Name: single-cell quantification of protein degradation rates by time-lapse fluorescence microscopy in adherent cell culture
Uploaded: 2018-02-04T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture at JoVE.com

在生物分子筛查设施 (BSF), 洛桑进行了时间推移显微镜实验。我们感谢马克 Delachaux (服务 Audiovisuel, 洛桑) 的录像和编辑的电影。

注: 本研究采用 E14 ES 细胞系。然而, 该协议直接适用于任何其他的小鼠 ES 细胞系, 表达一种与 SNAP 标签融合的感兴趣的蛋白质, 或者通过标记内源蛋白或使用过度表现。在结果部分所示的例子中, 使用了强力霉素-诱导的 snap 标记融合细胞系 (snap 标记融合到以下蛋白质: Nanog, Oct4, Srsf11, 或对荧光蛋白 mOrange2 和 sfGFP, 并置于控制下强力霉素诱导的启动子。有关用于生成强力霉素诱导的 SNAP 标记融合细胞系的质粒的进一步信息, 请参见11 。强力霉素诱导的系统可以特别有用, 因为它能够严格控制的时间和强度的表达的蛋白质的利益。建议采用 c-终端定位, 因为改变 n 端氨基酸序列更有可能改变目标蛋白的半衰期 (n 端规则12)。
1. e-钙黏附素涂层和细胞播种
<ol><li>生成一个 ES 单元格线, 表示与快照标记4融合的感兴趣的蛋白质。</li>
	<li>100毫米细胞培养皿4毫升0.1% 明胶 (稀释磷酸盐缓冲盐水 (PBS), 没有 Ca2 +/毫克2 +) 为 1 h. 除去明胶, 让干燥 1 h. 立即使用或将涂覆的盘子储存多达2月。</li>
	<li>培养 es 细胞培养基中的细胞系 (格拉斯哥最低必需培养基, 辅以 10% es 细胞合格胎牛血清, 2 毫米丙酮酸钠, 1% 非必需氨基酸, 1% 青霉素/链霉素, 2 毫米 l-谷氨酰胺, 100 µM 2-基, 白血病抑制因子 (LIF), 3 µM CHIR99021 和0.8 µM PD184352) 在明胶涂层的菜肴在37° c 和 5% CO2 2 天, 直到到达一个汇合 10-20 的宇达细胞每道菜。 注意: 在这里, LIF 是由 HEK 293T 细胞的瞬态转染产生的, 其次是上清收集和过滤。每批 LIF 测试的潜力, 以保持多, 但浓度没有确定。然而, 重组 LIF 也可以在商业上使用, 并且通常用于小鼠 ES 细胞培养的浓度是1000单位/毫升13。</li>
	<li>外套一个96井板适合成像与30µL 重组小鼠 e-钙黏蛋白 fc 嵌合体蛋白质 (e-cad-fc) 每井 (5 ng/µL, 稀释在 PBS 与 Ca2 +/毫克2 +。使用库存浓度 100 ng/µL, 储存在-80 ° c)。避免广泛的移, 因为电子 cad Fc 是非常脆弱的。孵育在37° c 为1.5 小时。 注: 视显微镜而定, 可使用其他格式。</li>
	<li>吸气电子 cad Fc, 用100µL 的 PBS 与 Ca2 +/毫克2 +, 并添加100µL 预热 ES 细胞培养基。</li>
	<li>用5毫升 PBS 清洗感兴趣的细胞而不需要 Ca2 +/毫克2 +。吸气并加入2毫升的胰蛋白酶-EDTA 0.25%。孵育4分钟, 在37° c。添加4毫升 es 细胞培养基, 重和旋转 1000 x g, 4 分钟, 在2毫升的新鲜 es 细胞培养基中吸取上清和重颗粒。 注: 不含 ca2 +/Mg2 +的 PBS 应用于在 trypsinization 之前清洗单元格, 以删除 ca2 +离子, 这是细胞黏附所必需的。相比之下, 对于 e cad fc 稀释和涂层 (步骤 1.4), 使用 PBS 与 ca2 +/Mg2 +, 因为 ES 细胞与 e-cad-fc 涂覆的盘子的黏附力是 ca2 +依赖14。</li>
	<li>在 ES 细胞培养基的1毫升中稀释悬浮细胞 1:10, 使用计数室 (载荷10µL 为 0.1 mm 深度的腔)。</li>
	<li>种子3万细胞/cm2在电子 cad-Fc 涂层成像板。用 ES 细胞培养基填充200µL。对于强力霉素诱导的细胞系, 诱导与适当剂量强力霉素 (优化剂量和时间诱导提前。在这里使用的细胞系治疗 500 ng/毫升强力霉素24小时前成像)。孵育在37° c 和 5% CO2 , 并进行脉冲标记和成像24小时后, 细胞播种。</li>
</ol>2. 标签的脉冲标记
注意: 对于蛋白质衰变实验来说, 使用适当的染料浓度是至关重要的。浓度应足够高, 以产生一个明亮的信号, 在开始时的时间推移, 因为荧光将减少随着时间的推移。然而, 使用太高的染料浓度可能会导致残留染料留在培养基或在细胞中, 即使在洗涤后。在电影的过程中, 游离染料可能会绑定到新产生的 SNAP 标记分子, 这将扭曲衰变曲线。所观察到的荧光信号将取决于染料的性质、所使用的细胞线以及相应蛋白质的表达水平。因此, 通过测试不同的稀释, 从制造商对活细胞成像的稀释建议开始, 对染料浓度进行优化是至关重要的。本研究采用了一种远红光荧光基板。测定了强力霉素诱导的过度表达细胞株的 12 nM 的最佳浓度。
<ol><li>在预热 ES 细胞培养基中, 将该染料稀释到适当浓度。使用吸管轻轻地从先前在96井板上播种的细胞中去除培养基, 并在每个井中添加50µL 的稀释的对齐染料。孵育30分钟, 在37° c。</li>
	<li>用吸管吸去稀释后的染料, 用200µL 预热 PBS (不含 Ca2 +/Mg2 +) 洗涤3x。添加200µL ES 细胞培养基, 孵育15分钟, 在37° c。</li>
	<li>重复前面的洗涤步骤两次以上。 注: 为了去除任何未粘合的染料, 广泛的洗涤是重要的。重复洗涤步骤与 PBS 去除残留的胞外染料在培养基中, 而15分钟孵化确保在开始成像实验之前, 对捕捉标签融合蛋白的鲁棒标记。然而, 通过温和的移来执行洗涤步骤, 不要使用自动器, 因为在电子 cad Fc 上播种的细胞倾向于容易分离。</li>
	<li>添加200µL 成像介质。为减少荧光背景, 使用酚红游离培养基 (辅以 10% ES 细胞合格胎牛血清, 2 毫米丙酮酸钠, 1% 非必需氨基酸, 1% 青霉素/链霉素, 2 毫米 l-谷氨酰胺, 100 µM 2-基, LIF, 3 µMCHIR99021 和0.8 µM PD184352)。</li>
</ol>3. 延时显微镜
<ol><li>使用适合实时成像的显微镜, 允许控制温度和 CO2。将温度设置为37° c, CO2到5% 之前使用, 并允许平衡为 1-2 h。</li>
	<li>把盘子放到显微镜下, 找到适合于成像的斑点。选择那些单元格分布均匀但不太密集的斑点, 这将有助于分析。将所选点的数目调整为分析所需的单元格数。在这项研究中, 每细胞线记录 3-5 点。</li>
	<li>选择目标。20X 目标适合 ES 细胞的大小。</li>
	<li>选择要记录的荧光通道的光照设置。根据感兴趣细胞的荧光信号强度调整激光功率和曝光率。确保获得一个强大的初始信号, 因为它会减少在电影的过程中, 但避免激光功率高于 30%, 以尽量减少光和漂白。本研究采用 632/22 nm (波长/带通) 的励磁滤波器、679/34 nm (波长/带通) 的发射滤波器、10% 的激光功率和 100 ms 的曝光时间。</li>
	<li>选择时间推移实验的成像间隔和周期。对于预期半衰期为 2-20 小时的蛋白质, 选择 12-24 h 的捕获时间, 间隔为15分钟。为更短的居住的蛋白质, 减少间隔时间和承购时间, 为更长的居住的蛋白质相应地增加。</li>
	<li>开始成像。</li>
</ol>4. 图像处理与分析
<ol><li>以 tiff 格式将获取的图像作为堆栈收集。要进行进一步的处理和分析, 请使用斐济软件15。如果显微镜软件未将时间间隔数据收集成堆栈, 请在软件中打开时间推移实验的所有帧 (通过单击文件|打开...或将相应的文件拖放到工具栏上), 然后单击图像|堆栈|要堆叠的图像。</li>
	<li>使用减法背景函数从堆栈中的所有图片中删除背景。为此, 请单击进程|减去背景。在弹出窗口中选择滚动球半径。使用的半径至少是所成像单元格的大小。若要将背景减法应用于堆栈中的所有单元格, 请在 "进程堆栈" 弹出窗口中选择是。</li>
	<li>选择一个感兴趣的单元格, 并使用工具栏在它周围绘制一个感兴趣的区域 (ROI)。对于核信号, 使用椭圆形选择, 首先单击工具栏中相应的选项卡, 然后单击感兴趣的核心并调整其周围的椭圆。对于细胞质信号或非常密集的区域使用徒手选择可以密切遵循细胞轮廓。避免包含太大的背景区域, 即使没有必要精确地跟踪单元格的轮廓, 因为所有背景像素的后续减法 (步骤 4.8-4.9)。</li>
	<li>通过单击分析 |工具 |投资回报率经理 |在键盘上添加或按T 。</li>
	<li>通过在 "堆栈" 窗口中移动选项卡, 或按键盘上的 "shift + <", 然后重复前面的操作, 继续到下一帧。在整个电影过程中跟踪感兴趣的单元格, 并将每个帧的 roi 添加到 roi 管理器中。单击其他| 保存每个跟踪单元格的最终 ROI 集在 ROI 管理器中保存..。 注意: 在细胞分裂之后, 分开地跟踪两个子细胞并且在背景减法之后总结他们的综合强度 (参见步骤 4.6-4.9) 在细胞分裂期间考虑蛋白质稀释。保存两个子单元格的 ROI 集。</li>
	<li>在整个影片中跟踪感兴趣的单元格后, 单击度量, 以获取平均强度和 roi 区域的值。将获得的值复制到电子电子表格程序中, 并计算每个时间点的单元格的原始集成强度, 如下所示: <img alt="Equation 1" src="/files/ftp_upload/56604/56604eq1.jpg"> 其中平均值c是平均强度和区域c相应 ROI 的区域。</li>
	<li>若要估计本地背景, 请在每个时间框架的感兴趣的单元格附近添加一个 ROI。避免包括任何细胞荧光信号。使用一个近似于单元格大小的循环 ROI, 如果需要, 可以相应地移动或收缩它,例如如果相邻的单元格干扰。按照前面所述的蜂窝 roi 集进行操作, 以便获得背景 roi 集并将测量的强度值复制到电子表格中。</li>
	<li>为了获得对单元格的综合强度的背景校正值, 首先计算每个时间点的背景的综合强度: <img alt="Equation 2" src="/files/ftp_upload/56604/56604eq2.jpg"> 其中平均值BG 是背景信号的平均强度,区域C 是环绕单元格的 ROI 区域。不要使用背景 ROI 区域, 除非两个区域的大小相同。</li>
	<li>计算每个时间点的单元格的最终背景减去集成强度: <img alt="Equation 3" src="/files/ftp_upload/56604/56604eq3.jpg">
</li>
	<li>为了使单个细胞衰变曲线正常化, 将每个时间点的强度值除以第一个时间点的强度值。 注意: 曲线拟合 (参见步骤 4.11) 可以在每个单个单元格上执行, 也可在总体平均值上进行。如果计算基于总体的平均值以避免细胞间不同荧光强度的偏差, 则需要进行规范化。因此, 正常化确保每个单元对最终衰变曲线具有相同的权重。此外, 规范化可以很有用地可视化单个细胞衰变, 独立于其绝对荧光强度 (参见图 3 a-3 c)。但是, 如果只确定单单元半寿命, 且数据不平均, 则可以省略规范化步骤, 并可直接对原始数据执行曲线拟合。</li>
	<li>对于蛋白质半衰期的估计, 使用曲线拟合工具。在这项研究中, 使用了 matlab 曲线拟合工具箱 3.4.1, 它位于 matlab 用户界面的应用程序部分的默认位置。通过单击导入数据选项卡, 将电子表格中的荧光强度和时间值导入到 MATLAB 中. 打开 "曲线拟合工具箱", 然后在X 数据和Y 中选择时间点和荧光衰减数据数据选项卡. 选择自定义公式在 "曲线拟合" 选项卡中, 输入指数衰减的公式: <img alt="Equation 4" src="/files/ftp_upload/56604/56604eq4.jpg"> 其中, f (t)是给定时间点的荧光强度, a初始强度和b衰变率。在适应选项... 选项卡中, 为a和b的下限选择0。然后, a和b的估计值将显示在结果窗口中。计算半寿命如下: <img alt="Equation 5" src="/files/ftp_upload/56604/56604eq5.jpg">
</li>
</ol>

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D., Lee, M. R., Broxmeyer, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Aminoimidazole-4-carboxyamide+Ribonucleoside+Induces+G1/S+Arrest+and+Nanog+Downregulation+via+p53+and+Enhances+Erythroid+Differentiation.">5-Aminoimidazole-4-carboxyamide Ribonucleoside Induces G1/S Arrest and Nanog Downregulation via p53 and Enhances Erythroid Differentiation.</a> Stem Cells. 30 (2), 140-149 (2012).</li><li>Lin, Y., 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=Reciprocal+Regulation+of+Akt+and+Oct4+Promotes+the+Self-Renewal+and+Survival+of+Embryonal+Carcinoma+Cells.">Reciprocal Regulation of Akt and Oct4 Promotes the Self-Renewal and Survival of Embryonal Carcinoma Cells.</a> Mol Cell. 48 (4), 627-640 (2012).</li><li>Wei, F., Scholer, H. R., Atchison, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sumoylation+of+Oct4+Enhances+Its+Stability,+DNA+Binding,+and+Transactivation.">Sumoylation of Oct4 Enhances Its Stability, DNA Binding, and Transactivation.</a> J Biol Chem. 282 (29), 21551-21560 (2007).</li><li>Gautier, A., 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=An+Engineered+Protein+Tag+for+Multiprotein+Labeling+in+Living+Cells.">An Engineered Protein Tag for Multiprotein Labeling in Living Cells.</a> Chem Biol. 15 (2), 128-136 (2008).</li><li>Los, G. V., 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=HaloTag:+A+Novel+Protein+Labeling+Technology+for+Cell+Imaging+and+Protein+Analysis.">HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis.</a> ACS Chem Biol. 3 (6), 373-382 (2008).</li></ol>

作者没有什么可透露的。

当使用 SNAP 标签来监测蛋白质衰变时, 最关键的一步是确保在培养基中或在洗涤后的细胞中没有残留的未粘合染料, 否则它可能在实验过程中与新产生的 SNAP 标签分子结合在一起。伊破坏衰变曲线。这是一方面通过仔细执行所有描述的洗涤步骤实现的。另一方面, 染料的浓度应该保持尽可能低, 而仍然在一个范围, 允许获得一个良好的信噪比。如图 2所示, 应通过测试不同的稀?...

众所周知, 细胞蛋白具有广泛的周转, 合成和降解率是特定的每个蛋白质和受生理调节。传统上, 蛋白质降解率已被测量使用散装方法, 如放射性脉冲追逐分析, 或涉及蛋白质合成抑制剂, 如放线菌1。最近, 与质谱联用的细胞培养 (SILAC) 中的氨基酸的稳定同位素标记已经建立, 以在全球范围内量化蛋白质周转量2。但是, 这些方法受人口平均的限制, 因此, 关于细胞间可变性的信息就会丢失。此外, 无法确定在整个细胞中不同步的蛋白质降解的瞬态变化。
另外, 蛋白质半衰期也可以通过荧光的方法来确定, 这通常具有提供单细胞分辨率的优点。例如, 一个 photoactivatable 的绿色荧光蛋白 (paGFP) 已经被用来确定早期哺乳动物胚胎中的 Oct4 半衰期3。另一种监测活细胞中蛋白质衰变的方法是使用捕捉标记与荧光延时成像相结合。SNAP 标签是一个变种版本的 dna 修复酶 O6-alkylguanine dna-alkyltransferase (AGT), 特别是与 benzylguanine (BG) 衍生物, 这可以耦合到分子探针4,5,6. 因此, 任何捕捉标签的融合蛋白都可以用荧光、细胞渗透染料进行不可逆转的标记。脉冲标记的兴趣蛋白融合到 SNAP 标签, 其次是残留染料的冲洗, 允许监测的标记蛋白的人口的退化, 从而确定蛋白质半生命。SNAP 标签已成功地用于蛋白质的脉冲追逐标记和确定蛋白半生命的黏附细胞培养和在体内5,7,8,9。在商业上有大量的对齐标签衬底覆盖常用的荧光光谱, 使每个特定应用的最佳染料的选择。因此, SNAP 标签也可以用于多色成像结合其他荧光融合蛋白或染料。细胞不渗透性染料适用于膜栓蛋白的标记, 而细胞通透性染料则适用于细胞内和膜结合蛋白的监测。此外, 这些探针中的一些几乎没有基本的荧光, 只有在绑定到 SNAP 标记10时才开始发出强烈的荧光信号。
本协议描述了如何使用 SNAP 标签测量单个细胞中不同蛋白质的降解率。我们将这种方法应用于小鼠胚胎干细胞培养的 e-钙黏蛋白, 但它应该有可能使用它与任何黏附培养的细胞类型。我们表明, 脉冲标记的快照标签融合蛋白, 其次是荧光时间推移成像, 允许确定的单个细胞半衰期的各种蛋白质的兴趣, 并提供了一个估计的细胞变化的一半生命在培养细胞的数量。

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ES cell line expressing a SNAP-tag fusion protein of interest</td><td>-</td><td>-</td><td></td></tr><tr><td>Falcon 100 mm TC-Treated Cell Culture Dish</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>96 Well, Black/Clear, Tissue Culture Treated Plate</td><td>Corning</td><td>353219</td><td></td></tr><tr><td>Neubauer-improved counting chamber, 0.1 mm</td><td>Marienfeld-superior</td><td>640030</td><td></td></tr><tr><td>CO2 Incubator</td><td>Panasonic</td><td>MCO-170AICUV-PE</td><td></td></tr><tr><td>Centrifuge 5804 R</td><td>Eppendorf</td><td>5804000528</td><td></td></tr><tr><td>InCell Analyzer 2200 Cell Imaging System</td><td>GE Healthcare Life Sciences</td><td>29027886</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>Glasgow Minimum Essential Medium</td><td>Sigma-Aldrich</td><td>G5154</td><td></td></tr><tr><td>Fetal Bovine Serum, embyonic stem cell-qualified</td><td>ThermoFisher</td><td>16141-079</td><td></td></tr><tr><td>Sodium pyruvate solution</td><td>Sigma-Aldrich</td><td>113-24-6</td><td></td></tr><tr><td>Minimum Essential Medium&amp;nbsp; Non-Essential Amino Acids</td><td>ThermoFisher</td><td>11140-035</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>BioConcept</td><td>4-01F00H</td><td></td></tr><tr><td>L-Glutamine 200mM</td><td>ThermoFisher</td><td>25030-024</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>63689-25ML-F</td><td></td></tr><tr><td>Leukemia Inhibitory factor</td><td>-</td><td>-</td><td>Produced in the lab by transient transfection of HEK-293T cells, followed by collection and filtering of the supernatant.&amp;nbsp;</td></tr><tr><td>CHIR99021 (GSK-3 Inhibitor XVI)</td><td>Merck Millipore</td><td>361559</td><td></td></tr><tr><td>PD 0325901</td><td>Sigma-Aldrich</td><td>391210-10-9</td><td></td></tr><tr><td>Gelatin from bovine skin</td><td>Sigma-Aldrich</td><td>9000-70-8</td><td></td></tr><tr><td>Dulbecco&#039;s PBS 10x concentrated</td><td>BioConcept</td><td>3-05K00-I</td><td></td></tr><tr><td>Dulbecco&#039;s PBS Without Ca++/Mg++</td><td>BioConcept</td><td>3-05F29-I</td><td></td></tr><tr><td>Trypsin-EDTA-Solution 0.25%</td><td>Sigma-Aldrich</td><td>T4049</td><td></td></tr><tr><td>Recombinant Mouse E-Cadherin Fc Chimera protein</td><td>R&amp;amp;D systems</td><td>748-EC-050</td><td></td></tr><tr><td>Doxycycline hyclate</td><td>Sigma-Aldrich</td><td>D9891</td><td></td></tr><tr><td>SNAP-Cell 647-SiR</td><td>New England BioLabs</td><td>S9102S</td><td></td></tr><tr><td>FluoroBrite DMEM</td><td>ThermoFisher</td><td>A18967-01</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;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FIJI</td><td>-</td><td>-</td><td>Open-source image analysis software</td></tr><tr><td>MATLAB R2014a</td><td>Mathworks</td><td>-</td><td></td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td>-</td><td></td></tr></tbody></table>

single-cell quantification of protein degradation rates by time-lapse fluorescence microscopy in adherent cell culture

蛋白质处于合成和降解的动态状态, 它们的半衰期可以在各种情况下进行调节。然而, 最常用的测定蛋白质半衰期的方法要么仅限于裂解细胞的总体平均值, 要么需要使用蛋白质合成抑制剂。该协议描述了一种方法来测量蛋白质半衰期在单一的生活粘附细胞, 使用捕捉标签融合蛋白结合荧光时间推移显微镜。任何有兴趣的蛋白质都可以通过与 benzylguanine 衍生物结合的荧光、细胞渗透染料与一个标签的共价键结合, 并且在残留染料被冲蚀后可以对标记蛋白的衰变进行监测。随着时间的推移, 细胞的跟踪和综合荧光强度的量化导致了每个跟踪细胞的指数衰减曲线, 从而可以通过曲线拟合确定单个细胞的蛋白质降解率。这种方法为培养细胞中半生命体的异质性提供了一个估计, 这是不能用其他方法来评估的。这里提出的方法适用于任何类型的培养粘附细胞表达的兴趣蛋白融合到一个 SNAP 标签。在这里, 我们使用小鼠胚胎干细胞生长在 e-钙黏附素涂层细胞培养板, 以说明如何单细胞降解率的蛋白质与广泛的半衰期可以确定。

所描述的协议提供了一个估计的细胞的可变性在半衰期的任何给定的蛋白质融合到一个 SNAP 标签。使用重组 e-钙黏附素-Fc 用于涂层的成像板允许单细胞分辨率在 ES 细胞, 否则生长在殖民地。可以在整个影片过程中单独跟踪单个单元格 (图 1A)。
为了确定每个单细胞的蛋白质半衰期, 重要的是要测量的集成, 背景扣除的 SNAP 标签荧光信号随着时间的推移 (<str...

Watch this Scientific Journal Video about Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture at JoVE.com

Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

该协议描述了一种方法, 以确定蛋白质半生命的单一生活粘附细胞, 使用脉冲标记和荧光时间推移成像的捕捉标签融合蛋白。

粘附细胞培养的时间推移荧光显微镜对蛋白质降解率的单细胞定量研究

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used ...

single-cell-quantification-protein-degradation-rates-time-lapse

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

8.6K 次观看.  École Polytechnique Fédérale de Lausanne (EPFL). 该协议描述了一种方法, 以确定蛋白质半生命的单一生活粘附细胞, 使用脉冲标记和荧光时间推移成像的捕捉标签融合蛋白。

视频: Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

科研

发育生物学

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer&#39;s and Parkinson&#39;s diseases. Cellular senescence is a state of permanent cell cycle arrest that typically occurs in response to exposure to sub-lethal stresses. However, like other non-dividing cells, senescent cells remain metabolically active and carry out many functions that require unique transcriptional and translational demands and widespread changes in the intracellular and secreted proteomes. Understanding how protein synthesis and decay rates change during senescence can illuminate the underlying mechanisms of cellular senescence and find potential therapeutic avenues for diseases exacerbated by senescent cells. This paper describes a method for proteome-scale evaluation of protein half-lives in non-dividing cells using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) in combination with mass spectrometry. pSILAC involves metabolic labeling of cells with stable heavy isotope-containing versions of amino acids. Coupled with modern mass spectrometry approaches, pSILAC enables the measurement of protein turnover of hundreds or thousands of proteins in complex mixtures. After metabolic labeling, the turnover dynamics of proteins can be determined based on the relative enrichment of heavy isotopes in peptides detected by mass spectrometry. In this protocol, a workflow is described for the generation of senescent fibroblast cultures and similarly arrested quiescent fibroblasts, as well as a simplified, single-time point pSILAC labeling time-course that maximizes coverage of anticipated protein turnover rates. Further, a pipeline is presented for the analysis of pSILAC mass spectrometry data and user-friendly calculation of protein degradation rates using spreadsheets. The application of this protocol can be extended beyond senescent cells to any non-dividing cultured cells such as neurons.

This protocol describes the workflow for metabolic labeling of senescent and non-dividing cells with pulsed SILAC, untargeted mass spectrometry analysis, and a streamlined calculation of protein half-lives.

使用代谢标记和质谱法测量衰老和非分裂培养细胞中的蛋白质周转率

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as ...

生物化学

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

免疫与感染

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 ...

生物学

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts 

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

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 ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

记者的增长分析的蛋白质降解的系统分析

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

测定法错误折叠的蛋白质在细胞中的降解

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

Measuring Diurnal Rhythms in Autophagic and Proteasomal Flux

Cells employ several methods for recycling unwanted proteins and other material, including lysosomal and non-lysosomal pathways. The main lysosome-dependent pathway is called autophagy, while the primary non-lysosomal method for protein catabolism is the ubiquitin-proteasome system. Recent studies in model organisms suggest that the activity of both autophagy and the ubiquitin-proteasome system is not constant across the day but instead varies according to a daily (circadian) rhythm. The ability to measure biological rhythms in protein turnover is important for understanding how cellular quality control is achieved and for understanding the dynamics of specific proteins of interest. Here we present a standardized protocol for quantifying autophagic and proteasomal flux in vivo that captures the circadian component of protein turnover. Our protocol includes details for mouse handling, tissue processing, fractionation, and autophagic flux quantification using mouse liver as the starting material.

We describe our protocol for measuring biological rhythms in protein catabolism via autophagy and the proteasome in mouse liver.

测量自噬和前列腺通量中的日节律

Cells employ several methods for recycling unwanted proteins and other material, including lysosomal and non-lysosomal pathways. The main ...

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high throughput. In general, the acquisition of single-cell time courses from cultured cells is hampered by cell motility and diversity of cell shapes. Adhesive micro-arrays standardize single-cell conditions and facilitate image analysis. LISCA combines single-cell microarrays with scanning time-lapse microscopy and automated image processing. Here, we describe the experimental steps of taking single-cell fluorescence time courses in a LISCA format. We transfect cells adherent to a micropatterned array using mRNA encoding for enhanced green fluorescent protein (eGFP) and monitor the eGFP expression kinetics of hundreds of cells in parallel via scanning time-lapse microscopy. The image data stacks are automatically processed by newly developed software that integrates fluorescence intensity over selected cell contours to generate single-cell fluorescence time courses. We demonstrate that eGFP expression time courses after mRNA transfection are well described by a simple kinetic translation model that reveals expression and degradation rates of mRNA. Further applications of LISCA for event time correlations of multiple markers in the context of signaling apoptosis are discussed.

Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics

We present a method for the acquisition of fluorescence reporter time courses from single cells using micropatterned arrays. The protocol describes the preparation of single-cell arrays, the setup and operation of live-cell scanning time-lapse microscopy and an open-source image analysis tool for automated preselection, visual control and tracking of cell-integrated fluorescence time courses per adhesion site.

单细胞阵列的活细胞成像 （LISCA） - 量化细胞动力学的多功能技术

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high ...

粘附细胞培养的时间推移荧光显微镜对蛋白质降解率的单细胞定量研究

摘要

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测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

使用代谢标记和质谱法测量衰老和非分裂培养细胞中的蛋白质周转率

单细胞荧光显微镜定量 Efferocytosis 的研究

裂殖酵母的活细胞定量荧光显微镜分析

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下

在单酵母细胞MAPK诱导表达的时空定量

记者的增长分析的蛋白质降解的系统分析

测定法错误折叠的蛋白质在细胞中的降解

测量自噬和前列腺通量中的日节律

单细胞阵列的活细胞成像（LISCA） - 量化细胞动力学的多功能技术

粘附细胞培养的时间推移荧光显微镜对蛋白质降解率的单细胞定量研究

摘要

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此视频中的章节

测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

使用代谢标记和质谱法测量衰老和非分裂培养细胞中的蛋白质周转率

单细胞荧光显微镜定量 Efferocytosis 的研究

裂殖酵母的活细胞定量荧光显微镜分析

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下

在单酵母细胞MAPK诱导表达的时空定量

记者的增长分析的蛋白质降解的系统分析

测定法错误折叠的蛋白质在细胞中的降解

测量自噬和前列腺通量中的日节律

单细胞阵列的活细胞成像 （LISCA） - 量化细胞动力学的多功能技术

单细胞阵列的活细胞成像（LISCA） - 量化细胞动力学的多功能技术