Name: light-mediated reversible modulation of the mitogen-activated protein kinase pathway during cell differentiation and xenopus embryonic development
Uploaded: 2017-06-15T15:00:00
Description: 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

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?...

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 border="0" cellpadding="0" cellspacing="0" width="748">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Glass coverslip&#160;</td>
			<td style="width:175px;">VWR</td>
			<td style="width:188px;">48393 230</td>
			<td style="width:167px;">Substrate for live cell imaging</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Coverslip holder&#160;</td>
			<td style="width:175px;">Newcomer Supply</td>
			<td style="width:188px;">6817B</td>
			<td style="width:167px;">Holder for coverslips</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Detergent&#160;</td>
			<td style="width:175px;">ThermoFisher</td>
			<td style="width:188px;">16 000 104</td>
			<td style="width:167px;">For cleaning coverslips</td>
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			<td height="21" style="height:21px;width:219px;">Boric acid&#160;</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:188px;">B6768-500G</td>
			<td style="width:167px;">For making PLL buffer</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Disodium tetraborate</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:188px;">71996-250G</td>
			<td style="width:167px;">For making&#160; PLL buffer</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Plastic beaker</td>
			<td style="width:175px;">Nalgene</td>
			<td style="width:188px;">1201-1000</td>
			<td style="width:167px;">For cleaning coverslips</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Sodium hydroxide&#160;</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:188px;">221465-2.5KG</td>
			<td style="width:167px;">For adjust pH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Poly-L-lysine hydrobromide</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:188px;">P1274-500MG</td>
			<td style="width:167px;">For coating coverslip</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Diethylpyrocarbonate (DEPC)-Treated Water</td>
			<td style="width:175px;">ThermoFisher Scientific</td>
			<td style="width:188px;">750024</td>
			<td style="width:167px;">For DNA preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Cover Glass Forceps</td>
			<td style="width:175px;">Ted Pella</td>
			<td style="width:188px;">5645</td>
			<td style="width:167px;">Cover glass handling</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Tissue cutlure dish</td>
			<td style="width:175px;">Thermofisher</td>
			<td style="width:188px;">12565321</td>
			<td style="width:167px;">Cell culture dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Sterile centrifuge tubes</td>
			<td style="width:175px;">ThermoFisher</td>
			<td style="width:188px;">12-565-271</td>
			<td style="width:167px;">Buffer storage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Transfection Reagent</td>
			<td style="width:175px;">ThermoFisher</td>
			<td style="width:188px;">R0534</td>
			<td style="width:167px;">Transfection</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:219px;">CO2-independent medium</td>
			<td style="width:175px;">ThermoFisher</td>
			<td style="width:188px;">18045088</td>
			<td style="width:167px;">For live cell imaging</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Polydimethylsiloxane (PDMS)</td>
			<td style="width:175px;">Ellsworth Adhesives</td>
			<td style="width:188px;">184 SIL ELAST KIT 0.5KG</td>
			<td style="width:167px;">Form make cell chamber</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Plasmid Maxiprep kit</td>
			<td style="width:175px;">Qiagen</td>
			<td style="width:188px;">12965</td>
			<td style="width:167px;">Plasmid preparation</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:219px;">DMEM medium</td>
			<td style="width:175px;">ThermoFisher</td>
			<td style="width:188px;">11965-084</td>
			<td style="width:167px;">Cell culturing medium component</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:219px;">F12K medium</td>
			<td style="width:175px;">ThermoFisher</td>
			<td style="width:188px;">21127022</td>
			<td style="width:167px;">Cell culturing medium component</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Horse serum</td>
			<td style="width:175px;">ThermoFisher</td>
			<td style="width:188px;">16050122</td>
			<td style="width:167px;">Cell culturing medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Fetal Bovine Serum</td>
			<td style="width:175px;">Signa-Aldrich</td>
			<td style="width:188px;">12303C-500 mL</td>
			<td style="width:167px;">Cell culturing medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Penicillin-Streptomycin-Glutamine</td>
			<td style="width:175px;">ThermoFisher</td>
			<td style="width:188px;">10378016</td>
			<td style="width:167px;">Cell culturing medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Trypsin (0.25%), phenol red</td>
			<td style="width:175px;">ThermoFisher Scientific</td>
			<td style="width:188px;">15050065&#160;</td>
			<td style="width:167px;">For mammalian cell dissociation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Agarose</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:188px;">BP1356-100</td>
			<td style="width:167px;">For DNA preparation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Ficoll PM400</td>
			<td style="width:175px;">GE Heathcare Life Sciences</td>
			<td style="width:188px;">17-5442-02</td>
			<td style="width:167px;">For embryo buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">L-Cysteine hydrochloride monohydrate</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:188px;">1.02839.0025</td>
			<td style="width:167px;">Oocyte preparation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">ApaI</td>
			<td style="width:175px;">ThermoFisher</td>
			<td>FD1414</td>
			<td style="width:167px;">For linearization of plasmids</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:219px;">Dnase I</td>
			<td style="width:175px;">ThermoFisher</td>
			<td style="width:188px;">AM2222</td>
			<td style="width:167px;">For removing DNA template in the in vitro transcription assay</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:219px;">Index-match materials (immersion oil)</td>
			<td style="width:175px;">Thorlabs</td>
			<td>MOIL-20LN</td>
			<td style="width:167px;">For matching the index between sample substrate and objective</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Blue LED</td>
			<td style="width:175px;">Adafruit</td>
			<td style="width:188px;">301</td>
			<td style="width:167px;">Light source for optogenetic stimulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Resistor kit</td>
			<td style="width:175px;">Amazon</td>
			<td style="width:188px;">EPC-103</td>
			<td style="width:167px;">current-limiting resistor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Aluminum boxes</td>
			<td style="width:175px;">BUD Industries</td>
			<td style="width:188px;">AC-401</td>
			<td style="width:167px;">light box&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">BreadBoard</td>
			<td style="width:175px;">Jekewin</td>
			<td style="width:188px;">837654333686</td>
			<td style="width:167px;">For making LED array</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Hook up Wire</td>
			<td style="width:175px;">Electronix Express</td>
			<td style="width:188px;">27WK22SLD25</td>
			<td style="width:167px;">For making LED array</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Relay Module</td>
			<td style="width:175px;">Jbtek</td>
			<td style="width:188px;">SRD-05VDC-SL-C</td>
			<td style="width:167px;">For intermittent light control</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">DC Power Supply&#160;</td>
			<td style="width:175px;">TMS</td>
			<td style="width:188px;">DCPowerSupply-LW-(PS-305D)</td>
			<td style="width:167px;">Power supply for LED</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Silicon&#160;Power&#160;Head</td>
			<td style="width:175px;">Thorlabs</td>
			<td style="width:188px;">S121C</td>
			<td style="width:167px;">For light intensity measurement</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Power meter</td>
			<td style="width:175px;">Thorlabs</td>
			<td style="width:188px;">PM100D&#160;</td>
			<td style="width:167px;">For light intensity measurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Microscope</td>
			<td style="width:175px;">Leica Biosystems</td>
			<td style="width:188px;">DMI8</td>
			<td style="width:167px;">For live cell imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">BioSafety Cabinet</td>
			<td style="width:175px;">ThermoFisher</td>
			<td style="width:188px;">1300 Series A2</td>
			<td style="width:167px;">For mammalian cell handling</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">CO2 incubator</td>
			<td style="width:175px;">ThermoFisher</td>
			<td style="width:188px;">Isotemp</td>
			<td style="width:167px;">For mammalian cell culturing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Stereo microscope</td>
			<td style="width:175px;">Leica</td>
			<td style="width:188px;">M60</td>
			<td style="width:167px;">For embryo micro-manipulation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Microinjector</td>
			<td style="width:175px;">Narishige</td>
			<td style="width:188px;">IM300</td>
			<td style="width:167px;">For embryo microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Micropipette puller</td>
			<td style="width:175px;">Sutter Instruments</td>
			<td style="width:188px;">P87</td>
			<td style="width:167px;">Needle puller</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:219px;">in vitro transcription kit</td>
			<td style="width:175px;">ThermoFisher</td>
			<td style="width:188px;">AM1340</td>
			<td style="width:167px;">For in vitro transcription. The kit includes nuclease-free water, SP6 RNA&#160; Polymerase, ribonucleotide mixture, cap analog, lithium choride precipitation solution, and spin column&#160;&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:219px;">RNA purfication kit</td>
			<td style="width:175px;">Qiagen</td>
			<td style="width:188px;">74104</td>
			<td style="width:167px;">Silica-membrane spin column for purification of synthesized RNA&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Convection oven</td>
			<td style="width:175px;">MTI corporation</td>
			<td style="width:188px;">&#160;EQ-DHG-9015</td>
			<td style="width:167px;">PDMS curing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Centrifugal mixer and teflon container</td>
			<td style="width:175px;">THINKY</td>
			<td style="width:188px;">AR310</td>
			<td style="width:167px;">For mixing PDMS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Silicon wafer</td>
			<td style="width:175px;">UniversityWafer</td>
			<td style="width:188px;">452</td>
			<td style="width:167px;">Base for making PDMS&#160; devices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Blade</td>
			<td style="width:175px;">Techni Edge</td>
			<td style="width:188px;">01-801</td>
			<td style="width:167px;">For cutting PDMS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Capillary glass</td>
			<td style="width:175px;">Sutter Instruments</td>
			<td style="width:188px;">BF100-58-10</td>
			<td style="width:167px;">For fabrication of injecting needles.&#160;</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（ ...

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

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