这项工作得到伊利诺伊大学厄瓜多尔香槟分校（UIUC）和美国国立卫生研究院（NIGMS R01GM111816）的支持。

动物研究是按照伊利诺斯机构动物保护和使用委员会（IACUC）和伊利诺伊州动物资源大学（DAR）制定的指导方针进行的。 蛋白定位在BHK21哺乳动物细胞培养中的光学诱导注意:步骤1.1-1.3提供了一种组装细胞培养室，用于通常具有较短工作距离的高倍率物镜（ 例如， 63X或100X）进行成像的方法。这些目标需要薄玻璃盖玻片（ 例如， ＃1.5,170μm厚度）作为成像基底。或者，可以使用玻璃底细胞培养皿/载玻片。在这种情况下，可以跳过步骤1.1-1.3。 <ol><li> 清洁玻璃盖玻片 <ol><li>将30个玻璃盖玻片放在盖玻片夹中。 </li><li>在600 mL塑料烧杯中加入20 g洗涤剂和400 mL温水自来水。的Plac将烧杯放在磁力搅拌器上并搅拌直到所有洗涤剂溶解。用棉纸去除泡沫。 </li><li>使用100 mm x 15 mm培养皿的底部作为搅拌棒的间隔物，并作为盖玻片支架的表面。为了在清洁过程中保持足够的流动，在培养皿中排放3-4个孔。 </li><li>为了做出排水孔，加热烙铁，并在排气罩中燃烧塑料。 小心:请勿用双手触摸加热的烙铁。 </li><li>将盖玻片夹放置在烧杯中的培养皿上。在室温下搅拌2小时至过夜。 </li><li>在1000 mL烧杯中用500 mL去离子水洗涤三次。可以重新使用洗涤剂溶液。 </li><li>用镊子拿起个别的盖玻片。将盖玻片浸入190（95％）乙醇中1分钟。 </li><li>将盖玻片干燥在无菌罩内。将盖玻片存放在无菌塑料容器中直到使用。 </li></ol&gt; </li><li> 制作聚-L-赖氨酸（PLL）涂层的盖玻片 <ol><li>通过将1.24g硼酸和1.9g四硼酸二钠溶于400mL水中来制备0.1M硼酸盐缓冲液。用10 M NaOH调节pH至8.5。 </li><li>将硼酸盐缓冲液中的PLL溶解至1 mg / mL的最终浓度。将PLL溶液等分于50 mL无菌离心管中。 </li><li>将50 mL PLL涂层溶液加入10 cm组织培养皿中。将盘放在37℃的培养箱中以加热PLL溶液。 </li><li>在无菌罩中打开盖玻片容器（步骤1.1.8）。 </li><li>将清洁的盖玻片一个接一个地浸入组织培养皿中预热的PLL溶液中。避免气泡形成，以便浸入所有表面。 </li><li>将盖玻片放在37℃CO 2培养箱中过夜。取出每个盖玻片并将其放在无菌的盖玻片夹中。用高压灭菌超纯水冲洗三次（18.2MΩ＆＃183厘米电阻率）在无菌罩中的1000毫升烧杯中。 </li><li>在无菌罩中的盖玻片支架上盖上盖玻片。将盖玻片存放在无菌塑料容器中直到使用。 </li></ol></li><li> 组装聚二甲基硅氧烷（PDMS）细胞培养室 <ol><li>在塑料容器中，重量为40g的PDMS基料，并加入4g固化剂以达到10:1w / w的PDMS和固化剂的比例。通过用移液管吸头搅拌混合PDMS /固化剂2分钟。或者，使用离心式混合器。 </li><li>使用4英寸直径的烧杯作为模板，制成一块铝箔的支架。将铝箔保持架放在15厘米的培养皿中。在这个铝箔保持架内放置4英寸的硅片。 </li><li>将混合的PDMS溶液倒入固定器中。使用薄玻璃棒轻轻地接触晶片的边缘。清除晶片下面的任何气泡。 </li><li>将15厘米培养皿插入真空口中r去除PDMS溶液。继续脱气约20分钟，直至不产生气泡。同时将对流烘箱预热至65°C。 </li><li>小心地将15厘米培养皿放入烤箱。孵育2小时。 </li><li>从烤箱中取出板。使用玻璃棒轻轻触摸PDMS的边缘，并确保完全交联。如果没有，培养更长时间，直到PDMS固体和不粘。 </li><li>从板上取下铝箔并将其从硅晶片上剥离下来。从边缘开始，小心地从硅晶片上剥离固化的PDMS。 小心:避免施加太大的力，因为它会破坏晶片。 </li><li>将PDMS修剪成20毫米×30毫米的矩形片，以使用剃刀刀片适合24毫米x 40毫米的盖玻片。 </li><li>使用凿形刀片从每个PDMS片的中心切出10mm×20mm的矩形开口。 </li><li>使用步骤1.1中描述的洗涤剂方案清洁PDMS室。 </li><li>将PDMS室清洁干净。将干燥室放入用铝箔覆盖的高压灭菌容器中。 </li><li>使用重力（121°C，15 psi）将容器高压灭菌30分钟，并将容器储存在室温下。 </li></ol></li><li> BHK21细胞培养和转染 <ol><li>通过将445mL DMEM与50mL FBS和5mL 100×Penn-Strep-Glutamine（10,000U / mL青霉素，10mg / mL链霉素和29.2mg / mL L）混合，制备500mL用于BHK21细胞培养的培养基谷氨酰胺）。确保FBS的最终浓度为10％。 </li><li>分装10 mL非HEPES CO 2 - 用于活细胞成像的独立培养基。 注意:CO 2 -独立培养基支持无CO 2培养箱的细胞生长，并且是在大气条件下成像细胞的理想选择。有关详细信息，请参阅材料清单。除了BHK21细胞系外，其他类型的哺乳动物细胞也应该给予类似的结果。 </li><li>通过在无菌罩中将一个高压灭菌的无菌PDMS室（步骤1.3）放置在无菌PLL涂覆的盖玻片上（步骤1.2）来组装细胞培养装置。 </li><li>将每个设备放在无菌的60 mm培养皿中。 </li><li>使用0.5mL的0.25％胰蛋白酶从12孔组织培养板的一个孔中分离细胞。用血细胞计数器计数细胞密度。用200μL细胞培养基将20000个BHK21细胞置于室中（约10,000个细胞/ cm 2 ）。 </li><li>用质粒制备试剂盒制备DNA质粒（参见材料清单）。 注:CRY2-mCherry-Raf1-2A-2CIBN-GFP-CaaX的设计和功能如图1A - 1B所示 。 </li><li>细胞培养24小时后，根据制造商的方案，用50-100ng的CRY2-mCherry-Raf1-2A-2CIBN-GFP-CaaX质粒转染细胞。 </li><li>转染后三（3）小时，更换m将其加入到200μL新鲜培养基（步骤1.4.1）中，并通过在37℃，5％CO 2培养箱中培养培养物使细胞恢复过夜。 </li></ol></li><li> 荧光活细胞成像 <ol><li>打开电脑，光源，显微镜和相机。在协议的其余部分使用共聚焦荧光显微镜（配备100X油浸物镜）。 注意:也可以使用倒置的荧光显微镜。 </li><li>用200μL的二氧化碳依赖培养基代替细胞培养基。 </li><li>在将细胞放在显微镜上之前设置数据采集协议。使用488nm激发FITC通道进行光遗传刺激。使用561 nm激发TRITC通道定位转染的细胞并追踪mCherry标记蛋白的细胞定位。 </li><li>将FITC和TRITC通道的增益分别设置为120和200。使用像素居住时间2.21; s和512×512像素的大小。 </li><li>通过将功率计放在物镜窗口附近来测量488nm光的功率。总功率为2μW（聚焦时约10,000 W / cm 2 ）足以引起CIBN-CRY2PHR缔合。 </li><li>设置5秒间隔的时间戳采集，总采集时间为2分钟。 </li><li>在目标窗口上应用适当的折射率匹配材料（浸油）。使用相位对比模式来聚焦在盖玻片表面上的单元格上。 </li><li>通过移动显微镜平台，在561nm光下定位转染的细胞。 注意:在此步骤中避免使用蓝光，因为它会激活光活化蛋白质之间的关联。 </li><li>一旦转染细胞定位，启动数据采集。 注意:成功转染的细胞应在FITC（来自2CIBN-GFP-CaaX）和TRITC（来自CRY2-mCherry-Raf1）通道中显示荧光（ 图1C-D </strong&gt;）。 </li><li>在FITC和TRITC通道中记录一系列时间戳的图像（ 图1D ）。 </li><li>为了探索CRY2-CIBN蛋白复合物的自发解离，用Txred通道单独记录20分钟，间隔30秒的另一个时间戳。 </li><li>保存时间戳的图像进行数据分析。 </li></ol></li><li> 图像分析 <ol><li>使用可以从图像40提取强度的图像分析软件打开图像。 </li><li>选择两个代表性的快照:一个在蓝光曝光之前，另一个是蓝光曝光。 </li><li>在跨越背景，质膜和细胞质的细胞上绘制线。沿着这条线投射强度。保存值并绘制出来，比较曝光前后的差异（ 图1D ）。 </li><li>分析蛋白质结合的动力学，选择在质膜中的一个代表性感兴趣区域（ROI），细胞质中的一个ROI和背景中的一个ROI。 </li><li>获取图像堆叠的质膜（I PM ），细胞质（I CYT ）和背景（I BKD ）的平均强度。 </li><li>使用以下公式计算每个图像的膜/胞质强度比: <img alt="公式1" src="/files/ftp_upload/55823/55823eq1.jpg" /></li><li>绘制比例对时间，并确定CRY2-CIBN的结合或解离动力学（ 图1E -F ）。 </li></ol></li></ol> 2.在二氧化碳培养箱中长期光刺激的LED阵列的构造注意:实验设置的总体原理图如图2A所示 。 <ol><li>通过将12个蓝色LED插入两个面包板来制作LED阵列ds和连接限流电阻。 注意:使用30 V电源时，可以串联连接四个LED，其亮度可由限流电阻控制。 </li><li>将面包板放入铝盒中。 注意:铝箱的高度应为2英寸，这个高度对于照亮12孔组织培养板是最佳的，因为发散光斑的大小与单个孔的大小相同。 </li><li>使用两根金属线连接电源。当灯箱放置在CO 2培养箱内时，确保电线的长度足够。 </li><li>使用透明光扩散器作为灯箱的盖子（ 图2C ）。 </li><li>在一定范围的电压输入端校准每个LED的功率输出。对于24小时PC12细胞分化测定，使用0.2 mW / cm 2的功率。使用5 mW / cm 2的活力来生活非洲爪蟾的胚胎或外植体试验。 </li></ol> 3. PC12细胞分化的光学诱导<ol><li> 细胞培养和转染 <ol><li>通过将407.5mL F12K与75mL马血清+ 12.5mL FBS + 5mL 100×Penn-Strep-Glutamine（马血清和FBS的终浓度）混合，制备500mL用于大鼠嗜铬细胞瘤（PC12）细胞培养物的培养基:分别为15％和2.5％）。通过在99体积的F12K培养基中混合1体积的全培养基来制备低血清培养基。 </li><li>平板PC12细胞在密度为30万个细胞/孔或75,000个细胞/ cm 2的12孔板中。 </li><li>细胞培养24小时后用CRY2-mCherry-Raf1-2A-2CIBN-GFP-CaaX转染细胞（类似于步骤1.4.7）。每个孔使用1.2μgDNA。允许细胞在培养基中在补充有5％CO 2的37℃培养箱中回收过夜。转染后16 h检查转染效率。 注意:30-50％的转染效率应该实现高血压以确保足够的细胞计数。 </li><li>转染后24 h将培养基更换为低血清培养基。每孔使用1 mL培养基的12孔板。 </li></ol></li><li> 光诱导的PC12细胞分化 <ol><li>将LED阵列放在CO 2培养箱中。使用一对电线将其连接到电源。将LED的功率设置为0.2 mW / cm 2 。将含有转染的PC12细胞的12孔板（步骤3.1.3）置于LED阵列的窗口上。在补充有5％CO 2的37℃培养箱中连续照明24小时。 </li></ol></li><li> 荧光活细胞成像 <ol><li>按照步骤1.5.1-1.5.4进行显微镜和样品制备。 </li><li>设置单快照数据采集。对GFP和Txred通道使用200 ms。 </li><li>在GFP和Txred通道中捕获转染细胞的图像。记录大约200个ls为每个条件。保存文件进行数据分析。 </li></ol></li><li> 图像分析 <ol><li>计算分化细胞在转染细胞总数中的百分比。 </li><li>在图像分析软件中使用任何细胞计数模块对细胞进行计数。 </li><li>手动计数分化细胞。 注意:分化细胞定义为其中至少一个神经突可明显长于细胞体的细胞（ 图3A- 3B ）。 </li><li>用未分化细胞重复步骤3.4.3。 </li><li>使用以下公式计算分化比: <img alt="公式2" src="/files/ftp_upload/55823/55823eq2.jpg" /></li></ol></li></ol> 4. 非洲爪蟾胚胎中激酶活性的光密控制<ol><li> 缓冲液的制备 <ol><li>准备1 L 1x Marc&#39;s Modified Ringer（MMR）:100 mMNaCl，2mM KCl，1mM MgCl 2，2mM CaCl 2和5mM HEPES; pH 7.5。 </li></ol></li><li> mRNA的制备 <ol><li>在37℃下将1μLApaI（50单位）的CRY2-mCherry-Raf1-2A-2CIBN-GFP-CaaX质粒DNA（2μg）消化2小时。 </li><li>在60μL100％乙醇中沉淀线性化DNA。在室温下以12,000×g旋转5分钟以沉淀DNA。仔细取出上清液，再次用60μL70％乙醇洗涤沉淀。在室温下旋转12,000×g另外5分钟。仔细取出上清液，将DNA沉淀重悬于6μL无RNase的H 2 O. </li><li>通过用2μL提供的SP6 RNA聚合酶，核糖核苷酸混合物（10mM ATP，CTP和UTP; 2mM GTP;和8mM cap类似物）孵育1μg线性化DNA和20μL核酸酶 -在室温下放置1小时。 </li><li>删除DNA模板通过DNA酶I在37℃下消化至少15分钟，并使用二氧化硅 - 膜旋转柱纯化合成的RNA。 </li><li>停止反应并通过加入30μL无核酸酶水和30μLLiCl沉淀溶液沉淀RNA。充分混合，-20°C冷却30分钟。 </li><li>以12,000×g离心4分钟15分钟以沉淀RNA。 </li><li>仔细清除上清液。用1 mL 70％乙醇清洗RNA一次，并重悬于40μL无核酸酶的水中。 </li><li>在旋转柱中加载40μLRNA。以12,000×g离心4分钟1分钟，以去除未掺入的核苷酸和帽。 </li></ol></li><li> 收获非洲爪蟾胚胎 <ol><li>按照Sive et al。 41获得非洲爪蟾胚胎。 </li></ol></li><li> mRNA显微注射 <ol><li>制造用毛细管拉拔玻璃毛细管用于显微注射的针头。将拉伸程序设置为:heat = 355，拉力= 80，速度= 50，时间= 100。 </li><li>通过用3％半胱氨酸（以0.2倍MMR稀释）处理它们从胚胎中除去果冻外壳。 </li><li>将胚胎转移到3％聚蔗糖和0.5x MMR溶液用于显微注射。向每个胚胎中注入500 pg至1ng的CRY2-mCherry-Raf1-2A-2CIBN-GFP-CaaX RNA。 注意:注射高于1ng的剂量会导致MAPK信号的蓝光独立激活。 </li></ol></li><li> 激酶活性的光致刺激 <ol><li>在室温下将3％聚蔗糖，0.5x MMR溶液中的显微注射胚胎培养直至达到中期胃肠期（12期）。然后，在0.2x MMR溶液中培养胚胎。 </li><li>将胚胎或外植体转移到12孔板上。将12孔板放在家用LED阵列上（步骤2.5），用于蓝色t（475nm）处理。 </li><li>在12孔板的顶部放置一个镜子，以确保胚胎或外植体的全蓝光照射。 </li><li>调节蓝光的功率为5 mW / cm 2 。 注意:蓝光处理可以在任何所需时间进行，在3％聚蔗糖，0.5x MMR溶液或0.2x MMR溶液中。 </li><li>在任何所需时间收获胚胎。用于组织学，Western印迹或基因表达分析。 </li></ol></li></ol>

作者宣称他们没有竞争的经济利益。

建造灯箱时，应测量各个LED的功率。根据以前的经验，由于制造差异，功率输出可能因个别LED而异。选择一组功率输出在10％以内的LED。可以针对不同类型的细胞培养容器（ 例如 6孔或24孔板）修改LED的数量，限流电阻和功率输入。功率为0.2mW / cm 2的 24小时的光照不会引起可检测的光毒性44 。如果使用更高的功率，请考虑使用间歇光来减少发热和光毒性。在本方案?...

生长因子参与广泛的细胞功能，包括增殖，分化，迁移和凋亡，并在许多生物学事件中发挥关键作用，包括胚胎发育，衰老和精神状态调节1,2,3,4 ， 5 。许多生长因子通过复杂的细胞内信号传导级联信号。这些信号事件通常以精确调节的方式可逆蛋白磷酸化操作6,7 。因此，对蛋白激酶的信号转导结果的理解，它们负责蛋白质磷酸化，这是非常重要的。 不同的生长因子通过相当普遍的细胞内信号网络起作用，即使它们刺激dist细胞反应8,9 。受体酪氨酸激酶的常见细胞内介质包括Ras，Raf，细胞外信号调节激酶（ERK），丝裂原活化蛋白激酶（MAPK）/ ERK激酶（MEK），磷酸肌醇3-激酶（PI3K），Akt和磷脂酶Cγ （PLCγ） 10,11 。积累的证据表明信号多样性和特异性取决于信号传导活动的空间和时间调节12 。例如，在大鼠嗜铬细胞瘤细胞（PC12）中，导致细胞增殖的表皮生长因子（EGF）刺激瞬时激活ERK通路9 。另一方面，导致细胞分化的神经生长因子（NGF）的刺激以持续的方式激活ERK通路9,13。在培养的r在海马神经元中，脑源性神经营养因子（BDNF）的瞬时信号传导促进原发神经突生长，而持续的信号传导导致神经突分支增加14 。在早期胚胎发育过程中，磷酸化的ERK活性在时间上是动态的，并且在胚胎中是广泛的6 。早期非洲爪蟾胚胎发生期间最近的遗传筛选显示ERK和Akt信号级联，两种下游原生生长因子途径，显示阶段特异性激活谱7 。因此，对激酶信号转导结果的理解需要能够以足够的分辨率探测激酶活性的空间和时间特征的工具。 探索发育过程中信号转导的动态特性的常规实验方法缺乏理想的空间和时间分辨率。例如，药理学方法使用小化学或生物分子刺激或抑制细胞和组织中的信号转导。这些小分子的扩散性质使得将其作用限制在特定的感兴趣区域15是有挑战性的。遗传方法（ 如转基因，Cre-Lox系统或诱变）通常导致靶基因表达或蛋白活性的不可逆活化或抑制16,17,18。 Tet-On / Tet-Off系统19提供改进的基因转录的时间控制，但是缺乏严格的空间控制，因为它依赖于四环素的扩散。化学诱导的蛋白质二聚化20或光解蛋白21,22,23,24中的最新发展有很大的增强作用信号网络的时间控制。然而，由于笼式化学品的扩散性质，空间控制仍然是具有挑战性的。 利用光的功能来控制蛋白质 - 蛋白质相互作用的最近出现的光生代谢途径允许以高时空精度和可逆性调节信号通路。在控制神经元激发25,26,27之后不久，光遗传学被扩展到控制其他细胞过程，如基因转录，翻译，细胞迁移，分化和凋亡28,29,30,31,32,33 ， 34 。使用p的策略最近开发了可光活化蛋白对拟南芥隐色素2（CRY2）蛋白和密码子相互作用的碱性 - 螺旋 - 环 - 螺旋（CIBN）的N末端结构域，以控制哺乳动物细胞和非洲爪蟾胚胎中的Raf1激酶活性。 CRY2在蓝光刺激下与CIBN结合，CRY2 / CIBN蛋白复合物在黑暗中自发解离34 。蓝光激发CRY2辅因子，黄素腺嘌呤二核苷酸（FAD），其导致CRY2的构象变化及其随后与CIBN的结合。可以通过FAD结合口袋中的突变产生CRY2的组成型活性（W374A）和黄素缺乏型（D387A）突变体:CRY2 W374A突变体独立于光而与CIBN结合，而CRY2 D387A突变体不结合蓝色的CIBN光刺激36,37 。光生系统描述了我该方案使用野生型CRY2和CIBN诱导活细胞中的蛋白质易位介导的Raf1活化。已知Raf1的膜募集增强其活性38 。在该系统中，串联CIBN模块锚定于质膜，CRY2-mCherry与Raf135的N末端融合。在没有蓝光的情况下，CRY2-mCherry-Raf1停留在细胞质中，Raf1不活跃。蓝光刺激诱导CRY2-CIBN结合，并将Raf1募集到质膜上，其中Raf1被激活。 Raf激活刺激Raf / MEK / ERK信号级联。 CRY2-和CIBN-融合蛋白均以双顺反子基因系统编码。该策略可以被推广以控制其他激酶，例如Akt，其激活状态也可以通过细胞中的蛋白质移位而被打开39 。这项工作提出了在哺乳动物细胞培养中实现这种光遗传策略的详细协议res和多细胞生物。

<table><tbody><tr><td>Glass coverslip&amp;nbsp;</td><td>VWR</td><td>48393 230</td><td>Substrate for live cell imaging</td></tr><tr><td>Coverslip holder&amp;nbsp;</td><td>Newcomer Supply</td><td>6817B</td><td>Holder for coverslips</td></tr><tr><td>Detergent&amp;nbsp;</td><td>ThermoFisher</td><td>16 000 104</td><td>For cleaning coverslips</td></tr><tr><td>Boric acid&amp;nbsp;</td><td>Sigma-Aldrich</td><td>B6768-500G</td><td>For making PLL buffer</td></tr><tr><td>Disodium tetraborate</td><td>Sigma-Aldrich</td><td>71996-250G</td><td>For making&amp;nbsp; PLL buffer</td></tr><tr><td>Plastic beaker</td><td>Nalgene</td><td>1201-1000</td><td>For cleaning coverslips</td></tr><tr><td>Sodium hydroxide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>221465-2.5KG</td><td>For adjust pH</td></tr><tr><td>Poly-L-lysine hydrobromide</td><td>Sigma-Aldrich</td><td>P1274-500MG</td><td>For coating coverslip</td></tr><tr><td>Diethylpyrocarbonate (DEPC)-Treated Water</td><td>ThermoFisher Scientific</td><td>750024</td><td>For DNA preparation</td></tr><tr><td>Cover Glass Forceps</td><td>Ted Pella</td><td>5645</td><td>Cover glass handling</td></tr><tr><td>Tissue cutlure dish</td><td>Thermofisher</td><td>12565321</td><td>Cell culture dish</td></tr><tr><td>Sterile centrifuge tubes</td><td>ThermoFisher</td><td>12-565-271</td><td>Buffer storage</td></tr><tr><td>Transfection Reagent</td><td>ThermoFisher</td><td>R0534</td><td>Transfection</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;-independent medium</td><td>ThermoFisher</td><td>18045088</td><td>For live cell imaging</td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Ellsworth Adhesives</td><td>184 SIL ELAST KIT 0.5KG</td><td>Form make cell chamber</td></tr><tr><td>Plasmid Maxiprep kit</td><td>Qiagen</td><td>12965</td><td>Plasmid preparation</td></tr><tr><td>DMEM medium</td><td>ThermoFisher</td><td>11965-084</td><td>Cell culturing medium component</td></tr><tr><td>F12K medium</td><td>ThermoFisher</td><td>21127022</td><td>Cell culturing medium component</td></tr><tr><td>Horse serum</td><td>ThermoFisher</td><td>16050122</td><td>Cell culturing medium component</td></tr><tr><td>Fetal Bovine Serum</td><td>Signa-Aldrich</td><td>12303C-500 mL</td><td>Cell culturing medium component</td></tr><tr><td>Penicillin-Streptomycin-Glutamine</td><td>ThermoFisher</td><td>10378016</td><td>Cell culturing medium component</td></tr><tr><td>Trypsin (0.25%), phenol red</td><td>ThermoFisher Scientific</td><td>15050065&amp;nbsp;</td><td>For mammalian cell dissociation</td></tr><tr><td>Agarose</td><td>Fisher Scientific</td><td>BP1356-100</td><td>For DNA preparation</td></tr><tr><td>Ficoll PM400</td><td>GE Heathcare Life Sciences</td><td>17-5442-02</td><td>For embryo buffer</td></tr><tr><td>L-Cysteine hydrochloride monohydrate</td><td>Sigma-Aldrich</td><td>1.02839.0025</td><td>Oocyte preparation</td></tr><tr><td>ApaI</td><td>ThermoFisher</td><td>FD1414</td><td>For linearization of plasmids</td></tr><tr><td>Dnase I</td><td>ThermoFisher</td><td>AM2222</td><td>For removing DNA template in the &lt;em&gt;in vitro&lt;/em&gt; transcription assay</td></tr><tr><td>Index-match materials (immersion oil)</td><td>Thorlabs</td><td>MOIL-20LN</td><td>For matching the index between sample substrate and objective</td></tr><tr><td>Blue LED</td><td>Adafruit</td><td>301</td><td>Light source for optogenetic stimulation</td></tr><tr><td>Resistor kit</td><td>Amazon</td><td>EPC-103</td><td>current-limiting resistor</td></tr><tr><td>Aluminum boxes</td><td>BUD Industries</td><td>AC-401</td><td>light box&amp;nbsp;</td></tr><tr><td>BreadBoard</td><td>Jekewin</td><td>837654333686</td><td>For making LED array</td></tr><tr><td>Hook up Wire</td><td>Electronix Express</td><td>27WK22SLD25</td><td>For making LED array</td></tr><tr><td>Relay Module</td><td>Jbtek</td><td>SRD-05VDC-SL-C</td><td>For intermittent light control</td></tr><tr><td>DC Power Supply&amp;nbsp;</td><td>TMS</td><td>DCPowerSupply-LW-(PS-305D)</td><td>Power supply for LED</td></tr><tr><td>Silicon&amp;nbsp;Power&amp;nbsp;Head</td><td>Thorlabs</td><td>S121C</td><td>For light intensity measurement</td></tr><tr><td>Power meter</td><td>Thorlabs</td><td>PM100D&amp;nbsp;</td><td>For light intensity measurement</td></tr><tr><td>Microscope</td><td>Leica Biosystems</td><td>DMI8</td><td>For live cell imaging</td></tr><tr><td>BioSafety Cabinet</td><td>ThermoFisher</td><td>1300 Series A2</td><td>For mammalian cell handling</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>ThermoFisher</td><td>Isotemp</td><td>For mammalian cell culturing</td></tr><tr><td>Stereo microscope</td><td>Leica</td><td>M60</td><td>For embryo micro-manipulation</td></tr><tr><td>Microinjector</td><td>Narishige</td><td>IM300</td><td>For embryo microinjection</td></tr><tr><td>Micropipette puller</td><td>Sutter Instruments</td><td>P87</td><td>Needle puller</td></tr><tr><td>&lt;em&gt;in vitro&lt;/em&gt; transcription kit</td><td>ThermoFisher</td><td>AM1340</td><td>For &lt;em&gt;in vitro&lt;/em&gt; transcription. The kit includes nuclease-free water, SP6 RNA&amp;nbsp; Polymerase, ribonucleotide mixture, cap analog, lithium choride precipitation solution, and spin column&amp;nbsp;&amp;nbsp;</td></tr><tr><td>RNA purfication kit</td><td>Qiagen</td><td>74104</td><td>Silica-membrane spin column for purification of synthesized RNA&amp;nbsp;</td></tr><tr><td>Convection oven</td><td>MTI corporation</td><td>&amp;nbsp;EQ-DHG-9015</td><td>PDMS curing</td></tr><tr><td>Centrifugal mixer and teflon container</td><td>THINKY</td><td>AR310</td><td>For mixing PDMS</td></tr><tr><td>Silicon wafer</td><td>UniversityWafer</td><td>452</td><td>Base for making PDMS&amp;nbsp; devices</td></tr><tr><td>Blade</td><td>Techni Edge</td><td>01-801</td><td>For cutting PDMS</td></tr><tr><td>Capillary glass</td><td>Sutter Instruments</td><td>BF100-58-10</td><td>For fabrication of injecting needles.&amp;nbsp;</td></tr></tbody></table>

light-mediated reversible modulation of the mitogen-activated protein kinase pathway during cell differentiation and xenopus embryonic development

激酶活性对于多种细胞功能至关重要，包括细胞增殖，分化，迁移和细胞凋亡。在早期胚胎发育过程中，激酶活性在胚胎中是高度动态和广泛的。药理学和遗传学方法通常用于探索激酶活性。不幸的是，使用这些策略来实现优越的空间和时间分辨率是很困难的。此外，在活细胞和多细胞生物体中以可逆的方式控制激酶活性是不可行的。这种限制仍然是在开发和分化期间实现对激酶活性的定量了解的瓶颈。这项工作提出了利用含有可光活化蛋白拟南芥隐色素2（CRY2）和密码子相互作用的基质 - 螺旋 - 环 - 螺旋（CIBN）的N-末端结构域的双顺反子系统的光遗传策略。黑白棋有丝分裂原激活的蛋白激酶（MAPK）信号通路的活化可以通过活细胞中的光介导的蛋白质易位实现。这种方法可以应用于哺乳动物细胞培养物和活的脊椎动物胚胎。这种双顺反子系统可以被推广以控制具有相似活化机制的其它激酶的活性并且可以应用于其它模型系统。

可光活化蛋白质对的比例表达: 图1A显示了基于猪teschovirum的双顺反子基因构建体CRY2-mCherry-Raf1-P2A-CIBN-CIBN-GFP-CaaX（称为CRY2-2A-2CIBN）的设计， 112A（P2A）肽，其在哺乳动物细胞系42中显示出最高的核糖体跳跃效率。在以前的工作中，已经确定CIBN-GFP-CaaX:CRY2-mCherry-Raf1的最佳比例为2:135。该配置允许CRY2-mCherry-Raf1的足够的膜募集和活化（ <str...

Watch this Scientific Journal Video about Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development at JoVE.com

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

该协议描述了在细胞分化和非洲爪蟾胚胎发育过程中调节丝裂原活化蛋白激酶（MAPK）活性的光遗传策略。这种方法允许哺乳动物细胞培养物和多细胞活生物体如非洲爪蟾胚胎中具有高空间和时间分辨率的可逆激活MAPK信号通路。

在细胞分化过程中光介导的可逆调节丝裂原活化蛋白激酶途径<em&gt;爪蟾</em&gt;胚胎发育

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

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Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Unit 1

Signaling Networks of Kinase Receptors

SCIENTISTS IN ACTION

8.7K 次观看.  University of Illinois at Urbana-Champaign. 该协议描述了在细胞分化和非洲爪蟾胚胎发育过程中调节丝裂原活化蛋白激酶（MAPK）活性的光遗传策略。这种方法允许哺乳动物细胞培养物和多细胞活生物体如非洲爪蟾胚胎中具有高空间和时间分辨率的可逆激活MAPK信号通路。 

视频: Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

利用共聚焦分析<em&gt;非洲爪蟾</em&gt;调查的Wnt和Shh形态发生素梯度的调节剂

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

科研

发育生物学

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

遗传学

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

This video article details a straightforward in vivo methodology that can be used to systematically and efficiently characterize components of complex signaling pathways and regulatory networks in many invertebrate embryos.

简单的力量:海胆胚胎作为<em&gt;体内</em&gt;对于研究复杂的细胞与细胞间信号传导网络交互开发模式

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency. The mechanisms that control na&#239;ve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify na&#239;ve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition.

通过高通量抑制剂筛选鉴定调节胚胎干细胞多能性的激酶的简单方法

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

A Microfluidics Approach for the Functional Investigation of Signaling Oscillations Governing Somitogenesis

Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and gradients are thought to control the timing and spacing of segment formation, respectively. While the involved signaling pathways have been studied extensively over the last decades, direct evidence for the function of signaling oscillations in controlling somitogenesis has been lacking. To enable the functional investigation of signaling dynamics, microfluidics is a previously established tool for the subtle modulation of these dynamics. With this microfluidics-based entrainment approach endogenous signaling oscillations are synchronized by pulses of pathway modulators. This enables modulation of, for instance, the oscillation period or the phase-relationship between two oscillating pathways. Furthermore, spatial gradients of pathway modulators can be established along the tissue to study how specific changes in the signaling gradients affect somitogenesis.
The present protocol is meant to help establish microfluidic approaches for the first-time users of microfluidics. The basic principles and equipment needed to set up a microfluidic system are described, and a chip design is provided, with which a mold for chip generation can conveniently be prepared using a 3D printer. Finally, how to culture primary mouse tissue on a microfluidic chip and how to entrain signaling oscillations to external pulses of pathway modulators are discussed.
This microfluidic system can also be adapted to harbor other in vivo and in vitro model systems such as gastruloids and organoids for functional investigation of signaling dynamics and morphogen gradients in other contexts.

A protocol for the culture and manipulation of mouse embryonic tissue on a microfluidic chip is provided. By applying pulses of pathway modulators, this system can be used to externally control signaling oscillations for the functional investigation of mouse somitogenesis.

一种微流体方法，用于控制体力生成的信号振荡的功能研究

Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and ...

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

使用浸泡珠测定分析鸡胚胎发生过程中的细胞分化，形态发生和图案化

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...

Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. These morphological changes are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. Here, two examples of physiologically-relevant contexts in which to study phosphorylation of proteins during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves. To overcome the common barrier of dephosphorylation during protein isolation, adaptations and modifications to standard laboratory methods that allow for isolation of phosphoproteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphoproteins following western blotting of whole cell protein lysates. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various phosphoproteins active during craniofacial development.

The protocol presents a method for isolating whole cell protein lysates from dissected mouse embryo facial processes or cultured mouse embryonic palatal mesenchyme cells and performing subsequent western blotting to assess phosphorylated protein levels.

从小鼠面部过程和培养的腭间充质细胞中分离全细胞蛋白质裂解物以进行磷酸化蛋白分析

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal ...

研究斑马鱼光遗传学信号转导操作的发育

Optogenetic Signaling Activation in Zebrafish Embryos

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally manipulate signaling are required to understand how signaling is interpreted in these wide-ranging contexts. Molecular optogenetic&#160;tools can provide reversible, tunable manipulations of signaling pathway activity with a high degree of spatiotemporal control and have been applied in vitro, ex vivo, and in vivo. These tools couple light-responsive protein domains, such as the blue light homodimerizing light-oxygen-voltage sensing (LOV) domain, with signaling effectors to confer light-dependent experimental control over signaling. This protocol provides practical guidelines for using the LOV-based bone morphogenetic protein (BMP) and Nodal signaling activators bOpto-BMP and bOpto-Nodal in the optically accessible early zebrafish embryo. It describes two control experiments: A quick phenotype assay to determine appropriate experimental conditions, and an immunofluorescence assay to directly assess signaling. Together, these control experiments can help establish a pipeline for using optogenetic tools in early zebrafish embryos. These strategies provide a powerful platform to investigate the roles of signaling in development, health, and physiology.

作者聚焦：操纵斑马鱼胚胎中的信号转导以解码细胞命运决定 

Optogenetic manipulation of signaling pathways can be a powerful strategy to investigate how signaling is decoded in development, regeneration, homeostasis, and disease. This protocol provides practical guidelines for using light-oxygen-voltage sensing domain-based Nodal and bone morphogenic protein (BMP) signaling activators in the early zebrafish embryo.

斑马鱼胚胎中的光遗传学信号激活

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally ...

在细胞分化过程中光介导的可逆调节丝裂原活化蛋白激酶途径

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利用共聚焦分析

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

简单的力量:海胆胚胎作为

通过高通量抑制剂筛选鉴定调节胚胎干细胞多能性的激酶的简单方法

一种微流体方法，用于控制体力生成的信号振荡的功能研究

使用浸泡珠测定分析鸡胚胎发生过程中的细胞分化，形态发生和图案化

从小鼠面部过程和培养的腭间充质细胞中分离全细胞蛋白质裂解物以进行磷酸化蛋白分析

斑马鱼胚胎中的光遗传学信号激活

斑马鱼胚胎中的光遗传学信号激活

斑马鱼胚胎中的光遗传学信号激活