这项工作得到了埃里森/阿法尔博士后研究员在老龄化研究项目 SJK, 和格伦基金会奖和 NIH 授予 PC (1P01AG034906, 1R01GM 090311, 1R01ES 020812)。所有的作者都出现在影片中。

1. 对细胞的多肽处理 <ol> <li> 板 200万 SH-SY5Y 或 HEK293 细胞 (2 x 10 6 ) 放入10厘米的盘中并长出2天 </li> <li> (可选) 第二天, 用无血清的 Dulbecco 和 #39 清洗细胞; s 修改鹰培养基 (DMEM) 培养基一旦需要在血清自由介质中进行, 可以在一夜之间用血清自由 DMEM. </li>
	<li> 在3天, 溶解 S14G-humanin 肽在0.2 和 #181; m 过滤, 蒸馏水, 并重组为1mM 库存解决方案. 
	注: 多肽应溶于适当的溶剂, 这可以由肽的序列特征 ( 例如 , 总电荷) 来确定, 如果多肽是商用的, 可以由制造商提供。例如, S14G-humanin, SHLP2, 和 SHLP6, 和妙语溶解在水中。在适当的体积 ( 例如 , 50 和 #956; L) 中分多肽, 以避免冻结和解冻循环。一旦解冻, 不要再使用它们. </li>
	<li> 分在50毫升锥形管中5ml 含血清或无血清培养基, 并添加室温1毫米的溶液, 使其进入工作浓度 ( 如 , 1 和 #181; m, 10 和 #181; m). </li>
	<li> 从步骤1.4 中用6毫升肽溶液替换介质. 并在适当的时间内孵育细胞. 
	注意: 根据激活 ERK 的条件选择孵育时间. </li>
</ol> 2。胞和核的亚细胞分馏 <ol> <li> 用10毫升的 ice-cold pbs 清洗两次电池, 在5毫升的 ice-cold pbs 中刮掉板上的细胞, 将细胞悬浮液转化为15毫升的锥形管. </li>
	<li> 离心 500 x g 的细胞5分钟在4和 #176; C. </li>
	<li> 在200和 #181 中吸出 PBS 并 re-suspend 颗粒; ice-cold, 分馏缓冲 (10 毫米 HEPES pH = 7.6, 3 毫米氯化镁 2 , 10 毫米氯化钾, 5% (v/v) 甘油, 1% 非离子表面活性剂 (c 13 h 22 o (c 2 h 4 o) n ), 蛋白酶/磷酸酶抑制剂). 
	注意: 为了保持蛋白质的磷酸化状态, 磷酸酶抑制剂和蛋白酶抑制剂应新鲜添加, 样品应保持在冰上的时刻。应避免重复冻结和解冻样品循环。将样本分为少量 ( 例如 50 和 #956; g), 然后将其冻结在-80 和 #176; C. </li>
	<li> 在冰上孵育悬浮颗粒15分钟, 然后在 250 x g 离心5分钟, 在4和 #176; C. </li>
	<li> 收集上清液为细胞质分数, 并保持颗粒为核分数. </li>
	<li> 离心上清液去除细胞碎片和其他污染物在 1.8万 x g 为10分钟在4和 #176; C 然后转移上清成一个新的离心管。这是胞的分数. </li>
	<li> 重200和 #956 中的颗粒; L ice-cold 洗涤缓冲器 (10 毫米 HEPES pH = 7.6, 1.5 毫米氯化镁 2 , 420 毫米氯化钠, 25% (v/v) 甘油, 0.2 毫米 EDTA, 蛋白酶/磷酸酶抑制剂) 和离心机在 250 x g 为 5 min 在4和 #176; C </li>
	<li> 在100和 #956 中除去上清和重颗粒; L ice-cold, 核萃取缓冲 (20 毫米 HEPES pH = 7.6, 1.5 毫米氯化镁 2 , 420 毫米氯化钠, 25% (v/v) 甘油, 0.2 毫米 EDTA, 蛋白酶/磷酸酶抑制剂) 和几种10次 (5 s on, 十年代关闭, 30% 安培)在冰上. </li>
	<li> 1.8万 x g 离心10分钟在4和 #176; C. </li>
	<li> 将上清液转移到新的离心管。这是核子分数. </li>
	<li> 使用一个免疫分析方法量化蛋白质量 </li> </ol>
 3. 粗的线粒体分数 <ol> <li> 用10毫升的 ice-cold pbs 洗涤每 10 cm 盘中的细胞, 加入5毫升 ice-cold pbs, 并使用细胞刮板分离细胞. 
	注: 使用三10厘米的细胞, 以获得良好的产量线粒体为西方印迹. </li>
	<li> 将电池悬浮液转换为15毫升锥形管, 并将所有从三10厘米的菜肴组合成一个15毫升的锥形管. </li>
	<li> 离心 600 x g 的细胞10分钟在4和 #176; C. </li>
	<li> 吸 PBS 和 re-suspend 1 毫升的 ice-cold 线粒体隔离缓冲 (10 毫米三拖把, 1 毫米 EGTA/三, 200 毫米蔗糖, 调整 pH = 7.4). </li>
	<li> 质25冲程2毫升均质机与聚四氟乙烯涂层杵在冰上的细胞. 
	注意: 这一步是关键的保持线粒体的完整性和最大的收益率的线粒体分数。应针对每个单元格类型优化笔画数。在开始操作前冷匀浆机. </li>
	<li> 将匀浆转移到离心管, 并将其离心于 600 x g, 用于10分钟的4和 #176; C 以除去细胞核和完整的细胞. </li>
	<li> 收集上清液, 转移到一个新的离心管, 并离心 7000 x g 为10分钟, 在4和 #176; C. 
	注: 颗粒松散, 用小心收集上清, 并尽量不扰乱颗粒. </li>
	<li> 去除上清液, re-suspend 200 和 #956 的颗粒; ice-cold 线粒体隔离缓冲, 并将溶液转移到新的离心管。离心管在 7000 x g 为10分钟在4和 #176; C. </li>
	<li> 重复步骤3.8 以清洗颗粒。在这一步中, 没有必要将上清液转移到新的离心管上. </li>
	<li> 从洗涤过的颗粒中去除上清液, 并 re-suspend 50 和 #956 的含有线粒体的颗粒; 帕缓冲. </li>
	<li> 在冰上孵育悬浮液10分钟. </li>
	<li> 离心悬浮在 1.6万 x g 为15分钟在4和 #176; C. </li>
	<li> 将上清液转移到新的离心管。这是线粒体的分数. </li>
	<li> 使用定量检测方法量化蛋白质量. </li>
</ol> 4。磷特异蛋白的西部印迹 <ol> <li> 执行 SDS-聚丙烯酰胺凝胶电泳 (8-16% 预制凝胶) 并将蛋白质转移到 PVDF 膜上 4 。
	<li> 在室温下孵育 5% BSA 的 TBST (0.1% 聚 20), 以阻止背景不特定的结合点. 
	注意: 对于磷酸化蛋白检测, 用 BSA 不加牛奶阻止细胞膜。牛奶含有酪蛋白, 一个丰富的磷, 导致高非特异性信号. </li>
	<li> 在一夜之间用主抗体 (anti-phospho-ERK1/2) 在4和 #176 孵育膜. </li>
	<li> 第二天, 在室温下用 TBST (0.1% 聚 20) 冲洗膜三次, 为5分钟. </li>
	<li> 室温下, 用二次抗体 (兔 HRP) 孵育膜1小时. </li>
	<li> 在室温下用 TBST (0.1% 聚 20) 清洗膜三次, 以5分钟. </li>
	<li> 用 ECL 溶液孵育膜, 并在图像分析仪中对膜进行图像处理. </li>
	<li> 在室温下剥去15分钟的剥离缓冲膜, 用 TBST 三次清洗5分钟. </li>
	<li> 使用 anti-total ERK1/2 抗体重复步骤4.2 到 4.7. </li>
	<li> 检查每个分数的纯度, 运行 SDS 页和执行 immunoblots 使用抗体识别每个舱室的标记 ( 如 , Lamin B1 核, GAPDH 胞, TOM20 线粒体). </li>
</ol>

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Pinchas Cohen 是 CohBar 的股东和顾问, Inc. 是 CohBar 的顾问, Inc。

在这里, 我们证明了 humanin 肽介导的 ERK1/2 活化发生在两种不同的细胞类型, 并根据条件 (例如, 肽的剂量, 时间点和细胞, 活化 ERK1/2 的亚单位的本地化可以是不同的。类型)。它已经表明, humanin 信号通过两个不同的受体22,23, 这可能解释两个细胞线之间的信号的差异, 以及对不同剂量的 humanin 的要求。这方面的生理意义还没有得到充分的研究, 但它可能建议一种可能的机制, 组织依赖反应的。
磷酸化 ERK1/2 在 ERK1/2 信号中起着至关重要的作用, 而 ERK1/2 化状态则表现出动态变化。这是很重要的, 以优化的条件, 以寻找时, ERK1/2 是磷酸化在特定条件/刺激。为了达到这个目的, 尝试不同的刺激条件 (例如, 剂量和培养基), 并进行时间课程, 以找到什么时候最高水平的磷酸化是实现。大多数的研究只集中在 ERK1/2 磷酸化的总细胞裂解。然而, 结果可能提供有限的功能信息, 因为 ERK1/2 可以与不同的目标和调节不同的下游叶栅在特定的亚细胞舱室。因此, 研究磷酸化 ERK1/2 的亚细胞定位能更好地了解 ERK1/2 活化的作用。
ERK1/2 活化后亚细胞分馏可作为进一步研究的来源 (如, co-immunoprecipitation), 以确定新的下游目标, 因为亚细胞分馏可以丰富的下游目标 ERK1/2。这种方法可以应用于其他的信号分子, 被移位到不同的亚细胞室后的刺激条件优化。
尽管在过去的几十年中, 已经提出了各种分离线粒体的方法, 但也有来自其他亚细胞隔离的部分污染。本文提出的粗线粒体分离方法也含有少量的细胞质污染物。由于污染的数量很小, 我们认为它仍然是一种有效的方法, 以检测 ERK1/2 的定位到线粒体激活时。由于肽在溶液中不一定是稳定的, 所以有时很难保持实验间的一致性。确保多肽溶液的小等分, 以避免冻融效果, 提高肽活性的一致性。

新兴研究表明, 线粒体衍生肽 (mdp) 在细胞和新陈代谢中起重要作用1,2,3。了解 mdp 存在下的信号转导通路, 可以让我们洞察 mdp 调节各种功能的机制。第一个确定的 MDP, humanin, 已证明增加细胞外信号调节激酶 1/2 (ERK1/2) 磷酸化通过其受体结合4,5。然而, ERK1/2 活化的下游效应仍 (。
ERK1/2 级联在各种细胞过程中起着重要的中介作用, 包括增殖、细胞迁移、细胞代谢、存活和凋亡6、7、8。为了协调所有这些不同的细胞过程, ERK1/2 的活性和亚型定位受到磷酸和支架蛋白的严格调控9,10。除了修饰修改, ERK1/2 的动态穿梭还调节其信号功能、活动和特异性11,12。ERK1/2 主要在胞13中进行本地化。一组锚和支架蛋白有助于保留 ERK1/2 在骨架元素, 在细胞器表面, 或在细胞质中扩散13。在刺激下, ERK1/2 磷酸化并与它的锚固蛋白分离, 允许 ERK1/2 转移到其他亚细胞室, 包括细胞核、线粒体、高尔基体和溶酶体14,15,16。
虽然 humanin 是已知的激活 ERK1/2 信号通路, ERK1/2 的激活只观察到总细胞裂解。如前所述, 由于 ERK1/2 亚细胞定位在其下游效应中起着至关重要的作用, 对亚细胞定位和磷酸化 ERK1/2 的总水平的分析是必要的, 以提供全面的理解humanin 诱导的 ERK1/2 活化和下游靶点的活化。
为了了解活化 ERK1/2 的靶细胞器, 进行了亚细胞分离, 其次为磷酸化 ERK1/2。这种方法可以很容易地实现, 因为它利用标准的实验室设备和试剂。分离的亚细胞室纯度高, 能使结果得到直截了当的解释。免疫的 ERK1/2 可能产生类似的结果。然而, 某些亚细胞隔间相对来说很难形象化, 需要特殊的固定和性方法。ERK1/2 的水平在亚细胞间不同, 这种变化可能导致假阳性和假阴性信号时, 看整个单元裂解。因此, 一个免疫使用孤立的亚细胞舱室让我们更好地了解 ERK1/2 的本地化。
该方法的通用性, 使该协议的修改, 以调查其他兴奋剂的影响, 包括其他 mdp 或其他信号分子的易位, 如 STAT3。最近发现的小 humanin 样肽 (SHLPs) 是编码从 16S rRNA 地区的 humanin 编码, 他们有相似的, 但独特的属性相比,17。例如, SHLP2 和 SHLP3 激活 ERK1/2 后8小时, 虽然 humanin 激活 ERK1/2 在5分钟内. 研究 ERK1/2 的亚细胞定位对不同肽的反应将使我们更好地了解这些肽的生物学。新的证据表明, 信号分子的亚细胞定位在其下游效应中起着至关重要的作用。例如, STAT3 传统上是已知的主要是在胞的休眠细胞, 然后它 translocates 到细胞核激活基因表达, 以响应细胞因子18。STAT3 也 translocates 线粒体和调节 TCA 循环和 ATP 生产19。关于自噬调控, 不同的亚细胞定位 STAT3 调节自噬的各种方式20。例如, 核 STAT3 转录调节自噬相关基因, 并充当自噬调制器。胞质 STAT3 组成通过与自噬信号分子相互作用抑制吞噬。线粒体 STAT3 抑制和防止 mitophagy 的氧化应激诱导自噬。因此, 这种亚细胞隔离方法对于理解其他信号分子以及 ERK1/2 的作用是至关重要的。

<table><tbody><tr><td>p44/42 MAPK (ERK1/2)</td><td>Cell signaling</td><td>9102</td><td>Dilution 1:1,000</td></tr><tr><td>phospho-p44/42 MAPK (ERK1/2)(Thr202/Tyr204)</td><td>Cell signaling</td><td>4370</td><td>Dilution 1:1,000</td></tr><tr><td>Lamin B1</td><td>Cell signaling</td><td>12586</td><td>Nuclear Marker, Dilution 1:1,000</td></tr><tr><td>GAPDH</td><td>Cell signaling</td><td>5174</td><td>Cytoplasmic marker, Dilution 1:2,000</td></tr><tr><td>Tom20</td><td>Santa cruz</td><td>SC-17764</td><td>Mitochondria marker, Dilution 1:2,000</td></tr><tr><td>anti-Rabbit-HRP conjugated</td><td>Cell signaling</td><td>7074</td><td>Dilution 1:30,000</td></tr><tr><td>RIPA Lysis and Extraction Buffer</td><td>ThermoFisher SCIENTIFIC</td><td>89900</td><td></td></tr><tr><td>100 mm Culture Dish</td><td>ThermoFisher SCIENTIFIC</td><td>12556002</td><td></td></tr><tr><td>HNG peptide</td><td>Genescript</td><td></td><td></td></tr><tr><td>25mm sylinge filter</td><td>ThermoFisher SCIENTIFIC</td><td>09-719A</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>MgCl2</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G9012</td><td></td></tr><tr><td>Triton X-100</td><td>ThermoFisher SCIENTIFIC</td><td>BP151-100</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>3609</td><td></td></tr><tr><td>MOPS</td><td>Sigma</td><td>M1254</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3889</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S7903</td><td></td></tr><tr><td>Tris-base</td><td>ThermoFisher SCIENTIFIC</td><td>BP152-1</td><td></td></tr><tr><td>HCL</td><td>Sigma</td><td>H1758</td><td></td></tr><tr><td>PBS</td><td>Lonza</td><td>17-512F</td><td></td></tr><tr><td>Cell Scraper</td><td>FALCON</td><td>353085</td><td></td></tr><tr><td>Halt&amp;trade; Protease and Phosphatase Inhibitor Cocktail (100X)</td><td>ThermoFisher SCIENTIFIC</td><td>78440</td><td></td></tr><tr><td>Thomas Pestle Tissue Grinder Assemblies with Smooth Pestles</td><td>Thomas Scientific</td><td>3432S90</td><td></td></tr><tr><td>Tween-20</td><td>ThermoFisher SCIENTIFIC</td><td>BP337-500</td><td></td></tr><tr><td>BSA</td><td>ThermoFisher SCIENTIFIC</td><td>BP1600-100</td><td></td></tr><tr><td>8-16% Mini-PROTEAN TGX Precast Protein Gels</td><td>BIO RAD</td><td>4561104</td><td></td></tr><tr><td>Mini Trans-Blot Module</td><td>BIO RAD</td><td>1658030</td><td></td></tr><tr><td>Trans-Blot Turbo Transfer System</td><td>BIO RAD</td><td>1704150</td><td></td></tr><tr><td>Trans-Blot Turbo RTA Mini PVDF Transfer Kit</td><td>BIO RAD</td><td>1704272</td><td></td></tr><tr><td>Clarity Western ECL Blotting Substrates</td><td>BIO RAD</td><td>1705060</td><td></td></tr><tr><td>Restore Western blot stripping buffer</td><td>ThermoFisher SCIENTIFIC</td><td>21059</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>ThermoFisher SCIENTIFIC</td><td>11965-092</td><td></td></tr><tr><td>Sonicator, Medel: FB120</td><td>ThermoFisher SCIENTIFIC</td><td>695320-07-12</td><td></td></tr></tbody></table>

subcellular fractionation for erk activation upon mitochondrial-derived peptide treatment

线粒体衍生肽 (mdp) 是一种新的肽类, 是由小开放阅读框架编码的其他已知基因的线粒体基因组。mdp 具有多种生物效应, 如保护神经细胞不受细胞凋亡, 改善代谢标志物, 以及保护细胞免受化疗。Humanin 是第一个被发现的 MDP, 是 MDP 家族中最受研究的肽。humanin 的膜受体和下游信号通路都经过了细致的表征。更多的 mdp, 如妙语和 SHLP1-6, 最近发现和信号机制尚未阐明。在这里, 我们描述了一种基于细胞培养的方法来确定这些肽的功能。特别是, 细胞分馏技术结合与西方印迹允许定量确定的激活和易位的重要信号分子。虽然还有其他的细胞分离方法, 这里描述的是一个简单明了的方法。这些方法可用于进一步阐明这些肽和其他治疗剂的作用机理。

使用这里介绍的过程, 我们对待 HEK293 和 SH-SY5Y 细胞与1μ m 和100μ m S14G-humanin, 一个强有力的 humanin 模拟21分别, 在完全媒介为被表明的时间期间 (图 1a和图 1 B). 然后, 我们检查了 ERK1/2 在202/Tyr204从总蛋白提取物的总和磷酸化形式。S14G-humanin 治疗显示, 在其监管的202/Tyr204站点上, ERK1/2 磷酸化的增加。ERK1/2 的亚细胞定位在其信号传递功能中起着重要作用, 特别是在获得其特异性方面。为了了解 S14G-humanin-mediated ERK1/2 活化的靶向性, 我们分析了 S14G-humanin 治疗后 ERK1/2 总和磷酸化的亚细胞定位。在 HEK293 细胞中, 磷酸化 ERK 在细胞质和细胞核中均有所增加, 但在线粒体 (图 2A) 中没有。有趣的是, 磷酸化 ERK 在 SH-SY5Y 细胞的细胞质、细胞核和线粒体中下降 (图 2B)。这些结果表明, S14G-humanin-mediated ERK 磷酸化可能在不同的细胞类型和不同的条件下发挥不同的作用。因此, 需要进一步的研究, 以分析 S14G-humanin-mediated 磷酸化的 ERK1/2 在其他亚细胞室, 作为磷酸化 ERK1/2 translocates 到细胞核, 线粒体, 体/溶酶体, 和各种膜。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56496/56496fig1.jpg"> 
图 1:S14G-humanin 增加 HEK293 和 SH-SY5Y 细胞中 ERK1/2 的磷酸化.在 (A) HEK293 和 (B) SH-SY5Y 细胞中, ERK 激活的量化和有代表性的印迹。总细胞裂解以下 (A) 1 µM 或 (B) 100 µM S14G-humanin 治疗 DMEM 补充与 10% FBS 的指定时间段 immunoblotted 使用 anti-phospho-和总 ERK 1/2 (Thr202/Tyr204)。此图已从 Kim et al.中修改4 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56496/56496fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56496/56496fig2.jpg"> 
图 2:磷酸化 ERK1/2 在 HEK293 和 SH-SY5Y 细胞中 S14G-humanin 治疗后, 被定位到胞、细胞核和其他亚单位.有代表性的西方印迹显示 ERK1/2 亚细胞定位 (n = 3)。(A) HEK293 细胞用1µM S14G-humanin 治疗。胞浆 (15 µg), 核 (15 µg 和50µg), 线粒体 (15 微克) 裂解 immunoblotted 使用磷和总 ERK 抗体。(B) 用100µM S14G-humanin 治疗 SY5Y 细胞。S14G-humanin 治疗后获得30分钟的线粒体分数。胞浆, 核和线粒体 (15 µg) 裂解 immunoblotted 使用磷和总 ERK 抗体。抗 GAPDH、抗 Lamin B、anti-Tom20 抗体分别用作胞浆、核、线粒体分数标记。此图已从 Kim et al.中修改4.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56496/56496fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Table 1" src="/files/ftp_upload/56496/56496table1v2.jpg"> 
表 1:胞、核和线粒体分数的缓冲.

Watch this Scientific Journal Video about Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment at JoVE.com

Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

该协议描述了如何刺激细胞线粒体衍生肽, 并评估信号级联和定位的磷蛋白。

线粒体源肽治疗中 ERK 活化的亚细胞分离

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the ...

subcellular-fractionation-for-erk-activation-upon-mitochondrial

Nanyang Technological University

Research

JoVE Journal

Biology

7.8K 次观看.  University of Southern California. 该协议描述了如何刺激细胞线粒体衍生肽, 并评估信号级联和定位的磷蛋白。

视频: Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

用放射性标记的ATP测定蛋白激酶活性

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

科研

生物化学

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

在人类胚胎干细胞的线粒体复杂的活动初探

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

生物学

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

在Active ERK的时空分析<em&gt;℃。线虫</em&gt;生殖系

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

发育生物学

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

高速离心条件下 u937 细胞的细胞分裂

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

萌芽酵母酿酒酵母中软骨蛋白定位的评估

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

对缝隙连接检查功能测定;贴壁细胞的氧化铟锡电

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

实验方法来研究线粒体定位和核细胞周期激酶CDK1功能

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

用于分析神经科学中线粒体网络的 MATLAB 优化

Optimized Automated Analysis of Live Neuronal Mitochondria Homeostasis Modulation by Isoform-Specific Retinoic Acid Receptors

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in timelapse files, considerably simplifying and speeding up the process of image processing. Nonetheless, existing tools produce a large output volume, requiring individual manual attention, and basic experimental setups have an output of thousands of files, each requiring extensive and time-consuming handling. 
To address these issues, a routine optimization was developed, in both MATLAB code and live-script forms, allowing for swift file analysis and significantly reducing document reading and data processing. With a speed of 100 files/min, the optimization allows an overall rapid analysis. The optimization achieves the results output by averaging frame-specific data for individual mitochondria throughout time frames, analyzing data in a defined manner, consistent with those output from existing tools. Live confocal imaging was performed using the dye tetramethylrhodamine methyl ester, and the routine optimization was validated by treating neuronal cells with retinoic acid receptor (RAR) agonists, whose effects on neuronal mitochondria are established in the literature. The results were consistent with the literature and allowed further characterization of mitochondrial network behavior in response to isoform-specific RAR modulation. 
This new methodology allowed rapid and validated characterization of whole-neuron mitochondria network, but it also allows for differentiation between axon and cell body mitochondria, an essential feature to apply in the neuroscience field. Moreover, this protocol can be applied to experiments using fast-acting treatments, allowing the imaging of the same cells before and after treatments, transcending the field of neuroscience.

作者聚焦:研究神经元线粒体中视黄酸受体的优化自动化方法

The mitochondrial network is extremely complex, making it very challenging to analyze. A novel MATLAB tool analyzes live confocal imaged mitochondria in timelapse images but results in a large output volume requiring individual manual attention. To address this issue, a routine optimization was developed, allowing for speedy file analysis.

通过亚型特异性视黄酸受体对活神经元线粒体稳态调节进行优化的自动分析

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in ...

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通过亚型特异性视黄酸受体对活神经元线粒体稳态调节进行优化的自动分析

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