我们感谢下院实验室的成员提供咨询和协助。得到美国国立卫生研究院 (R01-AI087644 M.H. 和 P30-CA008748 的支持, 纪念斯隆-凯特林癌症中心)。

1. 制备刺激性玻璃表面
<ol><li>涂层八井腔 coverglass 与生物素化聚 l-赖氨酸 (生物锁相环) 稀释1:500 在蒸馏, 去离子水 (ddH2 O)。室温下孵育30分钟 (RT)。</li>
	<li>使用 H2进行清洗。</li>
	<li>在 RT 干燥2小时。</li>
	<li>块生物锁相环涂层表面与阻塞缓冲 (HEPES 缓冲盐水 [10 毫米 HEPES pH 值 7.4, 150 毫米氯化钠], 与 2% BSA) 为30分钟在 RT。<ol>
<li>将5毫克的聚 l-赖氨酸氢溴酸盐溶于1毫升的10毫米矫正假肢4, pH 8.5。</li>
		<li>添加125μmol (0.55 毫克) 的 NHS-生物素 (从100毫克/毫升的股票在亚砜) 和检查的 pH 值的反应。如果 ph 值低于 8.5, 则添加 2 ul 4 N 氢氧化钠, 将其提高到 ph 值8.5 和9.5 之间。</li>
		<li>涡流为30分钟。</li>
		<li>淬火反应与 50 ul 100 毫米甘氨酸溶解在20毫米三 pH 值8.0。</li>
		<li>全功率旋转10分钟, 将上清液转移到新管上。 注: 生物锁相环的质量通常与以前的制剂相比, 通过将材料的两倍稀释涂敷在96井 ELISA 板上, 并用碱性磷酸酶结合链亲和素进行孵化后定量化素含量。</li>
	</ol>
</li>
	<li>删除阻止缓冲区。不要让水井干。添加链亲和素 (100 µg/毫升在阻塞缓冲区)。在4摄氏度孵育1小时。</li>
	<li>在哈佛商学院洗。填充和反转室滑井 4-5 次, 从井中删除哈佛商学院。不要让水井干。</li>
	<li>将生物素化抵押贷款公司配体和黏附分子添加到表面。 注意: 可通过将邻硝基保护组 (例如, nitrophenylethyl (photocaged) 或 nitroveratryloxycarbonyl (NVOC)) 添加到关键 lysines (mcc, K12 在卵中 K7) 的ε氨基组中, 来 MCC 和卵子。Photocaged MCC 和卵子可以从商业供应商获得。重建后的水缓冲, 他们被折叠到细菌表达的 I Ek和 H2-Kb使用已建立的方法11,12。</li>
	<li>photoactivatable MCC 或卵肽的验证<ol>
<li>Decage NVOC 保护肽通过紫外线照射与手持紫外线灯 (见材料表) 在 RT 20 分钟。</li>
		<li>脉冲 1.0 x 105 apc 与1µM nonstimulatory 肽 (阴性控制,例如Hb), 非辐照 photoactivatable 肽, 辐照的 photoactivatable 肽, 和激动肽 (正控制, 例如, MCC) 为 1 h 到隔夜在37°c。</li>
		<li>刺激 T 细胞表达5C。C7/2B4/AND TCR, 使用 CH12 或 CH27 B 细胞。为了刺激 OT1 T 细胞, 使用 RMA 或 EL4 胸腺瘤细胞。</li>
		<li>将脉冲 apc 与相同数量的 T 细胞在96井圆底板上混合, 最终体积为200µL. 孵育37°c 为 12-24 h。</li>
		<li>用链亲和素-辣根过氧化物酶比色检测法, 从各井中恢复上清液, 并进行酶联免疫分析 IL-2。 注: 强烈建议通过电喷雾质谱法确认所有肽的锁定状态, 然后再复性为 MHC 复合体。</li>
	</ol>
</li>
	<li>对 MHC 进行折叠和纯化, 以尽量减少光照射。例如, 用铝箔包裹折叠反应, 用 UV 灯关闭凝胶过滤层析。 注: 发现 photoactivatablek和卵 H2-Kb在高密度下诱导紫外独立 T 细胞活化, 可能是由于功能不完全的锁定。因此, 在固定化步骤中, photoactivatable 抵押贷款公司被稀释成 10 30x 以上的 nonstimulatory 抵押贷款公司 (例如I -e k,其中包含血红蛋白64-76 肽 (Hb))。这提高了后续成像实验的信噪比。</li>
	<li>若要 photoactivate CD4+ T 单元格, 请集体使用生物素化k (3 µg/毫升)、生物素化 photoactivatableMCC -e k (0.1 µg/毫升) 的混合物, 并对 i类MHC 生物素化 k (0.5 H2-K/mL). anti-H2-KK抗体鼓励5C。C7/2B4/AND T 细胞, 表达 H2-KK, 扩散到玻璃表面而不进行激活。</li>
	<li>对于 CD8+ T 单元格, 请使用生物素化H2-D b 的混合体, 其含肽 KAVYDFATL (1 µg/毫升)、生物素化photoactivatable OVA-H2-K b (0.1 µg/毫升) 和黏附分子 ICAM-1 的胞外域 (2 µg/毫升由昆虫细胞培养产生的13)。ICAM-1 通过将整合素 LFA1 在 T 细胞表面上, 鼓励紧密的接触形成。</li>
	<li>将所有蛋白混合物应用于阻塞缓冲液中, 然后在 RT 中孵化1小时, 或至少2小时4摄氏度。 注: 此类表面的分子密度为每µm2的 8000, 以前已确定为6。鉴于 photoactivatable 抵押贷款公司代表了生物素化蛋白在表面上的~ 1/30 th, 它的密度将是 decaging之前每µm 2 的267个分子。</li>
	<li>在步骤1.6 中进行清洗, 并在哈佛商学院离开, 直到准备使用。</li>
	<li>将 20万 CD4+或 CD8+ T 单元格表示为每个井中适当的 TCR, 并允许单元格在37摄氏度内坚持15分钟。一旦细胞在表面上附着并扩散, 它们就可以 photoactivation 和成像。 注: 荧光成像探针对效应 T 细胞的逆转录病毒转导, 详细描述 elswhere6,7。钙信号也可以研究使用 untransduced T 细胞加载的钙敏感染料 Fluo-46。比例染料 Fura-2 是不推荐的, 因为它需要在紫外线范围内的激发, 这也诱导 decaging photoactivatable 抵押贷款公司。</li>
</ol>2. 图像采集
<ol><li>使用带有 UV 兼容150X 物镜的倒置 TIRF 显微镜进行图像采集。紫外线照射用户定义的区域使用一个数字隔膜系统连接到 100 W 汞灯 (HBO)。使用 400 nm 长的后视镜将这盏灯上的紫外线直接照射到样品上。</li>
	<li>使用图像分析软件进行局部 photoactivation 和时间推移采集。在大多数实验中, 分别使用 488 nm 和 561 nm 激发激光器对绿色和红色通道中的探头进行监测。激光光被定向到样品上, 使用双通带分色镜, 也传输在紫外线范围 (以使 decaging)。有关显微镜配置的其他信息, 请参阅材料表和图 6 。</li>
	<li>安装包含 T 单元格的会议厅幻灯片后, 根据需要调整设置以获得 TIRF 或荧光照明。在 "实时" 模式下, 选择表示感兴趣的荧光探针的单元格字段。使用软件控制建立单个细胞下 photoactivation 的微米尺度区域。</li>
	<li>开始时间推移获取。通常, 获取 80 timepoints, 每个时间点之间间隔为5秒。在双色实验的情况下, 这给连续 488 nm 和 563 nm 曝光留下了足够的时间。</li>
	<li>10 timepoints 后, 通过打开数字隔膜快门 1-1.5 s, photoactivate 选定区域。</li>
	<li>完成时间后, 选择一个新的单元格字段并重复该过程。</li>
</ol>3. 数据分析
注: 固定化配体的局部 photoactivation 创建一个定义良好的固定刺激区域, 可用于定量分析信号响应和骨架重塑事件。分析协议通常涉及在辐照区域内量化荧光强度, 或者使用辐照区域作为距离测量的位置端点 (e. g. 用于评估中心体的偏振度到辐照区域)。下面介绍两种分析协议。各种交互式图像分析程序可用于进行强度和距离测量, 然后可以输出以进行额外的处理和分析。
<ol><li>
荧光强度
	<ol>
<li>确定单元格外部的背景区域 (fib) 的荧光强度 (fi)。这将用于背景更正。<ol>
<li>在单元格外绘制一个微米大小的正方形区域并制作一个面具。要确定荧光强度, 请单击分析 |掩码统计信息。选择平均值荧光强度并导出值。 注意: 过程详细信息 (例如3.1.1.1) 引用 Slidebook 软件 (请参阅材料表)。使用其他软件包 (e. g) 实现此协议将稍有不同。</li>
		</ol>
</li>
		<li>确定每个时间点的光活化作用区域内的 FI。<ol>
<li>选择掩码以突出显示光活化作用的区域。要确定荧光强度, 请单击分析 |掩码统计信息。选择平均值荧光强度并导出值。</li>
		</ol>
</li>
		<li>从区域内的实测 fi 值中减去背景 fi。然后, 通过在 photoactivation 前9帧的平均、背景校正 fi 划分, 使校正后的 fi 测量正常化。ΔF = (fib)/平均 (fi1-9)-fib))。 注: 图ΔF 作为时间的函数。</li>
	</ol>
</li>
	<li>
距离
	<ol>
<li>获取光活化作用区域中心的 x 和 y 坐标。<ol>
<li>要确定光活化作用区域中心的 x 和 y 坐标, 请选择掩码以突出显示光活化作用的区域。突出显示 photoactivation 区域后, 选择分析 |面具统计 |区域中心并导出值。</li>
		</ol>
</li>
		<li>确定每个时间点感兴趣的荧光探针的 x 和 y 坐标。这通常是通过手动或自动粒子跟踪实现的。<ol>
<li>要确定感兴趣的荧光探针的 x 和 y 坐标, 请选择手动粒子跟踪, 然后单击感兴趣的荧光探针。跟踪所有时间点后, 选择分析 |面具统计 |区域中心并导出值。</li>
		</ol>
</li>
		<li>使用等式计算每个时间点的荧光蛋白与光活化作用区域中心的距离: 距离 = √ (x2-x1)2 + (y2 1)2),其中 x2和 y2是感兴趣的荧光蛋白的坐标, x1和 y1是光活化作用区域中心的坐标。</li>
		<li>将距离图作为时间的函数。</li>
	</ol>
</li>
</ol>

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Organ. 105, 59-72 (2014).</li><li>Grusch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporally+precise+activation+of+engineered+receptor+tyrosine+kinases+by+light.">Spatio-temporally precise activation of engineered receptor tyrosine kinases by light.</a> EMBO J. 33, 1713-1726 (2014).</li><li>Yi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome+repositioning+in+T+cells+is+biphasic+and+driven+by+microtubule+end-on+capture-shrinkage.">Centrosome repositioning in T cells is biphasic and driven by microtubule end-on capture-shrinkage.</a> J. Cell Biol. 202, 779-792 (2013).</li></ol>

作者没有什么可透露的。

近年来, 光已成为 spatiotemporally 控制激活细胞过程的绝佳工具。开发了各种方法, 各有关联的优缺点。这里描述的系统, 这是基于 decaging 的固定化, 胞外配体, 是非常适合分析快速, 亚细胞, 极化信号响应。这种方法已被应用于检测 T 细胞的形成, 如上文所述。此外, 其他受体的笼型配体已被设计, 使我们能够应用同样的策略来评估自然杀伤细胞中的抑制信号和转录因子 NF-nf-κb 的核易位, 以回应像受体...

t 淋巴细胞 (t 细胞) 通过识别细胞表面 MHC 的抗原肽, 在细胞免疫中起重要作用。抗原识别是由 TCR 介导的, 它驱动了幼稚 T 细胞的分化, 促进了溶细胞的传递, 并推动了效应人群的交际反应。TCR 的参与也诱发了细胞结构的戏剧性变化。在几分钟内, T 细胞 gloms 到抗原呈现细胞 (APC) 的一侧, 形成一个极化接口, 称为免疫突触 (是)1,2。增强 t 细胞效应反应通过使细胞因子的定向释放, 或者, 在细胞毒性 T 淋巴细胞 (CTLs), 裂解蛋白, 破坏 APC。
TCR 参与抵押贷款公司诱导了多个下游适配器分子的快速磷酸化, 包括连接器的活化 T 细胞 (LAT), 这最终促进强健的重塑突触细胞骨架2。皮质丝状肌动蛋白 (F 肌动蛋白) 驱动 T 细胞传播在 APC 表面, 然后解决成环状结构的特点是 F 肌动蛋白积累在外围和耗尽从中心。F-肌动蛋白环形成紧密耦合到重新定位微管组织中心 (MTOC, 也称为中心体在 T 细胞) 到一个位置只是在界面的中心。这两种事件都发生在初始抗原识别的几分钟内, 并建立了随后激活事件和效应响应发生的体系结构上下文。
研究的是形成, 各种实验室已经开发的方法, 其中 APC 被一个玻璃表面取代, 其中包括固定化 TCR 配体或支持脂双层, 本身包含配体3,4。t 细胞形成类似的接触在这些表面上, 可以通过全内反射荧光显微镜 (TIRF) 或共聚焦显微成像, 使高分辨率研究早期 T 细胞活化和形成。
虽然这些方法已经允许充分组装的优秀可视化是, 许多信号跟随 TCR: 抵押贷款公司结扎发生在几秒钟之内, 复杂化努力确定事件序列在 TCR 活化以后准确地.为了规避此问题, 开发了一种 photoactivation 方法, 其中 photoactivatable 抵押贷款公司用于实现 TCR激活5、6、7的时空控制.在这一系统中, T 细胞附着在含有 photoactivatable 抵押贷款公司的玻璃表面上, 而非刺激 TCR 直到紫外线 (UV) 光照射。在 t 细胞下的微米尺寸区域的紫外线照射会使 photocage 产生一个可由 t 细胞识别的刺激区。随后的信号事件和骨架重塑, 然后监测使用基因编码荧光记者。两个 photoactivatable 版本的抗原肽, 蛾细胞色素 c88-103 (MCC) 和卵清蛋白257-264 (卵), 这是在第二类 mhc I-Ek和类 I mhc H2-Kb分别提出, 已已开发 (图 1)。这样, 就可以对 CD4k中特定的+ T 单元格进行分析 (表示5C。C7、2B4 或 TCRs) 和 CD8 OVA-H2-Kb (表示 OT1 TCR) 的+ T 单元格。
在过去的十年中, TCR photoactivation 和成像方法已被用来建立早期 TCR 信号步骤的精确动力学, 并确定的分子路径控制极化骨架重塑5,6,7,8,9,10. 例如, 这项化验有助于确定中心体转向 APC 的方向是由甘油中心的脂质第二信使的局部梯度介导的。预计这种方法将继续对要求高分辨率的 T 细胞功能成像分析的应用有价值。

<table><tbody><tr><td>Nunc Lab-Tek Chambered Coverglass</td><td>Thermofischer Scientific</td><td>155361</td><td></td></tr><tr><td>Poly-L-lysine hydrobromide</td><td>Sigma-Aldrich</td><td>P2636</td><td>Will need to make Biotinylated Poly-L-Lysine</td></tr><tr><td>EZ-Link NHS-Biotin</td><td>Thermofischer Scientific</td><td>20217</td><td>Will need to make Biotinylated Poly-L-Lysine</td></tr><tr><td>Streptavidin</td><td>Thermofischer Scientific</td><td>434301</td><td></td></tr><tr><td>BirA-500: BirA biotin-protein ligase standard reaction kit</td><td>Avidity</td><td>BirA500</td><td>Will be used to biotinylate proteins</td></tr><tr><td>Biotinylated Hb I-E&lt;sup&gt;K&amp;nbsp;&lt;/sup&gt;</td><td></td><td></td><td>For protein folding, see reference 6. For biotinylation, use BirA kit</td></tr><tr><td>Biotinylated NPE-MCC I-E&lt;sup&gt;K&amp;nbsp;&lt;/sup&gt;</td><td>Anaspec</td><td></td><td>Custom NPE-MCC (H-ANERADLIAYL-K(Nvoc)-QATK-OH) can be purchased from Anaspec</td></tr><tr><td>Biotinylated &amp;alpha;H2-K&lt;sup&gt;k&lt;/sup&gt; antibody</td><td>BD Biosciences</td><td>553591</td><td></td></tr><tr><td>Biotinylated NPE-OVA H2-K&lt;sup&gt;b&lt;/sup&gt;</td><td>Anaspec</td><td></td><td>Custom NPE-OVA (H-SIINFE-K(Nvoc)-L-OH) can be purchased from Anaspec</td></tr><tr><td>Biotinylated KAVY H2-D&lt;sup&gt;b&lt;/sup&gt;&amp;nbsp;</td><td>Anaspec</td><td></td><td>Custom synthesized protein (KAVYDFATL) can be purchased from Anaspec</td></tr><tr><td>Biotinylated ICAM1&amp;nbsp;</td><td></td><td></td><td>For protein folding, see reference in protocol. For biotinylation, use BirA kit</td></tr><tr><td>Hand held UV lamp</td><td>UVP</td><td>UVGL-25</td><td>Lamp is held &amp;lt; 1 cm from the sample.&amp;nbsp; 30 s of 365 light is sufficient for detectable decaging, 20 min for quantitative decaging.</td></tr><tr><td>Olympus IX-81 OMAC TIRF system.</td><td>Olympus</td><td></td><td>Additional information about the imaging system can be found in &lt;strong class=&quot;xfig&quot;&gt;Figure 6&lt;/strong&gt;</td></tr><tr><td>Mosaic digital diaphragm</td><td>Andor</td><td></td><td></td></tr><tr><td>Slidebook software</td><td>Intelligent Imaging Innovations</td><td></td><td></td></tr></tbody></table>

spatial and temporal control of t cell activation using a photoactivatable agonist

T 淋巴细胞参与快速, 极化信号, 发生在几分钟后 TCR 激活。这诱发了免疫突触的形成, 一种定型的细胞间结, 调节 T 细胞活化和定向靶效应反应。为了有效地研究这些过程, 需要一种适合捕获快速、极化响应的成像方法。该协议描述了这样一个系统, 它是基于 photoactivatable 肽-主要的组织相容性复合物 (抵押贷款公司), 它是不刺激的, 直到它暴露在紫外线。该试剂的靶 decaging 在 videomicroscopy 实验中, 通过全内反射 (TIRF) 成像, 实现对 TCR 活化和对后续细胞反应的高分辨率监测的精确时空控制。这种方法也符合遗传和药理的摄动策略。这允许组装明确定义的分子通路, 将 TCR 信号与免疫突触的极化骨架结构的形成联系起来。

photoactivation 和成像方法允许观察和简便量化快速, 极化信号响应。为了说明它的能力, 这里转载的是一个实验, 研究了 TCR 诱导的中心体的时空关系。5C。C7 T 细胞爆炸是 retrovirally 转基因与两个荧光记者: 一项由蛋白激酶 C-θ链接到与 GFP (C1-GFP) 和 RFP 蛋白的串联 C1 域的一台检测生物传感器, 以监测中心体。然后将 T 细胞连接到包含 photoactivatable coverglassk的腔内。C1-GFP ...

Watch this Scientific Journal Video about Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist at JoVE.com

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

Photoactivatable 激动剂对 T 细胞活化的时空控制

该协议描述了一种基于成像的方法, 激活 t 淋巴细胞使用 photoactivatable 肽 MHC, 使精确的时空控制 t 细胞活化。

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

spatial-temporal-control-t-cell-activation-using-photoactivatable

Research

JoVE Journal

Immunology and Infection

6.2K 次观看.  Memorial Sloan-Kettering Cancer Center. 该协议描述了一种基于成像的方法, 激活 t 淋巴细胞使用 photoactivatable 肽 MHC, 使精确的时空控制 t 细胞活化。

视频: Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. 
To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac4, Cdc424, RhoA4 and Ras5, in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells6. Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level6.

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

在活细胞中的小GTPase活性时空操纵在亚细胞水平上几秒钟的时间刻度

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

科研

生物工程

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

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

免疫与感染

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

生物学

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

成像的CD4 T细胞间质迁移发炎真皮

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

一个神经元蛋白在神经元使用遗传编码的非天然氨基酸的光控制

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

神经科学

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

单链 mhc 技术在人 cd8+ t 细胞活化中的相关研究

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist ...

Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity

Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain unanswered. One requirement for advancements in our understanding of the underlying disease mechanisms and cellular dynamics is the precise coupling of the microanatomical location of cell subsets with downstream molecular or functional analyses; a goal that has traditionally been difficult to achieve. The development of stable photoactivatable biological fluorophores and their integration into reporter strains has recently enabled precise microanatomical labeling and tracking of cellular subsets in murine models. Here, we describe how the ability to analyze autoreactive lymphocytes from single germinal centers may help to provide novel insights into autoimmunity, using the combination of a novel chimeric model of autoimmunity with a photoactivatable reporter as an example. We demonstrate a procedure for generating mixed chimeras with spontaneous autoreactive germinal centers populated by lymphocytes carrying a photoactivatable green fluorescent protein reporter. Using in vivo labeling strategies, single germinal centers can be visualized in explanted lymphoid tissues and their cellular constituents photoactivated by two-photon microscopy. Photoactivated lymphocytes from single germinal centers can then be analyzed or sorted flow cytometrically, as single cells or in bulk, and may be subjected to additional downstream molecular and functional analyses. This approach may directly be applied to provide renewed insights in the field of autoimmunity, but the procedure for generating bone marrow chimeras and the photoactivation procedure may additionally find broad application in studies of infectious diseases and tumor metastases.

This protocol describes the generation of mixed murine bone marrow chimeras with spontaneous autoimmune germinal centers, in which autoreactive lymphocytes carry a photoactivatable green fluorescent protein (PA-GFP) reporter. This provides the ability to link cellular location in tissues with downstream molecular and functional analyses.

在自抗性混合奇美体模型中, 光激活询问个体自反应萌发中心

Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain ...

<meta charset="utf8"></head><body>使用光调节变构开关对酶活性进行光遗传学控制

Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation

Optogenetics offers the potential for mimicking complex spatiotemporal control of enzyme activity down to a subcellular resolution. However, most optogenetic approaches often face significant challenges in integrating multiple capabilities in a single tool applicable to a wide range of target proteins. Achieving precise control over ON/OFF kinetics, ensuring minimum leakiness in the dark, and demonstrating efficient performance in mammalian cells with subcellular precision are some of the most common challenges faced in this field. A promising solution lies in the application of rationally designed light-sensitive domains to allosterically control protein activity. Using that strategy, we generated an optogenetic method combining all the desired features. The approach involves the incorporation of the Light-regulated allosteric switch module (LightR) in the target protein to regulate enzyme activity using blue (465 nm) light. The LightR domain is generated by linking two Vivid (VVD) photoreceptor domains, creating a light-sensitive clamp that can be incorporated into a small flexible loop within the catalytic domain of an enzyme. In its dark state, LightR clamp is open, thus distorting the enzyme&#39;s catalytic domain and inactivating it. Upon exposure to blue light, the LightR domain closes and restores the catalytic domain&#39;s structure and enzyme activity. In this manuscript, we discuss design strategies&#160;to generate a light-regulated protein kinase and demonstrate its control by blue light, reversibility, kinetics, and precise regulation at the subcellular level, enabling tight spatiotemporal precision. Utilizing Src tyrosine kinase as a model, we showcase a protocol for effectively regulating LightR-Src kinase activity. We also demonstrate LightR applicability across different enzyme classes, expanding the utility of the tool system in addressing mechanistic questions of signaling pathways in different diseases.

<meta charset="utf8"></head><body>通过光遗传变构调控对蛋白质活性进行时空调控

This protocol describes the application of an engineered blue-light-activated allosteric switch (LightR) domain for reversible spatiotemporal control of protein activity. Utilizing Src tyrosine kinase as a model, this study offers an elaborate protocol for developing and characterizing light-regulated Src (LightR-Src). It demonstrates the versatility of this approach across enzyme classes.

Photoactivatable 激动剂对 T 细胞活化的时空控制

摘要

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此视频中的章节

在活细胞中的小GTPase活性时空操纵在亚细胞水平上几秒钟的时间刻度

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

一个实时，同步成像的TCR和其相关的信号转导蛋白的TIRF显微镜技术

T细胞实时成像实时信号复合物的形成

在现场的斑马鱼胚胎的基于双光子Photoactivation

成像的CD4 T细胞间质迁移发炎真皮

一个神经元蛋白在神经元使用遗传编码的非天然氨基酸的光控制

单链 mhc 技术在人 cd8+ t 细胞活化中的相关研究

在自抗性混合奇美体模型中, 光激活询问个体自反应萌发中心

通过光遗传变构调控对蛋白质活性进行时空调控

Photoactivatable 激动剂对 T 细胞活化的时空控制

摘要

探索更多视频

此视频中的章节

在活细胞中的小GTPase活性时空操纵在亚细胞水平上几秒钟的时间刻度

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

T细胞实时成像实时信号复合物的形成

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

成像的CD4 T细胞间质迁移发炎真皮

一个神经元蛋白在神经元使用遗传编码的非天然氨基酸的光控制

单链 mhc 技术在人 cd8+ t 细胞活化中的相关研究

在自抗性混合奇美体模型中, 光激活询问个体自反应萌发中心

通过光遗传变构调控对蛋白质活性进行时空调控

一个实时，同步成像的TCR和其相关的信号转导蛋白的TIRF显微镜技术

在现场的斑马鱼胚胎的基于双光子Photoactivation