这项工作得到了 NIH R01 EB014869 的支持。

1. 准备用于成像的细胞
注: 直接力探头 (DFP) 可用于任何粘附单元类型。在这里, NIH 3T3 小鼠成纤维细胞被用作该协议的模型细胞线。
<ol><li>培养 NIH 3T3 成纤维细胞在 Dulbecco 的改良鹰的培养基 (DMEM) 补充10% 捐赠牛血清和1% 青霉素-链霉素在35毫米玻璃底部菜, 直到理想的融合。保持细胞在37°c 和 5% CO2。<ol>
<li>一定要涂上所有35毫米玻璃底菜与5µg/毫升纤维连接蛋白 (或类似的 ECM 蛋白质), 然后播种 NIH 3T3 细胞进行成像。 注: 这些细胞必须在盘子上充分传播和附着, 以进行实验。对于 DFP 方法的工作融合, 没有任何限制。</li>
	</ol>
</li>
	<li>在实验之前, 先用 PBS 冲洗细胞两次, 然后用完全生长培养基进行一次冲洗。</li>
	<li>添加3毫升完全生长培养基的玻璃底部菜。</li>
</ol>2. 显微和图像采集
注: 根据制造商的建议, 用机器人安装在侧臂上的倒置荧光显微镜 (或等效)。该显微镜还应配备一个环境室, 以保持37摄氏度的温度, 并2水平在5%。还需要机器人和 microinjector 显微镜。试验中建议采用油浸泡 40 x/1.3 na 或 60 x/1.49 na (或等效目标)。该显微镜应安装在隔振台上。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58038/58038fig1.jpg"> 图 1.核形变与显微镜聚焦 a. 最大的核形变和放松核形变。在计算最大核形变之前, 核形状的后缘首先与变形核的平移相吻合。在微尖端脱离时, 细胞核的形状被叠加在最初的核形状上, 然后拉起。两个形状之间的面积的差异被测量为Δ1。最大核形变被定义为Δ1 除以原核区。同样, 第二个参数, Δ2, 可以定义的叠加最终稳态核形状后, 微支队的原始核形状。b. 将单元格集中在平面 A 上, 然后将焦点平面移至 B 平面, 以找到微尖。在成像过程中, 微被翻译成右侧 (橙色箭头方向)。这个数字已经从处处长 neelam. 15.<a href="https://www.jove.com/files/ftp_upload/58038/58038fig1large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<ol><li>根据制造商的协议打开 microinjector。</li>
	<li>使用浸入式油吸管, 在物镜的顶端涂一滴浸入油。</li>
	<li>把盘子紧紧夹在盘子里, 把盘子放在台上。 注: 在整个实验中, 细胞必须保持在37摄氏度和 5% CO2 。</li>
	<li>调整目标的高度以使单元格成为焦点 (平面 A,图 1B)。</li>
	<li>移动显微镜阶段找到感兴趣的细胞。</li>
	<li>旋转机器人上的操纵杆, 将吸管保持架移动到顶部位置。将0.5 µm 直径尖端微加载到吸管支架上。<ol>
<li>为避免细胞黏附于微, 在室温下用0.3 毫克/毫升锁相环-g PEG 溶液对微尖端进行预处理。通过接触微到原子核而没有任何吸入压力来测试附着力, 然后将微从原子核中平移。缺乏附着力可以从完全缺乏核形变和翻译中看出。 注: 请按照生产建议打开包装。</li>
	</ol>
</li>
	<li>调整精细控制 (图 1B, 见步骤 2.4), 将平面 A 和单元格顶部的目标焦平面提升为平面 B。</li>
	<li>将机器人设置为粗控。通过观察微的剪影, 慢慢地把微下到飞机 B, 直到微完全进入焦点。</li>
	<li>一旦微提示集中, 将机器人设置为 "精细控制"。</li>
	<li>将目标降低到单元格的赤道平面 (平面 a、图 1B) 并将微降低到平面 a (图 1B、虚线微) 的大约15µm。</li>
	<li>将 microinjector 的补偿压力 (Pc) 设置为所需的压力;等待几秒钟的压力, 以稳定。 注: 最佳压力设定点取决于细胞类型和实验的具体目标。在大多数情况下, 300 的 hPa 将是一个很好的起点。</li>
	<li>确保微不堵塞, 使用机器人面板上的清洁设置, 并检查以确保气泡出现从微尖端。</li>
	<li>通过逐渐降低微直到尖端轻轻接触核表面, 将尖端插入到细胞中。 注意: 当降低微时, 微尖的轮廓将变得清晰, 因为它进入焦点。在微接触核之前, 提高目标焦点, 并将微尖端与核 (相同的 x y 坐标, 更高的 z 平面) 对齐。将焦点返回到原子核的赤道平面 (平面 A,图 1B), 并逐渐降低微尖端。</li>
	<li>通过从微注射系统中拔下压力供应管, 在微尖端和核膜之间建立一个密封, 从而打开微管的末端到大气层。这一步产生的负压等于在核表面上的Pc 。</li>
	<li>利用显微镜图像采集软件获取图像。在图像收集软件中设置 avi 捕获 (视频) 或 nd 捕获 (图像)。 注: 对于任何图像获取软件, 设置实时视频成像或时间推移图像采集短时间间隔。</li>
	<li>切换到相应的荧光成像通道 (如GFP、RFP等) 并开始成像。</li>
	<li>将微尖从细胞体 (右图 1B) 平移, 直到原子核与微分离。 注: 沿正向 x 方向 (在视野中的右侧) 拉出笔尖。拉速可通过计算机编程控制, 或者操纵杆可以手动移动。我们还没有发现拉速和核形变15之间的任何关联, 这暗示着对力的主要弹性反应。</li>
</ol>3. 数据分析
<ol><li>使用任何基本的图像处理软件进行图像分析。核形变的程度可以通过长度应变 (Ɛ) 或区域应变来量化 (图 1A)。用方程1来量化长度应变, 其中 l 和 l0分别表示最大变形和初始位置的原子核长度。 <img alt="Equation 1" src="/files/ftp_upload/58038/58038eq1.jpg">(等式 1)</li>
</ol>

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作者没有什么可透露的。

测量细胞核与细胞骨架的机械结合是目前大多数方法的挑战, 如微吸入16, 因为它们需要两个孤立的原子核 (在细胞核与骨架分离的地方) 或悬浮细胞中的细胞核 (如牵引力, 无细胞外力)。通过将双轴应变应用于附着在膜17、18的细胞中, 将力应用于细胞核;然而, 这种技术是有限的事实, 在原子核的力量是未知的。原子力显微镜 (AFM) 探针被用来在完整的细胞中缩进细胞核;这些提供了优势, 他们揭示了核的力学性质, 而它与细胞骨架结合。为了增加12、13、14核的现有体内技术, 我们开发了一种简单而健壮的方法来机械地探测活体黏附细胞中的细胞核, 而它仍然是整合到周围的细胞骨架。这项技术的一个关键特点是, 原子核上的力是精确已知和控制的。
通过应用 DFP, 我们估计纳米牛顿尺度力足以在活体、粘附细胞中变形和转化细胞核。此外, 通过系统研究各种骨架元素的贡献, 我们发现, 波形的中间丝是细胞细胞骨架的主要组成部分, 负责加强细胞核在细胞15。
一些注意事项适用于这些实验。重要的是定期检查微提示堵塞或破损在实验中, 因为两个堵塞的微或骨折的提示将导致未知的变化, 对应用于细胞的吸入压力。要检查微是否堵塞, 请使用机械手上的清洁设置来施加最大压力, 并检查从微尖端出现的气泡。如果微提示断开或断开, 请在开始下一个实验之前替换微。此外, 重要的是要确认, 核应变变化的力量, 并零的力量 (见处处长 neelam等。15). 如果在实验中没有观察到这一点, 那么微尖端与核表面之间的非特定粘附可能会导致核形变。这项技术的另一个局限性是, 将微插入细胞本身可能会导致局部破坏骨架结构。
测试核形变/平移程度与加载速率之间的相关性是有益的。缺乏相关性将意味着主要的弹性阻力。事实上, 这是我们发现的情况下, 成纤维细胞核15。当原子核的力已知时, 该方法不允许计算像杨氏模量这样的参数。我们主要用它来解剖细胞结构对核形变和细胞中的转化的抗性贡献。虽然我们已经报告了二维核形状15,19, 它可能是有用的, 以量化的完整的三维的形状, 原子核的力量。
微的吸入压力是已知的, 但它比实际压力大, 因为水流通过毛孔10的核表面。然而, 我们已经表明, 通过微, 通过核孔隙流动的阻力要大得多 (参见处处长 neelam等的计算支持信息。15) 合理的膜渗透和微尺寸。因此, 尽管存在流动, 外膜表面的压力应该等于吸力压力。
最后, DFP 允许使用者将粘附细胞中的核细胞骨架的机械反应量化为已知的和受控制的力。该方法可用于解剖细胞核和细胞质中分子成分对细胞内细胞核力学行为的贡献。

如癌症等病症涉及对核形态和结构1,2的改变, 通常伴随着 "软化" 的核心3,4。对机械变形的核阻力一般是将力应用于孤立原子核5。
细胞核在细胞是分子连接到细胞骨架由 Nucleoskeleton 和细胞骨架 (林肯) 复杂6,7,8,9的链接器。因此, 细胞核是机械地与细胞骨架结合, 并通过细胞基质粘连, 细胞外基质。机械地探测黏附细胞内的细胞核可以提供对这种机械整合的洞察力。在活细胞中操纵细胞核的方法包括微吸入10、11和原子力显微镜12、13、14。我们最近描述了一个直接的力量探针 (DFP), 直接地应用机械力量在原子核在活黏附力细胞15。
在这里, 我们概述了使用显微注射系统的程序, 通常是在显微镜的设施, 以应用已知的, 纳米级的机械力直接到核在黏附细胞。femtotip (0.5 µm 直径微尖端) 安装并连接到微注射系统由一个管。尖端, 定位在45°角度相对于养殖皿的表面, 被降低, 直到相邻的核表面。管子然后被断开并且被打开对大气, 在核表面创造一个负吸力压力并且封印微尖端反对核表面。通过微尖端的翻译, 原子核变形, 最终 (取决于施加的力的大小), 脱离微。当由原子核和细胞施加的恢复力 (抵抗) 力等于微所施加的吸力时, 这种分离就会发生。分析可以通过测量原子核的位移、长度应变 (方程 1) 或区域应变 (图 1A) 来进行。

<table border="0" cellpadding="0" cellspacing="0" style="width:843px;" width="843">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">FluoroDish</td>
			<td style="width:71px;">WPI</td>
			<td style="width:119px;">FD35</td>
			<td style="width:355px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:299px;">SYTO 59</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td style="width:119px;">S11341</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Femtotips&#160;</td>
			<td style="width:71px;">Eppendorf</td>
			<td align="right">930000043</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">InjectMan NI2</td>
			<td style="width:71px;">Eppendorf</td>
			<td style="width:119px;">NA</td>
			<td>discontinued, current equivalent model: InjectMan 4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">FemtoJet</td>
			<td style="width:71px;">Eppendorf</td>
			<td style="width:119px;">NA</td>
			<td>Current model FemtoJet 4i</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Plan Fluor oil immersion 40x</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Apo TIRF oil immersion 60x</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:299px;">Donor Bovine Serum (DBS)</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">16030074</td>
			<td>NIH 3T3 serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dulbecco&#39;s Modification of Eagle&#39;s (DMEM)</td>
			<td style="width:71px;">Mediatech cellgro</td>
			<td style="width:119px;">MT10013CVRF</td>
			<td>NIH 3T3 medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:71px;">Mediatech</td>
			<td style="width:119px;">MT30004CIRF</td>
			<td>NIH 3T3 medium supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Immersion Oil Type LDF Non-Fluorescing</td>
			<td style="width:71px;">Nikon</td>
			<td align="right" style="width:119px;">77007</td>
			<td>Immersion oil for objective lens&#160;</td>
		</tr>
	</tbody>
</table>

a direct force probe for measuring mechanical integration between the nucleus and the cytoskeleton

原子核的力学性质决定了它对细胞中产生的机械力的反应。由于细胞核是细胞骨架的连续分子, 因此需要方法来探讨其在黏附细胞中的力学行为。在这里, 我们讨论直接力探针 (DFP) 作为一个工具, 以力量直接向细胞核在一个活生生的黏附细胞。我们用吸力将一个狭窄的微附着在核表面。微从原子核中被翻译出来, 使原子核变形和转化。当恢复力等于吸力时, 原子核分离, 弹性松弛。因为吸入压力是已知的, 核表面上的力是已知的。该方法表明, 纳米尺度力足以使粘附细胞中的细胞核变形和转化, 并确定骨架元素, 使得原子核能够抵抗力。DFP 可用于解剖细胞和核成分对活细胞核力学性能的贡献。

图 2A显示了 NIH 3T3 小鼠成纤维细胞核的强迫。当微尖端被翻译成右侧时, 原子核会变形, 最终与微尖端分离。随着抽吸力的增加, 原子核的长度应变增加 (图 2B)。原子核的前缘 (微拉边) 形成核突起, 尾部边缘从原位置偏移。突起的长度远远大于尾部边缘位移 (图 2C), 表明细胞核与周围细胞质紧密结合。时间尺度是短期的放松的核前沿 (< 1 s) 和核后缘 (< 2 s) (图 2D)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58038/58038fig2.jpg"> 图 2.核形变的表征 DFP 的活体、黏附细胞中细胞核的变形和位移。NIH 3T3 成纤维细胞核被拉扯了与 6 nN 力量。图像显示核形状在指定的时间。刻度条: 5 µm b. 核形变按长度应变进行量化。核形变随作用力的增加而增大。误差条表示平均值的标准误差 (SEM);n > 6。在前缘的位移大于尾部的位移。长度应变松弛比后边缘松弛快得多。*P < 0.05;误差条表示 SEM;n=10。这个数字已经从处处长 neelam. 15.<a href="https://www.jove.com/files/ftp_upload/58038/58038fig2large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
我们使用了 DFP 方法来确定骨架力是如何导致核电阻在细胞中变形的。虽然在 F 肌动蛋白 (由细胞松弛素) 或微管中断 (nocodazole) 后发现核形变或翻译没有显著差异, 但通过 siRNA 的击倒减少波形的表达, 大大提高了核翻译和变形 (图 3)。这表明, 成纤维细胞中的波形中间丝是帮助细胞核抵抗局部力的主要骨架元素。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58038/58038fig3.jpg"> 图 3.核形变的荧光图像 DFP 被用来确定骨架力对核形变的贡献。细胞核被 SYTO 59 染料染色。荧光图像在所述条件下显示核的叠加 (红色、伪色) 和后 (绿色、伪色)。CTRL, 控制单元格;细胞, 细胞细胞松弛素治疗;用 nocodazole 治疗的细胞;滚, 用炒 siRNA 转染的细胞;vim siRNA, 细胞转染 siRNA 靶向波形。这个数字已经从处处长 neelam. 15.<a href="https://www.jove.com/files/ftp_upload/58038/58038fig3large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>

Watch this Scientific Journal Video about A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton at JoVE.com

A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

测量细胞核与骨架间机械一体化的直接力探针

在本协议中, 我们描述了一种微方法, 直接将受控力应用于活细胞中的细胞核。这项化验允许审讯活体, 黏附细胞的核机械性质。

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous ...

a-direct-force-probe-for-measuring-mechanical-integration-between

Nanyang Technological University

Research

JoVE Journal

Biochemistry

15.1K 次观看.  University of Florida. 在本协议中, 我们描述了一种微方法, 直接将受控力应用于活细胞中的细胞核。这项化验允许审讯活体, 黏附细胞的核机械性质。

视频: A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

科研

生物化学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

原子力显微镜对单蛋白质分子的力谱

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which may be essential for a given protein&#39;s role both physiologically and pathologically. Enrichment of PTMs is often necessary when investigating the PTM status of a target protein, because PTMs are often transient and relatively low in abundance. Many pitfalls are encountered when enriching for a PTM of a target protein, such as buffer incompatibility, the target protein antibody is not IP-compatible, loss of PTM signal, and others. The degree of difficulty is magnified when investigating multiple PTMs like acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation for a given target protein. Studying a combination of these PTMs may be necessary, as crosstalk between PTMs is prevalent and critical for protein regulation. Often, these PTMs are studied in different lysis buffers and with unique inhibitor compositions. To simplify the process, a unique denaturing lysis system was developed that effectively isolates and preserves these four PTMs; thus, enabling investigation of potential crosstalk in a single lysis system. A unique filter system was engineered to remove contaminating genomic DNA from the lysate, which is a problematic by-product of denaturing buffers. Robust affinity matrices targeting each of the four PTMs were developed in concert with the buffer system to maximize the enrichment and detection of the endogenous states of these four PTMs. This comprehensive PTM detection toolset streamlines the process of obtaining critical information about whether a protein is modified by one or more of these PTMs.

Investigating multiple, endogenous post-translational modifications for a target protein can be extremely challenging. Techniques described here utilize an optimized lysis buffer and filter system developed with specific PTM-targeting, affinity matrices to detect the acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation modifications for a target protein using a single, streamlined system.

利用综合沉淀富集系统识别目标蛋白的内源性后向转化剖面

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

测定植物铁含量的比色法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

原子力显微镜对细胞外囊泡的成像

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

使用光子芯片的高通量总内反射荧光和直接随机光学重建显微镜

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

一种高通量酶偶联活性测定法，用于探测小分子与 dNTP 酶 SAMHD1 的相互作用

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...