这项研究的部分得到了德意志 Forschungsgemeinschaft (SFB 1027, A6) 的资助。

注意: 一般实验工作流的概述如图 1所示。
警告: 对人类来说, 贸易协定是剧毒的。安全实验室许可 S2 (生物安全等级2当量) 是必要的。在整个实验中请戴上手套。
1. 在大肠杆菌中他标记的异种的异源表达
<ol>
	<li>用表达质粒 pET24-RTA(其) 6或空向量 pET24a(+)使用标准的电穿孔协议9,10转换大肠杆菌单元格。含有空质粒的细胞充当阴性对照。</li>
	<li>在含有卡那霉素的阳性克隆 (100 µg/毫升) lb 板后, 在5毫升kan培养基 (lb 培养基与100µg/毫升卡那霉素) 中接种含有区域内表达质粒或空向量的细胞, 并在37° c 和 220 rpm 下孵育 24 h。</li>
	<li>补充 1 L LB坎培养基与5毫升培养和孵育细胞在37° c 和 220 rpm 直到细胞达到了 OD600 0.8-1.0 (大约 3-4 h)。此后, 将培养温度降低到28° c, 并在 H2O 中准备1米的 isopropyl-β-D-1-thiogalactopyranoside (IPTG) 库存解决方案。</li>
	<li>通过在1毫米的最终浓度中加入 IPTG, 诱导大肠杆菌的表达。</li>
	<li>在 3.5 h 在28° c 和220转每分钟, 收获细胞由离心在 1万 x g 和4° c 为 10 min, 洗涤药丸二次以5毫升的捆绑缓冲 (500 毫米氯化钠, 10 毫米咪唑, 并且20毫米在2PO4, pH = 7.4) 在 1万 x g 和4° c 为 10 min, 和重颗粒在5毫升结合缓冲器。 注意: 协议在这个阶段可以暂停, 并且细胞可以被存放在80° c 几天。</li>
</ol>
2. 通过亲和层析纯化其标记的贸易量
<ol>
	<li>几种细胞在冰使用以下协议: 十五年代脉冲 (20 微米), 三十年代暂停。重复此步骤五次。</li>
	<li>离心分离器在 2.1万 x 和4° c 为15分钟和过滤上清液使用无菌注射器过滤系统 (0.2 µm 孔大小)。 注: 成功声样品的细胞颗粒显示透明边框。</li>
	<li>使用配备了5毫升 Ni2 +的亲和柱的自动净化系统, 从未消毒的过滤后的大肠杆菌上清纯化其标记的含量。一般而言, 使用1毫升/分钟的洗脱速度, 冷却整个净化系统, 以防止效率毒素的结合和毒素活性的丧失。 注意: 在表 1中列出了有效的贸易协定净化参数。参见贝克尔et al。有关详细信息,9。<ol>
		<li>简单地, 用 20 mL 的绑定缓冲区平衡关联列来删除存储缓冲区。用注射器将无菌过滤的上清液涂抹在亲和柱上。</li>
		<li>用25-35 毫升的结合缓冲器清洗柱, 从柱上移除未绑定的蛋白质。执行洗涤步骤, 直到 280 nm 的紫外线吸收接近初始 uv 值。</li>
		<li>洗在20-35 毫升的洗脱缓冲液 (500 毫米咪唑, 500 毫米氯化钠, 20 毫米,2PO4, pH = 7.2), 并保持样品在冰上 (图 2A和图 2B)。 注: 在紫外线吸收的情况下, 该组分的洗脱率明显增加。请注意, 专门收集这个分数, 以防止污染与非特异性结合蛋白。</li>
		<li>使用20µL 洗样品执行马斯亮蓝色染色11或西方印迹分析12,13 (可选)。使用初级抗他的抗体 (1:1, 000) 和第二 anti-mouse-IgG-HRP (1:10,000) 来检测和验证样品纯度 (图 3)。</li>
		<li>淡化洗在5毫升脱盐柱和平衡样品中的0.8 米山梨醇中的含量。<ol>
			<li>用脱盐柱取代纯化系统的亲和柱, 先平衡20毫升0.8 米山梨醇的柱。</li>
			<li>通过注射器将洗的比例分数应用到柱子上。当紫外线吸收开始增加 (图 2C) 时, 用100毫升0.8 米山梨醇和洗的盐水在15毫升管中清洗该柱。</li>
			<li>在4° c 下贮存洗的含量。 注: 当电导增加时, 直接停止含盐量的分数取样, 以避免盐分污染。脱盐过程的参数列于表1。</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>将洗 1万 x g 和4° c 的30-180 分钟集中度用 10 kDa 截止自旋柱到1-2 毫升的最终体积, 并在4° c 下储存样品3-4 周。 警告: 不要冻结样品, 因为冷冻会导致整个贸易协定活动的完全丧失。</li>
	<li>使用常规蛋白质测定试剂盒确定蛋白质浓度。蛋白质浓度应在 1-1. 5 毫克/毫升的范围内。 注: 如果蛋白质浓度太高 (#62; 5 毫克/毫升), 就会沉淀。</li>
</ol>
3. 酵母转化和细胞壁去除
<ol>
	<li>利用 gfp 报告质粒 pRS315-K28SP-gfp6使用标准醋酸锂转化方法14转换野生型或选定的酵母缺失突变体。在0.087% ° c 下孵育亮氨酸辍学 (d-/o) 葡萄糖板 (2% 葡萄糖, 1.5% 琼脂, 0.5% 硫酸铵, 0.17% 酵母氮基 (YNB) 和30的无亮氨酸混合) 的细胞, 用于阳性克隆选择的2-3 天。</li>
	<li>从每个板块挑选3不同的酵母克隆, 在100毫升的亮氨酸糖培养基中接种克隆 (2% 糖, 0.5% 硫酸铵, 0.17% YNB 和0.087% 的无亮氨酸的混合, 在 220 rpm 和30° c 到 OD600 = 1.0-2.0 (2-4 x 107细胞/毫升)。 注意: 不同酵母缺失菌株的细胞生长是不同的。通过分光光度计监视 OD600 。</li>
	<li>要计算 od600值, 请将样本稀释到 od600 = 0.1-0.3 (1:5 到1:10 稀释), 并在分光光度计中测量外径600 。作为参考, 使用 H2o 辅以相应数量的亮氨酸 raffionose 培养基。</li></ol>最后, 重 OD600 = 125 在5毫升的无菌水 (5 x 108细胞/毫升)。
	<li>离心细胞在25° c 为 5 min 在 1万 x g, 洗涤细胞两次与5毫升无菌水, 和重细胞在50毫升 spheroplasting 缓冲 (0.8 米山梨醇, 10 毫米三盐酸 (pH 7.5), 10 毫米 CaCl2, 2 毫米糖 (德勤), 和200µg/毫升溶解酶)。</li>
	<li>孵育50毫升培养在 100 rpm 和30° c 为 90 min. 可选: 每15分钟监测 spheroplast 形成的相衬显微镜。在显微镜下, 细胞应该有一个深半透明的灰色颜色。明亮 (折光) 细胞没有充分地被消化, 而鬼魂 (苍白灰色细胞与少许, 如果任何, 内部结构) 是 over-digested 的。</li>
	<li>检查每个样品的 spheroplast 制备效果。
	<ol>
		<li>完成步骤3.5 后, 混合4µL 的 spheroplasted 50 mL 文化与496µL spheroplasting 缓冲区 (约 2 x 106原生质) 和离心机为10分钟 400 x g。</li>
		<li>重颗粒在10毫升 H2O 蒸馏, 漩涡样品三十年代, 和板材出10µL 的样品上亮氨酸 d-/O 葡萄糖板 (2% 葡萄糖, 1.5% 琼脂, 0.5% 硫酸铵, 0.17% YNB, 和0.087% 的 d/o 混合没有亮氨酸)。</li>
		<li>孵育细胞3天在30° c。计算板上生长细胞菌落总数。对于数据评估, 只使用效率高于98% 的样本 (总细胞菌落数和 #60; 40 殖民地/板块)。</li>
	</ol>
	</li>
	<li>离心机细胞从步骤3.5 在 400 x g 和4° c 为10分钟和洗涤细胞两次与5毫升稳定亮氨酸 d/o 糖培养基 (0.8 M 山梨醇, 2% 糖, 0.5% 硫酸铵, 0.17% YNB, 和0.087% 辍学混合没有亮氨酸)。重细胞在5毫升稳定的亮氨酸 d/o 糖培养基 (1 x 108细胞/毫升), 并直接使用细胞的 GFP 记者化验测量 (4 节)。</li>

4. GFP 报告仪测定96井板
<ol>
	<li>将步骤3.7 中获得的酵母细胞原生质成 96-井板 (200 µL/井)。</li>
	<li>添加70µL 稳定的亮氨酸 d/o 糖培养基含有负控制 (洗的镍2 +亲和纯化细胞裂解 IPTG 诱导的大肠杆菌细胞表达空矢量) 或纯化的区域贸易协定在最后的贸易量集中5µM ( 对应于每井160克/升)。</li>
	<li>立即添加30µL 稳定的半乳糖溶液 (30% 半乳糖, 0.8 米山梨醇), 以诱导 GFP 的表达, 并随后开始测量。 注: 每次试验至少进行3技术复制, 每株酵母菌3次生物复制。</li>
	<li>完成样品制备后 (步骤 4.1-4.3), 把 96-井板在一个荧光阅读器, 并开始测量。使用 GFP 荧光检测所需的 475/509 nm 过滤器集。执行测量在30° c, 120 转每分钟, 并且以震动直径1毫米在时间窗口 20 h (测量的区间 10 min)。 注意: GFP 过滤器集通常在所有读卡器系统中都可用。温度、时间窗、测量间隔和浓度可根据自身需要进行调整。有代表性的结果如图 4所示。</li>
	<li>可选: 在质量控制的测量中使用额外的内部控制。通过添加30µL 稳定亮氨酸 d/o 糖培养基而不是30% 半乳糖 (无 GFP 诱导) 来准备负控制。此外, 添加70µL 0.8 米山梨醇稳定 G418 溶液 (300 µg/毫升)。蛋白质翻译抑制剂 G418 作为积极控制蛋白质抑制, 因为它防止 GFP 在酵母表达。</li>
	<li>根据以下公式计算 20 h 时间点的相对 GFP 荧光百分比 (请参见图 4A): <img alt="Equation" src="/files/ftp_upload/56588/56588eq1.jpg"> 其中 NC 为负控件
	<ol>
		<li>另外, 确定相对 GFP 荧光为每个测量点 (在这种情况下 10 min, 也参见步骤 4.4) 使用上述等式。如图 4B所示, 通过在时间 (x 轴) 上印迹 GFP 荧光强度 (y-axis) 来创建图形。</li>
	</ol>
	</li>
</ol>

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作者没有什么可透露的。

在执行上述协议时, 我们建议以下建议, 以取得成功的实验结果。
对于异源蛋白的表达, 重要的是不超过 IPTG 浓度的1毫米。IPTG 的浓度和 #62; 1 mM 抑制启动子诱导的贸易量表达, 导致毒素产量降低。此外, 细胞不应在高于28° c 的温度下培养, 以防止包涵体形成、低效折叠和毒素失活。在较低的温度 (例如, 20-28 ° c) 是可能的, 也导致生物活性毒素的生产。然而, 额外的温度降...

由毒素产生菌感染的病人是每个社会保健系统的严重的医疗和经济负担, 特别是因为有效的治疗治疗仍然很大程度上缺失。为了发展新的治疗策略, 在医学上相关的 a/b 毒素, 如霍乱毒素, 志贺毒素, 或蓖麻毒的复杂中毒机制, 需要在分子水平上充分了解, 基于新的强有力的化验, 必须实施。
近年来, 一些研究试图通过使用耗时和成本密集型的方法, 如放射性毒素标记1,2以及 siRNA-based 筛选来分析酵母和哺乳动物细胞中的 a/b 毒素转运接近3。在某些情况下, 毒素贩运已被可视化的在体内荧光显微镜后, 与荧光, 量子点, 或荧光蛋白的单个毒素亚基的化学和/或遗传耦合4,5。不幸的是, 这种修改往往导致不活泼和/或改变的自然性质的毒素。另一种可以间接回答各种科学问题的优雅方法是使用基于酶的记者系统, 如lacZ、荧光或荧光蛋白 (如GFP 或Discosoma sp. 红色荧光蛋白 (dsRed))。
在这篇手稿中, 一个简单的协议被描述为 extracellularly 在S. 酿酒酵母中的细胞内传输所需的细胞成分进行识别。因此, 荧光-报告质粒含有一个 N 终端 ER 输入信号, 其次是 gfp 作为蛋白质生物合成传感器, 这间接地测量了在体内的 gfp 荧光发射后的蛋白质的翻译抑制. 翻译6。如果与野生型相比, 在特定的酵母缺失突变体中, 吞和/或胞内贩运是消极的 (或积极的), 这可以通过 GFP 荧光发射的增加 (或减少) 检测6。
目前, 所有分析酵母细胞中的区域内转运的方法都被限制在 ER-to-cytosol 的转运过程中。在这样的人工系统中, 含有 ER 输入信号的含氟贸易协定是由一个可诱导的启动子表达的, 导致自杀型表现为1,7。虽然在本手稿中所描述的实验装置中, 蓖麻蛋白的细胞结合 B 亚基同样缺失, 因此, 不完全代表蓖麻毒素 holotoxin 中毒的自然情况8, 从等离子体中运毒膜通过高尔基体对 ER 的仪器可以与这种新的检测严密模仿。有趣的是, 在试点研究中取得的初步结果表明, 贸易协定所使用的贩运途径显示出与蓖麻毒 holotoxin 的中毒路线有惊人的相似之处。
总之, 所描述的方法可以用来确定特定的作用, 选择细胞蛋白在吞和贩运酵母。此外, 这种实验装置可以很容易地适应其他的核糖体灭活毒素产生和分泌的各种酵母和细菌物种, 如 zymocin 或志贺毒素。

<table><tbody><tr><td>&lt;strong&gt;Bacterial and yeast strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BL21 DE3(Gold)</td><td>Aligent Technologies</td><td>230130</td><td></td></tr><tr><td>&lt;em&gt;S. cerevisiae&lt;/em&gt; BY4742</td><td>Euroscarf</td><td>Y10000</td><td></td></tr><tr><td>&lt;em&gt;S. cerevisiae&lt;/em&gt; BY4742 deletion mutants</td><td>Dharmacon</td><td>YSC1054</td><td>whole collection</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Plasmids used in this protocol&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pET24a&lt;sup&gt;(+) &lt;/sup&gt;(Novagen)</td><td>Millipore</td><td>69772-3</td><td></td></tr><tr><td>pET-RTA&lt;sub&gt;(His6)&lt;/sub&gt;</td><td>Becker &lt;em&gt;et al.&lt;/em&gt; (2016)&lt;sup&gt;3&lt;/sup&gt;</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Zymolyase 20T</td><td>USBio</td><td>Z1000.250</td><td>lytic enzyme for cell wall removal</td></tr><tr><td>LB broth medium</td><td>Thermo Scientific</td><td>10855021</td><td>15 g agar was added for plate production</td></tr><tr><td>YNB</td><td>Thermo Scientific</td><td>DF0335-15-9</td><td></td></tr><tr><td>Ammonium sulfate</td><td>Sigma-Aldrich</td><td>A4418-100G</td><td></td></tr><tr><td>Yeast drop-out mix supplemts without leucine</td><td>Sigma-Aldrich</td><td>Y1376-20G</td><td></td></tr><tr><td>Agar</td><td>Sigma-Aldrich</td><td>05040-100G</td><td></td></tr><tr><td>D-glucose</td><td>Sigma-Aldrich</td><td>G8270-100G</td><td></td></tr><tr><td>DTT</td><td>Sigma-Aldrich</td><td>10197777001</td><td></td></tr><tr><td>D-raffinose</td><td>Sigma-Aldrich</td><td>83400-25G</td><td></td></tr><tr><td>D-sorbitol</td><td>Sigma-Aldrich</td><td>S1876-1KG</td><td></td></tr><tr><td>D-galactose</td><td>Sigma-Aldrich</td><td>G0750-10MG</td><td></td></tr><tr><td>G418</td><td>Thermo Scientific</td><td>11811031</td><td></td></tr><tr><td>IPTG</td><td>Sigma-Aldrich</td><td>I6758-1G</td><td></td></tr><tr><td>Imidazole</td><td>Roth</td><td>3899.1</td><td></td></tr><tr><td>PAGE ruler prestained</td><td>Fermentas</td><td>26616</td><td>protein ladder used for Western analysis</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Material for RTA purification, desalting and reader measurements&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Spectrophotometer Ultrospec 2100 pro</td><td>Amersham</td><td></td><td></td></tr><tr><td>Soniprep 150</td><td>MSE</td><td></td><td>old model, other models available</td></tr><tr><td>Fluoroskan Ascent</td><td>Thermo Scientific</td><td>5210470</td><td>old model, not available anymore</td></tr><tr><td>&amp;Auml;KTAPurifier</td><td>Thermo Scientific</td><td>28406266</td><td>Product is discontinued and replaced</td></tr><tr><td>HisTRAP HP column</td><td>GE Healthcare</td><td>17-5248-02</td><td></td></tr><tr><td>HiTRAP desalting column</td><td>GE Healthcare</td><td>11-0003-29</td><td></td></tr><tr><td>Midisart sterile filter</td><td>Sartorius</td><td>16534K</td><td>0.2 &amp;micro;m pore size</td></tr><tr><td>BCA protein assay kit</td><td>Pierce</td><td>23225</td><td></td></tr><tr><td>660 nm assay kit</td><td>Thermo Scientific</td><td>22660</td><td></td></tr><tr><td>96 well plates</td><td>Thermo Scientific</td><td>260860</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies (optional)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-Tetra-His</td><td>Qiagen</td><td>34670</td><td>primary antibody; 1:1,000 dilution</td></tr><tr><td>Anti-mouse-HRP</td><td>Sigma-Aldrich</td><td>A9044-2ML</td><td>secondary antibody, 1:10,000 dilution</td></tr></tbody></table>

a simple fluorescence-based reporter assay to identify cellular components required for ricin toxin a chain (rta) trafficking in yeast

细菌和植物 A/B 毒素利用真核细胞的自然贩运途径达到其在胞的细胞内目标, 并最终杀死。这种/B 类毒素一般由酶活性 Asubunit (如、蓖麻毒素 A ()) 和一个或多个细胞结合 Bsubunit (s) 组成, 后者负责对特定细胞表面受体的毒素结合。我们目前对甲乙毒素如何能有效地令人陶醉的细胞的知识帮助科学家了解基本的细胞机制, 如吞和细胞内蛋白在更高的真核细胞中排序。从医学的角度来看, 同样重要的是要找出主要的毒素贩运路线, 为病人找到适当的治疗方案, 或最终发展治疗毒素的癌症治疗应用。
由于对哺乳动物细胞的 a/b 毒素贩运的全基因组分析是复杂、耗时和昂贵的, 因此在酵母模型生物体酿酒酵母中进行了关于 a/b 毒素转运的几项研究。尽管不那么复杂, 酵母和高真核细胞中的基本细胞过程是相似的, 而且在酵母中获得的结果往往可以转移到哺乳动物的情况。
在这里, 我们描述一个快速和易于使用的记者化验分析的细胞内贩运的酵母。这种新的检测方法的一个重要优点是, 不仅有机会从内质网 (er) 中研究胞, 而且从细胞膜到 ER 的吞和逆行毒素的转运。该分析利用了一个记者质粒, 允许间接测量在体内的绿色荧光蛋白 (GFP) 的荧光发射后,在活体翻译。由于贸易协定有效地防止了蛋白质生物合成的 28S rRNA depurination, 这一分析可以通过检测荧光发射的变化来识别细胞内的细胞间贸易中所涉及的宿主细胞膜蛋白。

本手稿中所描述的协议的一般工作流程在图 1中作了说明, 大致总结了成功的贸易协定纯化和随后的 GFP 报告实验的单一步骤。可以在协议中找到每个单独步骤的更详细的描述。图 2说明了通过亲和层析 (图 2A) 和空矢量控件 (图 2B) 成功进行的贸易协定净化的预期结果。...

Watch this Scientific Journal Video about A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast at JoVE.com

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

一个简单的荧光记者的检测方法, 用于识别蓖麻毒素所需的细胞成分 a 链 (贸易协定) 贩运酵母

在手稿中, 我们描述了使用模型荧光记者分析, 以确定涉及的细胞成分的贩运和杀伤过程中的毒素 a 亚基的植物毒蓖麻。

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

a-simple-fluorescence-based-reporter-assay-to-identify-cellular

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

7.9K 次观看.  Saarland University. 在手稿中, 我们描述了使用模型荧光记者分析, 以确定涉及的细胞成分的贩运和杀伤过程中的毒素 a 亚基的植物毒蓖麻。

视频: A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

一个现场的固定化酵母细胞可视化的快速技术

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

科研

生物学

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is particularly relevant for survival in fluctuating environments such as those found in beer fermentation. However, sugar transitions are an environmental variable that is challenging to predict and detect, impacting the outcome of beer fermentations. This protocol describes an in vivo system to monitor transcriptional activation associated with the glucose-to-maltose metabolic shift in Saccharomyces eubayanus that applies to different wild Saccharomyces yeast strains. 
The system employs an episomal bioluminescent transcriptional reporter for maltose metabolism, focusing on MAL32, since it provides a good readout for metabolic shifts, as studied in S. cerevisiae. For this, yeast strains were transformed with plasmids containing the MAL32 regulatory region from S. eubayanus, controlling the expression of a gene encoding for a destabilized version of firefly luciferase1, and a hygromycin resistance gene used exclusively during transformation to ensure plasmid acquisition. Following selection, transformed yeast cells can be cultured under non-selective conditions, as the episomal plasmid remains stable in culture conditions for up to 7 days. 
This system was validated under a complex sugar environment in microfermentation assays, confirming the effectiveness of the luciferase reporter in informing metabolic transitions. Samples were collected regularly and analyzed with a luminometer, providing continuous insights into yeast responses. While broadly applicable, this protocol is particularly valuable for assessing yeast performance under fermentation conditions, where metabolic changes pose a significant challenge. Additionally, this methodology can be adapted by selecting alternative promoters to explore a broader range of responses to environmental changes, allowing characterization as well as optimization of wild yeast strains for diverse industrial applications.

This protocol employs a bioluminescent reporter, allowing measurements of transcriptional activity in Saccharomyces eubayanus to monitor the glucose-to-maltose transition, enabling real-time analysis of metabolic adaptations and supporting strain optimization for industrial fermentation under diverse conditions.

体内研究 使用酵母中的生物发光报告基因监测代谢转变过程中的转录活性

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is ...

遗传学

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

分析<em&gt;小肠结肠炎耶尔森菌</em&gt;效应易位到宿主细胞中使用β-内酰胺酶效应融合体

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

免疫与感染

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

在单酵母细胞MAPK诱导表达的时空定量

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

成长型测定和遗传需求生化确认的蛋白质降解<em&gt;酿酒酵母</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

pHluorin的申请在芽殖酵母细胞内吞的定量，动力学和高通量分析

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

荧光蛋白对酵母中多谷氨酰胺毒性测定的融合伙伴的影响

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

一个简单的荧光记者的检测方法, 用于识别蓖麻毒素所需的细胞成分 a 链 (贸易协定) 贩运酵母

摘要

探索更多视频

此视频中的章节

一个现场的固定化酵母细胞可视化的快速技术

体内研究使用酵母中的生物发光报告基因监测代谢转变过程中的转录活性

分析

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

裂殖酵母的活细胞定量荧光显微镜分析

在单酵母细胞MAPK诱导表达的时空定量

成长型测定和遗传需求生化确认的蛋白质降解

pHluorin的申请在芽殖酵母细胞内吞的定量，动力学和高通量分析

荧光蛋白对酵母中多谷氨酰胺毒性测定的融合伙伴的影响

通过流动细胞测定检测和描述 Vivo 中的蛋白质自组装

一个简单的荧光记者的检测方法, 用于识别蓖麻毒素所需的细胞成分 a 链 (贸易协定) 贩运酵母

摘要

探索更多视频

此视频中的章节

一个现场的固定化酵母细胞可视化的快速技术

体内研究 使用酵母中的生物发光报告基因监测代谢转变过程中的转录活性

分析

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

裂殖酵母的活细胞定量荧光显微镜分析

在单酵母细胞MAPK诱导表达的时空定量

成长型测定和遗传需求生化确认的蛋白质降解

pHluorin的申请在芽殖酵母细胞内吞的定量，动力学和高通量分析

荧光蛋白对酵母中多谷氨酰胺毒性测定的融合伙伴的影响

通过流动细胞测定检测和描述 Vivo 中的蛋白质自组装

体内研究使用酵母中的生物发光报告基因监测代谢转变过程中的转录活性