Name: methods to study mrp4-containing macromolecular complexes in the regulation of fibroblast migration
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Description: Watch this Scientific Journal Video about Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration at JoVE.com

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?...

<ol>
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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 fibroblasts...

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 border="0" cellpadding="0" cellspacing="0" width="950">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Lipofectamine 2000</td>
			<td style="width:324px;">Invitrogen(Carlsbad, CA)&#160;</td>
			<td style="width:187px;">11668-027</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Invitrogen (Carlsbad, CA)&#160;</td>
			<td style="width:187px;">11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IncuCyte Zoom</td>
			<td style="width:324px;">Essen BioScience</td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well IncuCyte Image-Lock microplates&#160;</td>
			<td>Essen BioScience</td>
			<td style="width:187px;">4493</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Latrunculin B</td>
			<td style="width:324px;">Sigma-Aldrich (St. Louis, MO).</td>
			<td>L5288</td>
			<td>Stock in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">H-89</td>
			<td style="width:324px;">Enzo Life Sciences (Farmingdale, NY)</td>
			<td>BML-EI196</td>
			<td>Stock in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35-mm glass-bottomed dishes&#160;</td>
			<td style="width:324px;">(MatTek Corporation; Ashland, MA)</td>
			<td style="width:187px;">P35G-1.5-20-C&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fibronectin</td>
			<td style="width:324px;">Sigma-Aldrich (St. Louis, MO).</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Opti-MEM Reduced Serum Media</td>
			<td>Invitrogen (Carlsbad, CA)&#160;</td>
			<td style="width:187px;">31985-088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">FRET microscopy system</td>
			<td>Olympus inverted microscope (IX51)</td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">CCD camera&#160;</td>
			<td style="width:324px;">Hamamatsu, Japan</td>
			<td style="width:187px;">ORCA285</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">SlideBook software 5.5</td>
			<td style="width:324px;">Intelligent Imaging Innovation ( Denver, CO)</td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ingenuity Pathway Analysis software</td>
			<td>IPA, QIAGEN Redwood City,</td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forskolin</td>
			<td>Tocris (Ellisville, MO).&#160;</td>
			<td style="width:187px;">1099</td>
			<td>Stock in100% EtOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM F-12&#160;&#160;</td>
			<td>Invitrogen (Carlsbad, CA)&#160;</td>
			<td style="width:187px;">11330-057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">HBSS</td>
			<td>Invitrogen (Carlsbad, CA)&#160;</td>
			<td style="width:187px;">14025-134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Excel</td>
			<td style="width:324px;">Microsoft</td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">PBS</td>
			<td style="width:324px;">Invitrogen(Carlsbad, CA)&#160;</td>
			<td style="width:187px;">10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Trypsin/EDTA Solution (TE)</td>
			<td style="width:324px;">Invitrogen(Carlsbad, CA)&#160;</td>
			<td>R-001-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Penicillin-Streptomycin</td>
			<td style="width:324px;">Invitrogen(Carlsbad, CA)&#160;</td>
			<td style="width:187px;">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）。 / -...

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 ...

Research

JoVE Journal

Developmental Biology

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

斑马鱼角膜植研究集体细胞迁移和再上皮化在皮肤伤口愈合

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

科研

发育生物学

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

伤口划痕试验的优化来探测在小鼠间充质干细胞迁移的可溶性香烟烟雾提取物受损后

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

方法来检查淋巴腺和血细胞中<em&gt;果蝇</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

光片为主的生活的荧光显微镜或固定和染色<em&gt;赤拟谷盗</em&gt;胚胎

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Methods for the Study of Regeneration in Stentor

Methods for the Study of Regeneration in Stentor

Stentor再生的研究方法

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

发展中雏鸡光学顶盖中切向细胞迁移的可视化

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

Methods to Test Endocrine Disruption in Drosophila melanogaster

检测果蝇黑色素的内分泌干扰的方法

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...