CHEMOSENSORY SIGNAL TRANSDUCTION

化学感应信号转导

基本信息

批准号：
2668224
负责人：
JUDITH L VAN HOUTEN
金额：
$ 16.19万
依托单位：
UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-03-01 至 2000-02-29
项目状态：
已结题

项目摘要

This is an application for renewal of funding of work that is exploring chemical sensing using Paramecium as a chemosensory cell. This organism presents the opportunity to apply multiple techniques in addressing hypotheses: biochemistry, molecular biology, transmission genetics, electrophysiology behavioral analysis. While cellular and molecular genetic approaches to some extent must disrupt the intact receptor cell or impose unphysiological conditions, it is often possible with Paramecium to return the study to the whole cell and ask what physiologically relevant changes have been made in chemosensory signal transduction. In the past funding period, we have adopted antisense technology to our study of the interaction between transduction components. Receptors in at least one pathway in Paramecium appear to activate the plasma membrane calcium pump, creating the sustained hyperpolarizing conductance that characterizes attractants. We have made use of antisense oligodeoxynucleotides (ODNs) for calmodulin, a regulator of the pump, to down regulate the levels of calmodulin in order to indirectly inhibit the pump as a test of its role in chemoresponse. We provided evidence that a conductance known to be calmodulin regulated is reduced in the antisense treated cells. More importantly, we have shown that chemosensory behavior is correspondingly and selectively inhibited. Response to acetate that initiates a pathway, thought to involve the pump, is inhibited while response to NH4Cl, utilizing a pathway without the pump, is not inhibited. We have prepared the plasmids necessary to stably transform cells to improve our physiological studies of these cells and we have developed the antibodies and immunocytochemistry that are necessary to monitor the transformation of cells with antisense ODNs or plasmids with genes for which there is no obvious behavioral, selectable phenotype. Other tests of interactions of transduction components using immunoprecipitation are on-going. We propose to test three related hypotheses: 1) Receptors for at least one of the three signal transduction pathways are coupled to the calcium pump. 2) Receptors, the calcium pump and other transduction components interact and are found within the same membrane domain. 3) The calcium pump and cyclases couple to GPI anchored receptors and possibly G proteins. We will use antisense and other transformation methods to produce phenocopies of mutants in the genes for the transduction components; confocal microscopy to locate components of the transduction pathways; perturbation of GPI anchored proteins by phospholipase C treatment.

这是续签工作资金的申请使用映射作为化学感应细胞的化学传感。这个有机体提出了应用多种技术解决的机会假设：生物化学，分子生物学，传播遗传学，电生理行为分析。而细胞和分子遗传方法在某种程度上必须破坏完整的受体细胞或施加非生理条件，通常可以将研究归还整个细胞并询问什么在化学感信号中已经进行了与生理相关的变化转导。在过去的资金期间，我们采用了反义技术研究转导组件之间的相互作用。受体在西苯第至少一条途径似乎激活了质膜钙泵，形成持续的超极化电导特征吸引者。我们使用了反义用于泵的调节剂钙调蛋白的寡脱氧核苷酸（ODN）下调调节钙调蛋白的水平，以间接抑制泵作为其在化学响应中的作用的测试。我们提供了证据表明在反义处理的细胞。更重要的是，我们已经表明化学感觉行为相应地有选择地抑制。对乙酸的反应，该途径涉及泵，在对NH4CL的响应时被抑制，利用没有的途径泵没有抑制。我们已经准备了所需的质粒稳定转化细胞以改善我们对这些细胞的生理研究细胞和我们已经开发了抗体和免疫细胞化学对于用反义ODN监测细胞的转化是必要的或具有没有明显行为的基因的质粒，可选表型。其他转导相互作用的测试使用免疫沉淀的组件正在进行中。我们建议测试三个相关假设：1）至少受体三个信号转导途径之一与钙耦合泵。 2）受体，钙泵和其他转导组件相互作用并在同一膜结构域中找到。 3）钙泵和循环酶夫妇到GPI锚定受体，可能是G 蛋白质。我们将使用反义和其他转换方法在基因中产生突变体的表载体进行转导成分;共聚焦显微镜以定位转导组件途径；磷脂酶C锚定GPI锚定蛋白的扰动治疗。