An open microfluidic device to study chemotaxis of eukaryotic cells

描述(申请人提供)：拟议的研究项目试图调查细胞极化的机制，并帮助我们进一步了解极化在细胞定向迁移中的作用。这些研究将使用聚二甲基硅氧烷制成的开放式微流控装置进行研究。这种设备有多个微米大小的通道，可以用来相互竞争形态上不同的细胞，这些通道还能够限制细胞进行极性研究。通过对盘状芽孢杆菌细胞的更多了解，这些发现可以与控制其他真核细胞(如中性粒细胞)的趋化过程相关联，也可以阐明调控癌症转移的途径。为了实现这些目标，已经确定了三个具体目标。第一个具体目标集中在进一步开发一种开放的微流控设备，该设备可以用于趋化分析，并允许可视化稳定的被动化学梯度。第二个特定的目标是通过比较极化和未极化的盘状芽孢杆菌细胞向各自的趋化物质的迁移，来研究极性在趋化细胞中的作用和机制。表达荧光细胞骨架和信号蛋白的细胞将暴露在微流控通道的一端的化学吸引剂中，之后梯度将被逆转，随着细胞的去极化和重新极化，这些荧光分子的重新分布将被分析。第三个具体目标是研究真核细胞中的梯度传感机制。梯度的浓度(输入)将与迁移速度和信号蛋白定位(输出)进行比较。根据国家普通医学科学研究所(NIGMS)的使命，这项研究计划试图了解真核细胞的趋化过程。这些实验的发现将有助于阐明细胞感知化学梯度和迁移的基本机制，并进一步了解癌细胞是如何转移的。拟议的研究计划将要求私人情报员应用在课堂上和从科学文献中学到的理论。适当的研究技术将在实验室进行，实验将在研究赞助商的指导下进行。Janetopoulos实验室的合格人员和范德比尔特大学医疗中心的工作人员将协助进行关于使用适当设备和技术的培训和实施。

DESCRIPTION (provided by applicant): The proposed research project seeks to investigate the mechanisms by which cells polarize and help further our understanding of the role of polarization in directed cell migration. These studies will use the social amoeba Dictyostelium discoideum and will be conducted using a newly developed open microfluidic device made out of Polydimethylsiloxane. This device has multiple, micron-sized channels which can be used to race morphologically distinct cells against each other and the channels are also capable of confining cells for polarity studies. By learning more about D. discoideum cells, these findings can be correlated with the chemotaxis processes controlling other eukaryotic cells such as neutrophils and also elucidate the pathways that regulate cancer metastasis. In order to achieve these goals, three specific aims have been set. The first specific aim focuses on the further development of an open microfluidic devices that can be used for chemotaxis assays, and allows the visualization of a stable, passive chemical gradient. The second specific aim targets the role and mechanisms of polarity in chemotaxing cells by comparing the migration of polarized and unpolarized D. Discoideum cells towards their respective chemoattractants. Cells expressing fluorescent cytoskeletal and signaling proteins will be exposed to chemoattractant at one end of a microfluidic channel, after which the gradient will be reversed and the redistribution of these fluorescent molecules will be analyzed as the cells de-polarize and re-polarize. The third specific aim focuses on the gradient sensing mechanism in eukaryotic cells. The concentration of the gradient (input) will be compared to the migration speeds and signaling protein localizations (output). In line with the mission of the National Institute of General Medical Sciences (NIGMS), this research proposal seeks to understand the chemotaxis processes of eukaryotic cells. The findings from these experiments will help elucidate the basic mechanisms by which cells sense chemical gradients and migrate and further our understanding of how cancer cells metastasize. The proposed research program will require the P.I. to apply theory learned in both the classroom and from scientific literature. Appropriate research techniques will be picked up in the laboratory and the experiments will be performed under the guidance of the research sponsor. Training and implementation on the use of appropriate equipment and techniques will be facilitated by qualified personnel in the Janetopoulos laboratory and the staff at Vanderbilt University medical center.