Name: analysis of the gap junction-dependent transfer of mirna with 3d-frap microscopy
Uploaded: 2017-06-19T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy at JoVE.com

德国联邦教育和研究部（FKZ 0312138A和FKZ 316159），梅克伦堡 - 西波美拉尼亚与欧盟结构基金（ESF / IVWM-B34-0030 / 10和ESF / IVBM-B35-0010 / 12），DFG（DA1296-1）和德国心脏基金会（F / 01/12）。此外，RD由罗斯托克大学医学中心（889001），DAMP基金会和BMBF（VIP + 00240）的FORUN计划支持。

根据罗斯托克大学医学中心动物护理的道德准则进行了涉及新生小鼠的本议定书中的所有步骤。 1.细胞培养皿和心肌细胞培养基的制备<ol><li>用PBS中的0.1％明胶包被细胞培养板，并在37℃下孵育4小时或在4℃下孵育过夜。取出明胶，使其在无菌层流空气流下干燥。 </li><li>准备由补充有10％胎牛血清（FBS）和1％青霉素/链霉素（P / S）的50mL的DMEM组成的细胞培养基。预热至37°C。 </li></ol> 2.新生儿心肌细胞的分离<ol><li>用无菌剪刀斩首新生儿小鼠（1-2天），沿胸骨打开胸部。使用镊子去除心脏，同时轻轻地将胸部按压在一起，并将心脏转移到含有冰冷HBSS的24孔板中（不含Ca 2+和Mg 2+ ）。 </li><li>使用无菌镊子去除非心脏组织和较大的血管。将清洁的心脏转移到含有冰冷HBSS的1.5mL管中。每管最多使用5颗心进行酶消化。 </li><li>用小剪刀将心脏夹碎成0.5至1毫米3片。 </li><li>使用1 mL微量移液管抽吸/添加HBSS，用HBSS洗涤切碎的心脏两次。加入酶前，彻底清除HBSS。 </li><li>对于新生儿心脏的酶消化，使用市售的试剂盒，并遵循制造商的方案（参见材料表 ）。每5分钟摇动含有切碎的心脏的管，同时用酶在37℃下孵育35分钟。 </li><li>将含有细胞培养基的未包被的细胞培养皿上的悬浮细胞悬浮1.5-2小时以允许非心肌细胞部分的粘附。如果高纯度重复此步骤需要心肌细胞。如果需要，进一步培养非心肌细胞部分。 注意:对于15个心脏的细胞悬浮液，可以在75cm 2的细胞培养瓶上进行预镀层步骤。通常，从一个新生儿心脏获得〜5×10 5个细胞。 </li><li>将上清液收集在15 mL锥形管中，并以300 x g离心10 min。将细胞重悬于1mL细胞培养基中。 </li><li>用Neubauer室血细胞计数器计数细胞，或使用等效方法。 </li><li>在密度为3×10 5个细胞/ cm 2的6孔板上平板分离的心肌细胞。在37℃和5％CO 2下孵育细胞过夜。 注意:任选地，细胞也可以在这一点被转染。然而，当在进行电穿孔之前将细胞培养一天时，观察到增加的活力。 </li></ol> 3.用荧光标记的miRNA进行转染<ol><li>预温水细胞培养基（参见步骤1.2）至37℃在水浴或等效装置中。 </li><li>在无RNase的无菌水中制备20μM的荧光miRNA储备溶液。 </li><li>制备含有90mM Na 2 HPO 4，90mM NaH 2 PO 4，5mM KCl，10mM MgCl 2和10mM琥珀酸钠的电穿孔缓冲液，并将pH调节至7.2;电穿孔缓冲液可以在-20°C储存几个月。 </li><li>使用0.05％胰蛋白酶从培养皿中分离细胞5分钟。通过添加细胞培养基灭活胰蛋白酶。 </li><li>用Neubauer室血细胞计数器计数细胞，或使用等效方法。将细胞以300xg离心10分钟。 </li><li>将细胞重悬于电穿孔缓冲液中以获得每100μL4×10 5个细胞的浓度。 </li><li> 将100μL细胞悬浮液与荧光miRNA（终浓度:0.25μM）在管中混合将混合物装入电穿孔试管。 <ol><li>根据细胞类型经验性地确定适当的miRNA数量。 </li></ol></li><li>使用"G-009"程序，使用电穿孔装置进行电穿孔（参见材料表 ）。 </li><li>加入500μL预热后的细胞培养液，将全细胞悬浮液（4×10 5个细胞）转移到4孔玻璃底部载玻片的孔中。在37℃，5％CO 2气氛中培养细胞1天。 注意:对于FRAP分析，细胞密度为〜80％是最佳的。标记的miRNA的转染应该仅使用电穿孔进行。由于标记的miRNA分子的均匀分布对于FRAP测量是有益的，因此不推荐基于试剂的转染。 </li></ol> 4.应用3D-FRAP显微镜（第3天） <ol><li>将显微镜孵化器预热至37°; C，并在FRAP测量前至少2小时开启共焦显微镜系统，建立热平衡并降低漂移的可能性。如果可能，保持5％二氧化碳气氛。 </li><li>将腔室载玻片插入平台样品架。 </li><li>使用1.4 NA油物镜（400倍放大）和561nm激光激发光在低激光功率下寻找转染的心肌细胞簇，检测范围为570-680nm。 注意:使用显微镜专用软件（参见"材料表" ）对FRAP设置，图像采集和分析进行了定义 。 </li><li> 激活"安装管理器"中的"z-Stack"，"时间序列"，"漂白"和"区域"按钮。 </li><li> 定义FRAP参数。 <ol><li>通过设置"帧大小"来定义"采集模式"菜单中的图像采集设置4;到512×512，"行步"为1，"扫描时间"为&lt;1秒。 </li><li>在"通道"菜单中，调整激光功率，偏移和增益设置，以从最小激光激发（ 例如，激光功率:1-5％）获得最大荧光;调整以避免强度饱和。将"针孔尺寸"设置为2μm。 </li><li>接下来，在"区域"菜单中，选择"ROI绘图工具"，并使用光标标记目标单元格，参考单元格和背景区域。如果需要，选择几个目标细胞进行光漂白。 </li><li>调整"漂白"菜单中的漂白设置（ 例如，迭代:9-14，光漂白激光功率:100％，图像采集间隔:60秒，周期:15）。定义3次初始扫描后的漂白开始。 </li><li>在"z-Stack"菜单中，根据单元格的厚度定义z-stack采集的限制。调整"数字of z层"至12 - 15。 </li><li>开始FRAP实验并记录荧光恢复。 注意:由于FRAP实验的设置取决于细胞类型，荧光染料和显微镜系统，最好进行试验性实验以确定FRAP的最佳参数。漂白应足以将初始荧光强度降低至少50％。通常，每30-60秒应记录荧光恢复，直到达到平台期。 </li></ol></li></ol>数据分析<ol><li> 创建所获得的z-叠层的最大投影并获得漂白的靶细胞，参考细胞和背景的荧光强度值。使用显微镜专用软件，点击"处理"→"最大强度投影"→选择文件→"应用"。 <ol><li>或者，使用等效图像分析工具（ <e例如，ImageJ）。 </li></ol></li><li> 将所有时间点的荧光强度数据从目标单元格，背景和参考单元复制到电子表格中。 <ol><li>从目标单元的强度值中减去参考单元的背景强度和强度，以校正在采集过程中产生的漂白。对每个时间点执行背景和参考单元校正。 </li><li>通过将每个值除以初始荧光，将校正的FRAP数据归一化为漂白前的初始荧光强度。 </li><li>为了获得FRAP曲线，通过从每个时间点的强度值中减去，将基线设置为漂白后立即的荧光强度。 </li></ol></li></ol>

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Y., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+delivery+of+miRNAs+for+cancer+therapy:+Challenges+and+strategies.">In vivo delivery of miRNAs for cancer therapy: Challenges and strategies.</a> Adv Drug Deliv Rev. 81C, 128-141 (2015).</li></ol>

作者宣称没有利益冲突。

<html> miRNA是细胞生理学中的关键参与者，并被证明通过使用G_s作为细胞间交换途径，作为信号分子11,12,22。目前的方案提出体外活细胞成像技术来表征使用荧光miRNA在细胞簇内的GJ依赖性穿梭。 该方案作为细胞模型系统在心肌细胞上开发。然而，如果电穿孔是miRNA转染的合适方法，则该方法可以应用于几种细胞类型。除了细胞间细胞交换，所提出的技术也适用于研究miRNA?...

小的非编码RNA是细胞基因调控的重要参与者。这些分子由与特异性靶mRNA结合的20-25个核苷酸组成，导致翻译的阻断或mRNA降解1,2 。由小RNA（例如miRNA和siRNA）进行的基因调控过程是许多不同物种中发现的高度保守的机制。特别地，miRNA分子对多种生理过程至关重要，包括增殖，分化和再生4,5 。此外，miRNA表达的失调归因于许多病理学障碍。相应地，miRNA已经被证明适合作为诊断的生物标志物和用作基因治疗的治疗剂6,7 </suP&gt;。 间隙连接（GJs）是两个相邻细胞的质膜中的专门的蛋白质结构，允许分子量高达1kD的扩散交换。已被证明对于组织发育，分化，细胞死亡和病理性疾病如癌症或心血管疾病8,9,10很重要 。已经描述了几个分子能够穿过GJ通道，包括离子，代谢物和核苷酸。有趣的是，还发现GJs为小RNA 11,12的细胞间移动提供了途径。因此，miRNA不仅可以在其产生的细胞内，而且在受体细胞内起作用。这突出了miRNA在细胞间信号转导系统中的作用。同时，数据显示该间隙连接细胞间通讯与miRNA功能密切相关。由于miRNA和GJs对组织稳态，病理学和诊断的重大影响，全面了解GJs的功能和miRNA的相关细胞间动力学有助于阐明基于miRNA的疾病的机制，并制定新的策略miRNA疗法。 根据间隙连接耦合的程度，miRNA分子在细胞间的转移可以是非常快速的过程。因此，需要一种允许可视化和量化这些调节信号分子的快速细胞间移动的方法。通常，已经应用流式细胞术和分子生物学技术来证明小RNA的穿梭11,12,13,14。然而，与FRAP微观相反复制，这些方法缺乏高时间分辨率，这是通过GJs分析miRNA的交换时强制的。此外，FRAP显微镜侵入性较小，因此代表了一种功能强大且新颖的活细胞成像技术，用于评估几种15,16,17型细胞中分子的GJ依赖性交换。 在这里，我们提出了一个详细的协议，描述了3D-FRAP应用于评估心肌细胞之间的miRNA穿梭。为此，用荧光标记的miRNA转染心肌细胞。用该miRNA标记的细胞被光漂白，并且以时间依赖的方式记录来自相邻细胞的间隙连接miRNA再流入。 FRAP实验的高时间分辨率提供了进行动力学研究以准确评估活细胞之间的miRNA和siRNA的细胞间转移的可能性。 Moreo由于小RNA可以通过具有高度不同动力学的不同机制进行交换，FRAP显微镜可以帮助澄清GJ在相应的穿梭过程18中涉及的程度。此外，3D-FRAP可用于研究GJ通透性的生理和病理学改变及其对小RNA转移的影响15,19。

<table><tbody><tr><td>neonatal NMRI mice</td><td>Charles River</td><td></td><td></td></tr><tr><td>Gelatin</td><td>Sigma Aldrich</td><td>G7041</td><td>0.1% solution in PBS, sterilized</td></tr><tr><td>PBS</td><td>Pan Biotech</td><td>P04-53500</td><td></td></tr><tr><td>Dulbecco&amp;acute;s modified medium</td><td>Pan Biotech</td><td>P04-03550</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>100 U/mL, 100&amp;micro;g/mL</td></tr><tr><td>Fetal bovine serum</td><td>Pan Biotech</td><td>P30-3306</td><td></td></tr><tr><td>Cell culture plastic</td><td>TPP</td><td></td><td></td></tr><tr><td>Primary Cardiomyocyte Isolation Kit</td><td>Thermo Fisher Scientific</td><td>88281</td><td></td></tr><tr><td>HBSS</td><td>Thermo Fisher Scientific</td><td>88281</td><td>included in primary cardiomyocyte isolation kit</td></tr><tr><td>Trypan blue</td><td>Thermo Fisher Scientific</td><td>15250061</td><td></td></tr><tr><td>miRIDIAN Dy547 labeled microRNA Mimic</td><td>Dharmacon</td><td>CP-004500-01-05</td><td>dissolve in RNAse free water, stock solution 20&amp;micro;M</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma</td><td>S3139</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Carl Roth</td><td>T877.2</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>Magnesium chloride</td><td>Serva</td><td>28305.01</td><td></td></tr><tr><td>Sodium succinate</td><td>Carl Roth</td><td>3195.1</td><td></td></tr><tr><td>0.05% Trypsin/EDTA solution</td><td>Merck</td><td>L2153</td><td></td></tr><tr><td>4-well- Glass bottom chamber slides</td><td>IBIDI</td><td>80827</td><td>coat with 0.1% gelatin solution</td></tr><tr><td>Amaxa Nucleofector II</td><td>Lonza</td><td></td><td>program G-009 was used for Electroporation&amp;nbsp;</td></tr><tr><td>LSM 780 ELYRA PS.1 system</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Excel software</td><td>Microsoft</td><td></td><td></td></tr><tr><td>&amp;alpha;-actinin antibody</td><td>Abcam</td><td>ab9465</td><td>dilution 1:200</td></tr><tr><td>Connexin43 antibody</td><td>Santa Cruz</td><td>sc-9059</td><td>dilution 1:200</td></tr><tr><td>goat anti-mouse Alexa 594 antibody</td><td>Thermo Fisher Scientific</td><td>A-11005</td><td>dilution 1:300</td></tr><tr><td>goat anti-rabbit Alexa 488 antibody</td><td>Thermo Fisher Scientific</td><td>A-11034</td><td>dilution 1:300</td></tr><tr><td>Connexin43 siRNA</td><td>Thermo Fisher Scientific</td><td>AM16708 ID158724</td><td>final concentration 250 nM</td></tr><tr><td>Near-IR Live/Dead Cell Stain Kit</td><td>Thermo Fisher Scientific</td><td>L10119</td><td></td></tr><tr><td>CellTrace Calcein Red-Orange</td><td>Thermo Scientific</td><td>C34851</td><td></td></tr><tr><td>DAPI nuclear stain</td><td>Thermo Scientific</td><td>D1306</td><td></td></tr></tbody></table>

analysis of the gap junction-dependent transfer of mirna with 3d-frap microscopy

小反义RNA（如miRNA和siRNA）在细胞生理学和病理学中起重要作用，而且可用作治疗几种疾病的治疗剂。开发miRNA / siRNA治疗新的创新策略是基于对基础机制的广泛了解。最近的数据表明，小细胞以间隙连接依赖的方式在细胞之间进行交换，从而在受体细胞中诱导基因调控作用。分子生物学技术和流式细胞仪分析通常用于研究miRNA的细胞间交换。然而，这些方法不提供高的时间分辨率，这在研究分子的间隙连接通量时是必需的。因此，为了研究miRNA / siRNA作为细胞间信号分子的影响，需要新的工具，这将允许在细胞水平上分析这些小RNA。本协议描述了ap光漂白后三维荧光恢复（3D-FRAP）显微镜的澄清，以阐明心脏细胞之间的miRNA分子的间隙连接依赖性交换。重要的是，这种直接和非侵入性的活细胞成像方法允许实时荧光标记的小RNA的间隙连接穿梭的可视化和定量，具有高的时空分辨率。通过3D-FRAP获得的数据证实了细胞间基因调控的新途径，其中小RNA作为细胞间网络内的信号分子。

在这里，我们提出3D-FRAP显微镜作为非侵入性技术研究新生儿心肌细胞荧光miRNA间隙连接穿梭。分离的心肌细胞显示典型的条纹α-肌动蛋白模式，并且沿着细胞 - 细胞边界包含大量的Cx43斑块（ 图1A ，白色箭头），这允许分子的高细胞间通量。通过显微定量α-肌动蛋白阳性细胞评估分离的心肌细胞的纯度。虽然培养物中存在一些非心肌细胞（α-肌动蛋?...

Watch this Scientific Journal Video about Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy at JoVE.com

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

在这里，我们描述了光漂白后三维荧光恢复（3D-FRAP）的应用，用于分析miRNA的间隙连接依赖穿梭。与通常应用的方法相比，3D-FRAP允许实时定量小RNA的细胞间转移，具有高的时空分辨率。

用3D-FRAP显微镜分析miRNA的间隙连接依赖性转移

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

analysis-gap-junction-dependent-transfer-mirna-with-3d-frap

Research

JoVE Journal

Biology

8.8K 次观看.  Reference and Translation Center for Cardiac Stem Cell Therapy (RTC). 在这里，我们描述了光漂白后三维荧光恢复（3D-FRAP）的应用，用于分析miRNA的间隙连接依赖穿梭。与通常应用的方法相比，3D-FRAP允许实时定量小RNA的细胞间转移，具有高的时空分辨率。 

视频: Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

科研

生物化学

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

活细胞成像的移植细胞在一个三维矩阵表示荧光标记的蛋白质

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

生物工程

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

神经科学

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

荧光技术结合测定活细胞中的力敏感蛋白动力学

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

细胞形态变化，从三维荧光图像量化的分析工具，

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

生物学

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Cell的传播分析单细胞显微注射

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET成像的三维水凝胶

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

基于3D 荧光显微镜的病毒转录工厂功能成像

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.