU/ml penicillin, and 100 mg/ml streptomycin. | Gibco | 15070063 | |
| PBS | Gibco | 10010049 | |
| 0.25% Trypsin/EDTA | Gemini | 50-753-3104 | |
| 12-well glass bottomed imaging dish | Cellvis | P12-1.5H-N | |
| Paclitaxel | TSZ Chem | RS036 | |
| Dimethyl Sulfoxide (DMSO) | Sigma | NC0215085 | |
| Environmental Chamber | In Vivo Scientific | ||
| 5% CO2 | Airgas | Z03NI7432000201 | |
| 15 mL Conical Tubes | Fisherbrand | 05-539-12 | |
| 10 cm polystyrene tissue culture plates | Falcon | 08772E | |
| 10 mL Disposable Serological Pipette | Fisherbrand | 4488 | |
| 5804R 15 amp Centrifuge | Eppendorf | 97007-370 | |
| 1000 microliters Pipette | Gilson | Pipetman Classic | |
| 20 microliters Pipette | Gilson | Pipetman Classic | |
| p1000 Pipette Tips | Sharp | p1126 | |
| p20 Pipette Tips | Sharp | P1121 | |
| RPE-1 Cells | ATCC | CRL-4000 | |
| Incubator Galaxy 170S | Eppendorf | CO17011005 | |
| Pipette Boy | Integra | 156401 | |
| Hemacytometer | Fisher Scientific | 0267110 | |
| Nikon Ti-E-PFS Inverted Microscope for Live Cell Imaging | Micro Video Instruments, Inc. | MEA53100 | |
| Prior X-Y Stage System and White Light LED Illuminator | Micro Video Instruments, Inc. | 500-H117P1N4 | |
| Prior Proscan 3 Controller XYZ with Encoders | Micro Video Instruments, Inc. | 500-H31XYZE | |
| Andor Digital Camera | Micro Video Instruments, Inc. | D10NLC | |
| Nikon NIS Elements AR Software | Micro Video Instruments, Inc. | MQS31000 | |
| SOLA LED Illuminator for Fluorescence | Micro Video Instruments, Inc. | SOLA-E | |
| InVivo Environmental Chamber with CO2 Flow Control | Micro Video Instruments, Inc. | Ti-IVS-100 | |
| Ti-ND6-PFS Perfect Focus Motorized Nosepiece | Micro Video Instruments, Inc. | MEP59391 | |
| Ti-C System Condenser Turret | Micro Video Instruments, Inc. | MEL51000 | |
| Ti-C LWD LWD Lens Unit for System Condenser Turret | Micro Video Instruments, Inc. | MEL56200 | |
| Te-C LWD Ph1 Module | Micro Video Instruments, Inc. | MEH41100 | |
| CFI Plan Fluor DLL 10X Objectivena 0.3 wd 16mm | Micro Video Instruments, Inc. | MRH10101 | |
| CFI Super Plan Fluor ELWD 20xc ADM Objective | Micro Video Instruments, Inc. | MRH48230 |
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