This work was supported by National Institutes of Health grants R01-DK080834 and R01-DK093045. We thank J. Denise Wetzel, CCHMC Medical Writer, for editing of the manuscript.

1.独创性途径分析<ol><li>上传蛋白相互作用组数据集<ol><li>插入目的蛋白质/基因在其独特的基因标识（如用质谱数据而获得优选的基因符号和基因标识号）的电子表格。 </li><li>指定在电子表格中的基因标识号一列，为观测值一列（ 例如 ，折叠变化或p值）。选择"包含列标题"选项来查看列标题。 </li><li>通过单击上传数据选项卡上，选择上面提到的电子表格上传的IPA的数据集。选择灵活的格式选项卡，选择基因标识的相应类别。 注:该数据集的灵活的格式克服了格式规范上传。 </li><li>出现的数据后，选择ID和观察列。确保按质量-SP标识的所有蛋白质（MRP4相互作用组ectrometry）由数据汇总选项卡上检查映射。数据集现在准备用于所需的分析。 </li></ol></li><li>数据分析 注:所描述的是被用于该研究结果表明的IPA功能部分的用途。 <ol><li>上传MRP4相互作用组与基因名称作为唯一标识符后，点击新的选择IPA软件左上角的菜单岩心分析。 </li><li>设置了根据实验式为强度表达截断值（被推荐用于分析100强度值）。 </li><li>计算中表达的倍数变化，同时准备数据集，如果对照和实验条件都参与，并且如果比较分析需要执行它作为数据集文件的一部分（1.5倍改变的截止值通常建议用于分析）。 注意:芯分析完成后，可以得到分析的多个部分。第一个选项卡显示日摘要E共分析和建议以及其他顶级经典途径，上游监管机构，分子和细胞的功能，和网络;基于所述置信水平（p值）的所有计算和设置在P值的升序排列。 </li><li>打开各大经典途径（使用开放路径选择）作为大分子网络，形象地展现跨说话，重叠，影响信号通路。 看所涉及基于p值majorly的规范途径的列表（第0.05被认为是&lt;作为显著）。 注:为规范途径的意义值由Fisher精确检验右尾计算。的意义表明分子协会从单独的随机几率典型途径数据集的概率。 </li><li>确认涉及每个途径蛋白的列表中输入的蛋白质（通过质谱MRP4相互作用组）。 注意:使用此OPTION，有人指出，蜂窝肌动蛋白细胞骨架的信令网是主要受影响的途径和方式输入蛋白装配到它（ 图1）。这种方法有助于确定哪些分子网络会密切相关，并受测试的蛋白质。 </li><li>使用在分析网络标签识别顶部的疾病和功能都潜在地链接到基于聚焦的分子数特定蛋白网络。 注意:得分的值是基于被确定为一个网络例如一部分的已知蛋白质定，细胞装配和组织网络，通过文献的知识和实验证据和聚焦个从数据集中鉴定的分子，并成为网络的一部分。 。此后，网络被过滤以基于聚焦的分子数之首。 </li></ol></li></ol> 2.高含量显微镜<ol><li>细胞的制备 注:所有的细胞培养工作水平流动的细胞培养罩下进行。 <ol><li>使用96孔微孔板的伤口愈合测定法。 </li><li>涂层用1％纤连蛋白溶液的微量培养板（在PBS中复原），并在37℃下保持所述板在一个标准的 CO 2培养箱中培养2小时。 </li><li>删除MRP4 - / -和MRP4 + / +小鼠胚胎成纤维细胞（MEFs）中从培养箱25平方厘米的细胞培养瓶中生长，洗1次，用PBS，并trypsinize细胞用1毫升4倍胰蛋白酶-EDTA溶液5分钟。 </li><li>洗细胞用完全培养基（含10％FBS和1％青霉素/链霉素的DMEM）并在5ml完全培养基重悬。 </li><li>吸从孔的纤连蛋白溶液。算使用血球细胞和种子30,000个细胞（需要根据细胞类型进行优化单元编号）到每个孔中。 </li><li>生长细胞为100％汇合在一个标准的 CO 2培养箱在37℃下24小时。 </li></ol></li><li>创建伤口<ol><li>确保所有的井都充满了解决方案。填充闲置孔用PBS，以防止伤口使工具损坏（ 例如 ，WoundMaker）。 </li><li>确认100％的细胞汇合后，放置在金属基底板的伤口使工具内的96孔板，然后按96孔销块向下。 </li><li>洗涤细胞三次，用PBS，以消除任何脱落细胞，并加入100微升完全培养基。 </li><li>添加试验化合物 - 的H-89（50μM; 1:1000稀释使用完全培养基中的DMSO的50mM股票）或Latrunculin B（1微米; 1:1000稀释使用1mM储备在DMSO完全培养基）在这个阶段。 </li><li>放置读取器内的测定板在37℃和监测在实验期间迁移。 </li></ol></li><li>监测细胞迁移 注意:使用显微镜和软件PR监测细胞迁移ovided具有高含量显微系统。使用96孔微量确保伤口自动显微镜监测和由软件登记。 <ol><li>设置软件扫描测定板每小时使用时间表扫描标签迁移测定。放置测定板读取器内，以允许成像之前平衡后至少15分钟选择一个开始时间。 </li><li>选择托盘和容器选择类型（96孔板）和实验类型（划痕）。 </li><li>选择10X客观选择相衬成像参数。选择需要使用编辑扫描模式选项进行扫描的孔中。 </li><li>通过选择属性选项卡，并设立一个板图指定的治疗组和任何重复。扫描可以在根据实验条件的任何时间被停止。 </li></ol></li><li>数据分析<ol><li>扫描完成后，选择检测板的容器视图选项卡。 GO操作分析工作的实用程序和选择推出新的分析工作。 </li><li>设置划痕的作业类型，并选择分析（0时间点到24小时的时间点）的时间段。 </li><li>由上井一次点击来选择的分析选择井，然后单击"确定"按钮来启动分析。一旦分析完成后，以图形方式在每个时间点使用出口到图形选项形象化相对卷绕密度（RWD）数据为每个孔中。 </li><li>查看相衬图像集的每一个孔对应使用视图图像标签不同的时间点。点击选择平板地图上的具体的好，选择时间范围选项卡中的特定时间点，点击查看图像选项卡上。 </li></ol></li></ol> 3.荧光共振能量转移（FRET） <ol><li>细胞的制备<ol><li>用100微升1％，纤维连接蛋白溶液（如2.1节所述），并让他们在大衣35毫米玻璃底菜一个标准的 CO 2培养箱中于37℃2小时。 </li><li>吸从HEK293细胞的板和种子数目相等于完全培养基纤连蛋白溶液（10,000个细胞/板）。 </li><li>生长细胞60-70％汇合在纤连蛋白涂覆的玻璃底菜在一个标准的 CO 2培养箱中在37℃下24小时。 </li></ol></li><li>转<ol><li>执行使用根据制造商的说明在商业转染试剂所有瞬时转染。 </li><li>分装250微升完全培养基分成两个独立的1.5毫升无菌离心管中每个35mm培养皿。 </li><li>添加DNA（pmAKAR3或pmAKAR3-TA）的一个管和5μl2微克在另一管（2.5倍的DNA浓度）转染试剂。 </li><li>孵育20分钟，将管在室温下，然后将稀释的DNA转移到转染试剂含有管。 </li><li>调匀，于37孵育°下30分钟。 </li><li>吸出细胞培养基，并用PBS洗一次他们。加入1ml含有10％FBS到细胞中不含抗生素的DMEM F-12培养基中。 </li><li>添加507微升与媒体在每个板中的细胞中的DNA脂质缀合物，轻轻混匀，并保持在CO 2培养箱中在37℃下48小时。使用2微克DNA（1微克/微升股票）和5微升脂质为10,000-50,000细胞每块板。 </li></ol></li><li>活细胞成像<ol><li>转染48小时后，除去生长培养基并洗涤细胞2次用Hank氏平衡盐溶液（HBSS，于37预热 C）。 1.9毫升HBSS的终体积加入到洗涤的细胞和它们安装的倒宽视场显微镜系统上用于FRET成像定做的37℃内保持室中。 注:在本系统中，激发光通过与50％的光输电工程中性密度滤镜衰减一个300瓦氙灯提供离子。 </li><li>使用60X的目标。手动聚焦在细胞上，用显微镜设置的视图的最佳场。使用软件上的焦点"F"选项激活的查看计算机屏幕上选定的领域。为了执行FRET测量，选择适当的过滤器设置（CFP / YFP过滤用二十五分之四百三十零nm激发过滤器，双二色分光镜设置和两个发射滤光片（三十零分之四百七十〇纳米时CFP和30分之535的FRET纳米）手动。 </li><li>通过在通道选项CFP / YFP / FRET依次点击打开氟标签首先检查可视化荧光。通过使用相机选项卡中的集约化工具提高了信号强度。 </li><li>选择细胞中表达pmAKAR3或pmAKAR3-TA避免了信号强度的饱和。使用捕获​​窗口开始FRET测量。 </li><li>选择时间推移选项，并设置一个时间扫描与30秒的时间间隔30分钟。检查FRET选项，并设置曝光时间为100毫秒。输入图片的标签d按启动开始测量。 </li><li>后五个时间点和建立一个基线，加入25μM的毛喉素（5微升10μM的库存在乙醇中，用100微升的HBSS稀释）至细胞中，而不会干扰板和监测细胞共30分钟。 </li></ol></li><li>数据分析 注:数据分析使用比例计算模块。 <ol><li>点击在图像窗口的左侧选择工具，然后从下拉菜单中选择一个坚实的矩形。拖动单元格中的空白区域上的图像场的矩形，并用鼠标右键单击它设置为背景背景扣除的目的。 </li><li>进入面膜和使用&#39;创建一个新的掩码"选项。进入选择工具，然后从下拉菜单中用笔手动绘制细胞周围口罩，选择它进行测量。选择每个条件至少有4-6个细胞。 </li><li>选择Mask选项并执行面具的统计数据。 </li><li>检查"整个面具"，拓展跨渠道选项，并选择捐助标准化FRET（N-FRET）（FRET / CFP）。的N FRET值是代表PKA活性的在质膜。 </li><li>添加时间戳，使用注释查表和比例尺。 </li></ol></li></ol>

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D., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+dynamics+of+protein+kinase+A+activity+visualized+by+FRET-based+reporters.">Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters.</a> Biochem Biophys Res Commun. 348 (2), 716-721 (2006).</li><li>Ananthanarayanan, B., Ni, Q., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+propagation+from+membrane+messengers+to+nuclear+effectors+revealed+by+reporters+of+phosphoinositide+dynamics+and+Akt+activity.">Signal propagation from membrane messengers to nuclear effectors revealed by reporters of phosphoinositide dynamics and Akt activity.</a> Proc Natl Acad Sci U S A. 102 (42), 15081-15086 (2005).</li><li>Yamaguchi, H., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+actin+cytoskeleton+in+cancer+cell+migration+and+invasion.">Regulation of the actin cytoskeleton in cancer cell migration and invasion.</a> Biochim Biophys Acta. 1773 (5), 642-652 (2007).</li><li>Lamalice, L., Le Boeuf, F., Huot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+migration+during+angiogenesis.">Endothelial cell migration during angiogenesis.</a> Circ Res. 100 (6), 782-794 (2007).</li><li>Ghosh, M. C., Makena, P. S., Gorantla, V., Sinclair, S. E., Waters, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCR4+regulates+migration+of+lung+alveolar+epithelial+cells+through+activation+of+Rac1+and+matrix+metalloproteinase-2.">CXCR4 regulates migration of lung alveolar epithelial cells through activation of Rac1 and matrix metalloproteinase-2.</a> Am J Physiol Lung Cell Mol Physiol. 302 (9), L846-L856 (2012).</li><li>Vicente-Manzanares, M., Webb, D. J., Horwitz, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+at+a+glance.">Cell migration at a glance.</a> J Cell Sci. 118 (Pt 21), 4917-4919 (2005).</li><li>Zaccolo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded,+fluorescent+indicator+for+cyclic+AMP+in+living+cells.">A genetically encoded, fluorescent indicator for cyclic AMP in living cells.</a> Nat Cell Biol. 2 (1), 25-29 (2000).</li></ol>

The authors have nothing to disclose.

Cell migration is an intricate process that plays indispensable roles in many important physiological events including wound healing1,2. Aberrant cell migrations may cause catastrophic events, such as tumor metastasis and angiogenesis24,25. Therefore, fine-tuned regulation of cell migration is required to maintain normal body function.
Using high-content microscopy18, we demonstrated that MRP4-deficient MEFs migrate faster compared to wild-type fibroblasts14. In contrast to the conventional scratch wound assay, the microscopy here conducts the wound healing assay in an automated convenient, consistent and high-throughput manner. The software analyzes the cell migration rate based on three separate metrics: i) change in wound width, ii) change in cell confluence within the wound region, and iii) relative wound density (RWD). RWD is a self-normalized metric that measures the spatial cell density inside the wound area relative to the spatial cell density outside of the wound area. Therefore, it is not affected by changes in cell density due to cell proliferation and provides very specific information regarding cell migration which is otherwise difficult to be obtained by the conventional scratch wound assay26. Initially, at the 0 time point, the RWD will be 0% and upon complete wound healing, the RWD will be 100%. All of these metrics are calculated by custom-made software with inbuilt algorithms and the migration information is automatically generated for every time point. The assay is easy to perform but proper washing (70% ethanol) and handling (inside the hood) of the wound-maker is important to prevent contamination. Our data suggested that the RWD kinetics for Mrp4-/- MEFs are significantly higher compared to Mrp4+/+ MEFs.
During migration, cells polarize into leading and trailing edges that ultimately pull the cell body toward the direction of migration1,27. Distinct and segregated signaling events at different regions of a moving cell ensure the polarization process. Polarized accumulation of cAMP and subsequent activation of PKA at the leading edge is a key early step in directional cell migration5. Since MRP4 has very high affinity for cAMP (Km = 45 &#181;M)12, we hypothesize that the effect of MRP4 on cell migration is cAMP-dependent. To identify the proteins acting downstream of MRP4 and simultaneously interacting with MRP4, we characterized MRP4-containing macromolecular complexes by mass-spectrometry. The MRP4 interactome was subjected to multiple analyses including generation of protein networks, path maps, and functional integration to the canonical pathways of the cellular signaling and their respective pictorial representation through the use of IPA. In general, IPA allows scientific users to recognize the molecular and physiological contexts of their genes and proteins of interest. However the analysis is completely based on the literature and experimental evidences. Novel interactions cannot be suggested by IPA. But it can identify which network, the proteins of interest, can potentially form. Additionally users can identify the top diseases and functions that are potentially linked to a particular protein network based on the confidence level (P value). Of interest to our study, the actin cytoskeleton pathway was a major affected pathway with a P value of 6.75 x 10-4. This comprehensive approach also revealed that F-actin is a major protein target for MRP4 and cAMP is the key mediator17. Based on these data, we further studied the underlying molecular mechanisms.
To understand the effect of MRP4 on cAMP dynamics and PKA activity during the course of cell migration, we used FRET-based live-cell imaging techniques. Using FRET-based sensors for cAMP and PKA activity, we confirmed higher cAMP accumulation and higher PKA activity at the leading edge of migrating and polarized fibroblasts22,28. We further demonstrated that in the absence of MRP4, MEFs have more polarized cAMP and PKA activity, which in turn facilitates cortical actin formation and cell migration. The high-content microscopy-based wound-healing assay showed that the effect of MRP4 on cell migration is completely abolished by PKA inhibition or actin disruption, which indicates a direct role of PKA and actin as downstream targets17. Unlike conventional cell population-based assays such as ELISA, use of a FRET-based sensor allows us to identify the downstream effector kinases that regulate various signaling processes and detect the correlation between cyclic nucleotide dynamics and their corresponding kinase activity in real time and space. Additionally it can discriminate intracellular and intercellular heterogeneity during the signaling events. For example it can detect the difference in PKA dynamics in the cells at the wound edge compared to the cells inside the monolayer and away from the wound edge5, whereas ELISA based assays can only detect total intracellular cyclic nucleotide or PKA level in a sample14,17. However the transfection efficacy of particular cell types can be a limiting factor for conducting FRET based assays but the highly efficient transfection reagents can overcome this problem.
Together, our results indicate that in addition to conferring drug resistance, MRP4 also plays important physiological roles by modulating intracellular cAMP dynamics. Using three unique approaches, i) high-content microscopy18, ii) IPA15, and iii) FRET5,28, we have begun to unravel the previously undefined role of MRP4 in cell migration. In general, these useful scientific techniques will allow us to identify new downstream targets of any protein of interest and explore novel molecular mechanisms involved in particular pathological or physiological cell responses. Where IPA provides useful information regarding downstream effectors of the protein of interest and potential regulatory networks; FRET-based live imaging can monitor compartmentalized signaling in real time. High-content microscopy is a convenient high-throughput screening tool to monitor and analyze physiological events, such as cell migration and cell proliferation, over a period of time as a final readout.

细胞迁移是一个复杂的多步骤过程。有研究显示，在迁移细胞分化为前缘和后缘。通过坚持细胞外基质的领先优势提供了必要的牵引细胞体向前移动。最后，后缘释放后部附件和完成迁移周期1,2。 高效细胞迁移细胞极化是由细胞内信号的空间隔离监管。细胞第二信使，如环磷酸腺苷，调解的微调定向细胞迁移3,4所需的信号事件的条块分割。在前沿的cAMP和cAMP依赖性激酶PKA活性的优惠积累起到定向细胞迁移5,6关键的作用。通过磷酸化小GTP酶如Ras相关C3肉毒杆菌毒素底物（RAC）和细胞分裂抑制蛋白同源42或Cdc42的，PKA以前缘激活肌动蛋白相关蛋白2/3（阿普2/3）并诱导伪足7-9的形成。 PKA也磷酸化抗封端剂，血管扩张剂刺激的磷蛋白（VASP），从而调节膜延伸和缩回10,11的振动周期。 ⅰ）合成由腺苷酸环化酶，ⅱ）由磷酸二酯酶降解，以及iii）运输通过膜结合的流出转运3:在细胞中，cAMP水平由三个主要过程调节。多药耐药蛋白4（MRP4），膜转运，功能的ATP结合盒（ABC）家族的成员，环核苷酸的内源性流出转运。因此，MRP4能调节细胞内cAMP水平和cAMP依赖的细胞信号11-13。我们以前曾表明，在MRP4 - / - ，成纤维细胞含有相对较高水平的环核苷酸和迁移速度更快çompared到MRP4 + / +成纤维细胞14。我们还报道环核苷酸对成纤维细胞迁移的双相效应。根据以往的研究，我们的发现，MRP4 - / -成纤维细胞迁移包含的过程中，更多的偏光阵营中，我们假设成纤维细胞迁移的这一MRP4介导的调控是依赖于cAMP的。为了了解下游的机制，我们采取了直接的还多面的方法。 为了确定相关的和与MRP4相互作用蛋白，我们从免疫，超过表达MRP4 HEK293细胞含有MRP4-大分子复合物。使用质谱分析，我们确定了多个MRP4相互作用蛋白，并使用独创性途径分析（IPA），分析了它们的互连。 IPA是分析蛋白质 - 蛋白质相互作用（结构和功能），并探讨其特别的生理和病理的贡献的有用工具根据文献和实验依据15,16事件。 IPA表示F-肌动蛋白是在细胞迁移的情况下MRP4的主要下游靶，其中cAMP和cGMP是关键信号分子17。这些数据由高含量显微镜下进一步证实。高含量显微镜可以捕获和分析细胞行为，如细 ​​胞迁移以更方便，准确，高通量的方式18。高含量显微镜数据显示，MRP4对成纤维细胞迁移的影响在17 PKA的肌动蛋白骨架或抑制的破坏被完全取消。 此外，我们使用了荧光共振能量转移（FRET）为基础的PKA传感器来监视PKA动力学实时迁移细胞。基于FRET激酶传感器通常由被CFP和YFP荧光两侧19-21具体磷酸化底物肽。 pmAKAR3是一种改进和我mbrane针对性包含叉头相关域1（FHA1）和PKA底物序列LRRATLVD 5,22基于FRET-PKA传感器。通过PKA催化亚基增加pmAKAR3的磷酸化FRET CFP和YFP 19之间的信号。脂质修饰域到传感器插入其定位于质膜监测PKA动力学，尤其在膜室23。 使用pmAKAR3，我们证明了迁移MRP4的前沿- / -表现出更加两极化PKA活性比MRP4 + / +成纤维细胞，这反过来又增加了在小区的领先优势17皮层肌动蛋白的成纤维细胞形成。一起，这些事件导致在没有MRP4更好蜂窝偏振和更快的定向细胞迁移。我们的具体和直接的方式确定了MRP4关键下游的目标，并提供一个重要的，但作为的还未知机制成纤维细胞迁移的MRP4相关规定。

<table><tbody><tr><td>Lipofectamine 2000</td><td>Invitrogen(Carlsbad, CA)&amp;nbsp;</td><td>11668-027</td><td></td></tr><tr><td>DMEM</td><td>Invitrogen (Carlsbad, CA)&amp;nbsp;</td><td>11965-092</td><td></td></tr><tr><td>IncuCyte Zoom</td><td>Essen BioScience</td><td></td><td></td></tr><tr><td>96-well IncuCyte Image-Lock microplates&amp;nbsp;</td><td>Essen BioScience</td><td>4493</td><td></td></tr><tr><td>Latrunculin B</td><td>Sigma-Aldrich (St. Louis, MO).</td><td>L5288</td><td>Stock in DMSO</td></tr><tr><td>H-89</td><td>Enzo Life Sciences (Farmingdale, NY)</td><td>BML-EI196</td><td>Stock in DMSO</td></tr><tr><td>35 mm glass-bottomed dishes&amp;nbsp;</td><td>(MatTek Corporation; Ashland, MA)</td><td>P35G-1.5-20-C&amp;nbsp;</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich (St. Louis, MO).</td><td>F1141</td><td></td></tr><tr><td>Opti-MEM Reduced Serum Media</td><td>Invitrogen (Carlsbad, CA)&amp;nbsp;</td><td>31985-088</td><td></td></tr><tr><td>FRET microscopy system</td><td>Olympus inverted microscope (IX51)</td><td></td><td></td></tr><tr><td>CCD camera&amp;nbsp;</td><td>Hamamatsu, Japan</td><td>ORCA285</td><td></td></tr><tr><td>SlideBook software 5.5</td><td>Intelligent Imaging Innovation ( Denver, CO)</td><td></td><td></td></tr><tr><td>Ingenuity Pathway Analysis software</td><td>IPA, QIAGEN Redwood City,</td><td></td><td></td></tr><tr><td>Forskolin</td><td>Tocris (Ellisville, MO).&amp;nbsp;</td><td>1099</td><td>Stock in 100% EtOH</td></tr><tr><td>DMEM F-12&amp;nbsp;&amp;nbsp;</td><td>Invitrogen (Carlsbad, CA)&amp;nbsp;</td><td>11330-057</td><td></td></tr><tr><td>HBSS</td><td>Invitrogen (Carlsbad, CA)&amp;nbsp;</td><td>14025-134</td><td></td></tr><tr><td>Excel</td><td>Microsoft</td><td></td><td></td></tr><tr><td>PBS</td><td>Invitrogen(Carlsbad, CA)&amp;nbsp;</td><td>10010-023</td><td></td></tr><tr><td>Trypsin/EDTA Solution (TE)</td><td>Invitrogen(Carlsbad, CA)&amp;nbsp;</td><td>R-001-100</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Invitrogen(Carlsbad, CA)&amp;nbsp;</td><td>15140-122</td><td></td></tr></tbody></table>

methods to study mrp4-containing macromolecular complexes in the regulation of fibroblast migration

多药耐药蛋白4（MRP4）是ATP结合盒家族的膜转运的成员，并且是环核苷酸的内源流出转运蛋白。通过调节细胞内环核苷酸浓度，MRP4可以调节多循环核苷酸依赖性细胞活动，包括细胞迁移。先前，我们表明，在不存在MRP4的，成纤维细胞含有较高水平的细胞内环核苷酸的，并且可以迁移更快。要理解这一发现的基本机制，我们通过直接还多方面的办法。首先，我们分离MRP4的潜在相互作用蛋白复合物使用免疫沉淀，随后通过质谱一个MRP4过表达细胞系。识别所述MRP4相互作用组独特的蛋白质后，我们利用独创性途径分析（IPA），探讨在信号转导的情况下，这些蛋白 - 蛋白相互作用的作用。我们阐明宝在细胞迁移的MRP4蛋白复合物，并确定了F-actin为的MRP4对细胞迁移的影响的主要调停者的角色势。这项研究还强调cAMP和cGMP的作用，在迁徙现象的关键球员。使用高含量显微镜，我们进行了细胞迁移测定和观察到MRP4对成纤维细胞迁移的影响完全被cAMP依赖性激酶A（PKA）的肌动蛋白细胞骨架或抑制的破坏废除。为了显现在实时迁移细胞信号的调制，我们利用用于测定PKA活性的基于FRET的传感器，并发现，更偏振光PKA活性的存在迁移MRP4的前缘附近- / -成纤维细胞相比，MRP4 + / +成纤维细胞。这反过来又增加皮质肌动蛋白的形成和增强迁移的过程。我们的方法使下游采取行动，MRP4蛋白质的鉴定和为我们提供了一个过鉴于涉及的成纤维细胞迁移MRP4依赖的调节机制。

研究MRP4对成纤维细胞迁移的影响，我们使用利用高含量显微镜14伤口愈合测定。精确伤口上从分离的MEF的汇合单层制成或MRP4 - / -或MRP4 + / +小鼠，和图像在1小时的时间间隔为24小时拍摄的。我们观察到了更高的迁移率MRP4 - / -的MEF相比MRP4 + / +的MEF（ 图2）。 / - -该伤口为MRP4不到15小时完全愈合的MEF，而所需的MRP4 + / + MEF中几乎20小时覆盖伤口。 在细胞迁移的前缘PKA活性的偏振积累为定向迁移的关键的早期事件。 PKA活性可以实时全光照监视克PKA 5,17基于FRET的pmAKAR3传感器。要检查pmAKAR3对PKA活性的特殊性，我们处理的HEK293细胞过度pmAKAR3或点突变pmAKAR3-TA，其中包含了PKA的基底区苏氨酸到丙氨酸突变，因此irresponsive到PKA磷酸化25μM毛喉素，一个营地诱导剂14。我们发现，在细胞过度pmAKAR3增加FRET信号，但细胞过度pmAKAR3-TA保持不变（ 图3）。基础FRET水平也表达pmAKAR3相比表达pmAKAR3-TA细胞的细胞高。这些数据表明，pmAKAR3为PKA活性非常具体的。 总之，在协议部分中描述的方法是有用的工具来研究与特定的细胞事件相关的分子机制。 <img alt="图1"的src ="/文件/ ftp_upload / 53973 / 53973fig1.jpg"/&gt; 图1:MRP4相互作用组独创性途径分析（IPA）使用的IPA的肌动蛋白细胞骨架通路被鉴定为受MRP4相互作用组的主要规范途径之一。提出肌动蛋白网络的信令连接指示蛋白（白色），并从文献和实验证据推断出MRP4相互作用组（粉红色）鉴定蛋白质的交叉通讯。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53973/53973fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53973/53973fig2.jpg" /> 图2:伤口愈合使用高含量测定显微镜 MRP4 + / + 和 MRP4 - / - 小鼠胚胎成纤维细胞（MEF）上生长fibroneCTIN包被的96孔培养皿中，并在单层伤口作了精确使用96针卷绕机。在不同时间点的代表图像显示有10X的放大倍率。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53973/53973fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53973/53973fig3.jpg" /> 图3:荧光共振能量转移（FRET）的使用pmAKAR3传感器为基础的测量的PKA活性的 N-二FRET的代表性伪彩色图像用60X放大率对前和毛喉素处理后与pmAKAR3传感器和pmAKAR3-TA传感器转染的HEK293细胞。示（顶部小图）。在每个面板图像从同一视场捕获。彩条表示的N FRET的幅度。线图（下图）表示后处理方法的N-FRET水平的变化吨，毛喉素。数据代表至少有三个独立实验的平均值（平均值±SEM; N = 3）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53973/53973fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration at JoVE.com

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

MRP4调节各种循环依赖于核苷酸信号事件，包括细胞迁移最近阐明的作用。我们描述一个直接的，而是多方面的方法解开导致该起主要作用的成纤维细胞迁移的微调调控独特MRP4相互作用组鉴定MRP4下游分子的目标。

方法在成纤维细胞迁移的规律研究大分子复合物含MRP4，

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

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Nanyang Technological University

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Developmental Biology

6.7K 次观看.  Cincinnati Children’s Hospital Medical Center.  MRP4调节各种循环依赖于核苷酸信号事件，包括细胞迁移最近阐明的作用。我们描述一个直接的，而是多方面的方法解开导致该起主要作用的成纤维细胞迁移的微调调控独特MRP4相互作用组鉴定MRP4下游分子的目标。 

视频: Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

白三烯B实时成像 4介导的细胞迁移和BLT1与β- arrestin的相互作用

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

科研

免疫与感染

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

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

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

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

在活细胞中的小GTPase活性时空操纵在亚细胞水平上几秒钟的时间刻度

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

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

观察和量化纤维细胞介导的纤维素凝胶压实

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line.&#160;

基于阻抗的癌细胞迁移和入侵的实时测量

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

癌症研究

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

同心凝胶系统来研究基质微环境的三维细胞迁移的生物物理作用

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

表征细胞迁移在三维体外伤口环境

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

在体外细胞迁移和侵袭实验

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

生物学

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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.

方法在成纤维细胞迁移的规律研究大分子复合物含MRP4，

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白三烯B实时成像₄介导的细胞迁移和BLT1与β- arrestin的相互作用