对急性机械刺激的网柄反应的评估
Summary
在这里, 我们描述了评估急性机械刺激的细胞反应的方法。在 microscopy-based 试验中, 我们研究了荧光标记的生物传感器的局限性, 并对剪切流进行了简单的刺激。我们还测试了各种感兴趣的蛋白质的活化反应的急性机械刺激生化。
Abstract
趋化性, 或迁移上升的梯度, 是最了解的定向迁移模式。研究使用社会阿米巴虫网柄显示, 一个复杂的信号转导网络的平行路径放大的反应, 化, 并导致有偏见的肌动蛋白聚合和突起的伪足在渐变的方向。相比之下, 其他类型的定向迁移的分子机制, 例如, 由于接触剪切流或电场, 是未知的。许多趋化性的调控者在迁移细胞的前缘或滞后边缘表现出定位, 以及在全球刺激与趋化因子的作用下, 显示局部或活化的瞬态变化。为了了解其他类型的定向迁移的分子机制, 我们开发了一种方法, 允许在短 (2-5 秒) 接触剪切流的基础上检测细胞对急性机械刺激的反应。这种刺激可以提供一个通道, 而成像细胞表达荧光标记的生物传感器, 以检查个别细胞的行为。此外, 细胞的数量可以刺激在一个板块, 裂解, 和 immunoblotted 使用抗体, 识别活性蛋白的兴趣。通过结合这两种化验, 你可以检查一个广泛的分子组的变化激活的亚细胞定位和/或磷酸化。使用这种方法, 我们确定急性机械刺激触发激活的趋信号转导和肌动蛋白细胞骨架网络。检测细胞对急性机械刺激的反应的能力对于理解剪切流诱导运动所需的启动事件是非常重要的。该方法还为研究趋信号转导网络提供了工具, 而不影响趋化因子受体的混杂作用。
Introduction
真核细胞的迁移在环境中有不同的化学和物理暗示, 包括可溶解的或基质结合的化的梯度, 基板的变刚度, 电场, 或剪切流。虽然我们对推动趋化的分子机制的理解有许多进展, 但对于其他类型的定向迁移以及这些不同的信号是如何整合在细胞层面以产生统一的迁徙响应.
定向迁移向日益集中的趋化因子涉及三行为成分: 运动, 定向传感, 和极性1。运动是指由伪足突起所实现的细胞的随机运动。定向传感是细胞检测趋化因子的来源的能力, 即使在固定细胞中也能发生。极性是指在细胞的前缘和后缘之间更稳定的胞内成分的不对称分布, 从而导致运动中的持续性增加。
细胞趋化反应取决于四概念上定义的调控网络的活动: 受体/G 蛋白, 信号转导, 肌动蛋白骨架, 和极性1。趋化与 g 蛋白偶联受体结合, 通过聚 g 蛋白α和βγ向下游信号转导网络传递信号, 从而放大方向性信号。信号传导网络中的多个通路在平行的作用下, 进入肌动蛋白骨架网络, 以偏压肌动蛋白聚合, 并随之伪足突起, 在梯度方向。在趋化的重要调控者中有 Ras 酶、TorC2、肌3激酶 (PI3K)、磷酸酶和张力同源 (PTEN) 和 guanylyl 苷。信号传导网络中的反馈机制以及信号转导与肌动蛋白细胞骨架网络之间的联系进一步放大了反应。最后, 定义差的极性网络接收来自肌动蛋白骨架的输入, 并进一步偏向信号转导网络, 以促进梯度方向的持续迁移。
我们对趋化性的许多机械理解之所以成为可能, 是因为开发了荧光标签的生物传感器, 用于监管网络的各个组成部分。许多趋化调控分子本身或其活动的不对称分布。例如, 能够识别小酶 ras 和 Rap1 的激活版本的生物传感器 (此处称为 RBD) 和 RalGDS 的 ras 绑定域, 分别定位到 chemotaxing 单元的前导边缘2,3。同样, PI3K 及其产品醇 (34、5)-trisphosphate (PIP3), 由 pleckstrin 同源 (PH) 域识别, 也显示在单元格45前面的本地化。相比之下, 3-磷酸酶 PTEN, 它将 PIP3 回醇 (45)–1,6-, 本地化的滞后边缘的细胞6。重要的是, 这些生物传感器改变他们的本地化响应全球刺激与趋化。领先的边缘标记, 这是胞浆或在休息细胞突起的提示, relocalize 到皮层, 而滞后的边缘标记, 其中有皮质定位, 并没有从突起的提示在休息细胞, 成为胞浆后刺激.生物传感器在全球刺激作用下的分布分析最小化运动和极性的贡献, 这往往混淆的意见。全球或统一的刺激细胞悬浮与趋化因子也被用来作为一个工具, 以评估整个种群的蛋白质活化变化, 通常检测蛋白磷酸化7,8,9.这种生化检测主要用于获取时间信息, 而显微镜则用于收集有关监管网络各组成部分行为的时间和空间信息。
信号转导网络包含易激动的系统的特征10,11。对超阈值趋刺激的反应是 “全有-无” 和显示耐火期。响应也会随机触发, 并且可以显示振荡行为。信号转导事件是本地化的皮层的区域, 传播为波12,13,14,15。前面, 或后面, 标记被招募, 或脱离, 活动区域的传播波。由于在活动区尾部的耐火区域, 相反的定向波湮灭, 因为他们满足。传播信号传导波的基础上的细胞突起, 调解细胞迁移10。
上述关于趋化性的许多信息来自于对社会阿米巴的研究网柄, 虽然类似的调节机制也适用于中性粒细胞和其他哺乳动物细胞类型16 .网是一个成熟的模型生物体, 在饥饿时有一个强健的趋反应, 当数以千计的单细胞向聚集中心迁移时, 最终形成一个含有孢子的多细胞子实体。在这个有机体的单细胞生长阶段, 趋化性也是至关重要的, 用于定位细菌的食物来源。重要的是, 单个网细胞的迁移非常类似于哺乳动物中性粒细胞或转移性癌细胞的迁移, 所有这些都经历了非常迅速的变形型迁移。事实上, 在网和哺乳动物白细胞17之间, 调控网络的整体拓扑以及参与趋化的各个信号转导通路都是守恒的。此外, 其他细胞, 如成纤维蛋白, 使用受体酪氨酸激酶 (RTK) 而不是受体;然而, 受体可能会进入类似的网络。
与趋化性相反, 对驱动各种其他定向迁移模式的信号机制的透彻理解是缺乏的。类似于在趋化梯度渐变中迁移的细胞, 一些研究报告了典型的前缘标记的活化和/或定位, 包括肌动蛋白聚合、PIP3 和/或胞外信号调节激酶 (ERK) 1/2, 在在响应剪切流或电场变化的情况下接受定向偏移的单元格前面18,19,20,21。然而, 在这些研究中, 持续接触刺激物也会导致细胞迁移, 这就导致了一个问题, 例如, 领先的边缘标记是否专门针对刺激的反应而定位, 或者它们是否只是在前沿因为迁移单元前面的 pseudopods 数量增加。
我们开发的化验, 让我们观察细胞对急性机械摄动的反应, 作为剪切流在人口水平和作为单个细胞22。类似于全球刺激与趋化, 急性 stimula剪切流允许研究的细胞反应的机械刺激, 而不是混杂的贡献, 从运动或极性。结合这些生物化学和微观的检测与遗传或药理的扰动, 让我们了解如何机械刺激的感知和传播。此外, 这种方法还提供了一种新的方法, 以攻入系统下游的趋化因子受体在没有一个趋化因子, 从而隔离信号转导和肌动蛋白骨架网络从受体/G蛋白质网络。
使用下面描述的技术, 我们最近证明, 急性剪应力导致趋信号转导和肌动蛋白骨架网络的多个组成部分的激活22。通过在不同的时间间隔应用急性机械刺激, 我们证明, 类似于化, 对机械刺激的反应也表现出兴奋系统的特征, 包括在饱和条件和不耐期的存在。最后, 通过结合机械和化学刺激, 我们发现, 这两个刺激共享信号转导和肌动蛋白细胞骨架网络, 这可能允许集成的多重刺激的偏见细胞迁移。
Protocol
Representative Results
Discussion
这里描述的方法提供了一个方便的方法来评估人口和个别细胞的行为, 以响应剪切流。重要的是, 虽然以前的研究分析了在迁移过程中领先和滞后边缘标记的定位, 但目前的方法可以通过应用切变流来研究直接的影响。使用这种方法我们证明, 事实上, 细胞对切变流的初始响应不需要细胞迁移。相反, 对初始切变流刺激的快速和瞬态响应之前, 长期暴露于切变流的方向迁移, 类似于初始的全球响应, 以?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生研究院的资助, R35 GM118177 PND。
Materials
| Reagents | |||
| Dextrose | Fisher | D16-1 | Use to prepare HL-5 media and SM plates (steps 1.1, 1.2) |
| Proteose peptone | Fisher | DF0120-17-6 | Use to prepare HL-5 media (step 1.1) |
| Yeast extract | Fisher | 50-550-445 | Use to prepare HL-5 media and SM plates (steps 1.1, 1.2) |
| Na2HPO4·7H2O | Fisher | S373-500 | Use to prepare HL-5 media, and 10X Phosphate Buffer (steps 1.1, 1.3) |
| KH2PO4 | Fisher | P386-500 | Use to prepare HL-5 media, SM plates, and 10X Phosphate Buffer (steps 1.1-1.3) |
| Streptomycin (dihydrostreptomycin sulfate) | Fisher | ICN10040525 | Use to prepare HL-5 media (step 1.1) |
| Peptone (Bacto) | Fisher | DF0118-17-0 | Use to prepare SM plates (step 1.2) |
| K2HPO4 | Fisher | P288-100 | Use to prepare SM plates (step 1.2) |
| Agar | Fisher | BP1423-500 | Use to prepare SM plates (step 1.2) |
| MgSO4 | Fisher | M65-500 | Use to prepare DB Buffer (step 1.4) |
| CaCl2 | Fisher | C79-500 | Use to prepare DB Buffer (step 1.4) |
| Caffeine | Fisher | O1728-500 | Use to prepare caffeine (step 1.5) |
| cAMP (EMD Millipore Calbiochem Adenosine 3 ft.,5 ft.-cyclic Monophosphate, Sodium Salt) | Fisher | 11-680-1100MG | Use to prepare cAMP (step 1.6) |
| Folic acid | Sigma | F7876 | Use to prepare folic acid (step 1.7) |
| Tris base | Fisher | BP152-500 | Use to prepare 3X sample buffer (step 1.8) |
| Sodium dodecyl sulfate (SDS) | Fisher | BP166-500 | Use to prepare 3X sample buffer (step 1.8) |
| Glycerol | Fisher | G33-500 | Use to prepare 3X sample buffer (step 1.8) |
| Dithiothreitol (DTT) | Bio-Rad | 1610610 | Use to prepare 3X sample buffer (step 1.8) |
| Bromophenol blue | Bio-Rad | 1610404 | Use to prepare 3X sample buffer (step 1.8) |
| NaF | Fisher | S299-100 | Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8) |
| Na3VO4 | Fisher | 50-994-911 | Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8) |
| Sodium pyrophosphate decahydrate | Fisher | S390-500 | Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8) |
| Complete EDTA-free protease inhibitor cocktail (Roche) | Sigma | 11873580001 | Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8) |
| Latrunculin A | Enzo Life Sciences | BML-T119-0100 | Use to prepare Latrunculin A (step 1.9) |
| 4-15% Tris-HCl polyacrylamide gel | Bio-Rad | 3450029 | Use for immunoblotting (step 3.3) |
| Polyvinylidene fluoride membrane | Bio-Rad | 1620177 | Use for immunoblotting (step 3.3) |
| Phospho-PKC (pan) (zeta Thr410) (190D10) Rabbit antibody | Cell Signaling | 2060 | Primary antibody for use in immunoblotting (step 3.3) |
| Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP antibody | Cell Signaling | 4370 | Primary antibody for use in immunoblotting (step 3.3) |
| Rabbit IgG HRP Linked Whole Ab (from Donkey) | GE Healthcare | NA934-100UL | Secondary antibody for use in immunoblotting (step 3.3) |
| Enhanced chemiluminescence substrate (Clarity Western ECL Substrate) | Bio-Rad | 1705060 | Use for immunoblotting (step 3.3) |
| Stripping buffer (Restore Plus Western blot stripping buffer) | Pierce | PI46430 | Use for immunoblotting (step 3.3) |
| Coomassie Brilliant Blue | Fisher | PI20278 | Use for immunoblotting (step 3.3) |
| K. aerogenes strain (non-pathogenic) | Dicty Stock Center (dictybase.org) | ||
| Name | Company | Catalog Number | Comments |
| Materials and Equipment | |||
| Orbital shaker (model G-33, or a comparable alternative) | New Brunswick Scientific | Use to grow K. aerogenes (step 2.1.1), and to basalate and stimulate D. discoideum cells (step 3). The radius of gyration for this shaker is 9.5 mm. | |
| 10 cm Petri dish | Fisher | FB0875712 | Use to prepare SM plates (step 1.2) |
| Hemocytometer | Fisher | 02-671-51B | Use to count D. discoideum cells (step 2.1.2) |
| 35 mm dish (Corning Falcon Easy-Grip Tissue Culture Dish) | Fisher | 08-772A | Use to plate D. discoideumcells for mechanical stimulation followed by cell lysis (step 3.1.1) |
| Polystyrene cup (5 mL) | VWR | 13915-985 | Use to stimulate D. discoideum cell suspension with a chemoattractant (step 3.2.2) |
| Fluidic unit with pump (ibidi Pump System) | Ibidi | 10902 | Use to deliver mechanical stimulation to D. discoideum cells in a channel (steps 4.1, 4.2, and 5) |
| Slide with a channel (μ-Slide III 3in1 ibiTreat: #1.5 polymer coverslip, tissue culture treated, sterilized) | Ibidi | 80316 | Use to plate D. discoideum cells for mechanical stimulation delivered by a fluidic device (steps 4.1, 4.2, and 5) |
| 50 mL reservoirs (Ibidi Reservoir Holder for Fluidic Unit) | Ibidi | 10978 | Use to setup the fluidic device for delivering mechanical stimulation to D. discoideum cells in a channel (steps 4.1, 4.2, and 5) |
| Lines for the fluidic unit (Perfusion Set YELLOW-and-GREEN) | Ibidi | 10964 | Use to setup the fluidic device for delivering mechanical stimulation to D. discoideum cells in a channel (steps 4.1, 4.2, and 5). Note: the two lines can also be cut from the 1.6 mm tubing (catalog number 10842). |
| Line for the drain (1.6 mm ID tubing) | Ibidi | 10842 | Use to setup the fluidic device for delivering mechanical stimulation to D. discoideum cells in a channel (steps 4.1, 4.2, and 5). |
| Zeiss Observer.Z1 inverted microscope equipped with a 40X/1.3 oil objective and a 20X/0.3 air objective (or a comparable alternative) | Zeiss | Used to image D. discoideum cells with phase-contrast or fluorescence illumination (steps 4 and 5) | |
| UltraView spinning disk confocal microscope equipped with a 40×/1.25–0.75 oil objective (or a comparable alternative) | Perkin-Elmer | DM 16000 | An alternative used to image D. discoideum cells (step 4.1.4) |
| FemtoJet Microinjector (Eppendorf; model 5247, or a comparable alternative) with Eppendorf InjectMan | Eppendorf | 5252000021D and 5192000027 | Use to deliver mechanical stimulation to D. discoideum cells by a micropipette (step 4.3). Note: catalog number are for the newest version of the equipment (4i). |
| Micropipette (Femtotip; 1 μm outside diameter, 0.5 μm inside diameter ) | Eppendorf | 930000035 | Use to deliver mechanical stimulation to D. discoideum cells by a micropipette (step 4.3) |
| Microloader tips (Eppendorf Femtotips Microloader Tips for Femtojet Microinjector) | Eppendorf | 5242956.003 | Use to fill the micropipette (femtotip) with buffer (step 4.3.1) |
| 1-well chamber (Thermo Scientific Nunc Lab-Tek Chambered Coverglass) | Fisher | 12-565-472 | Use to plate D. discoideum cells for mechanical stimulation delivered by a micropipette (step 4.3.2) |
| 8-well chamber (Thermo Scientific Nunc Lab-Tek II Chambered Coverglass) | Fisher | 12-565-338 | Use to plate D. discoideum cells for mechanical stimulation delivered by bulk buffer addition (step 4.4.1) |
| Name | Company | Catalog Number | Comments |
| Software | |||
| Image Analysis Software (Fiji) | NIH | https://fiji.sc/ | Use to quantify response to mechanical stimulation (step 4.5) |
| Migration analysis software (Tracking Tool PRO) | Gradientech | http://gradientech.se/tracking-tool-pro/ | Use to quantify cell speed and persistence (step 5.4) |
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