SGER: Measurement Using Calcium-Specific Probes of Ca in a Diatom Treated With Chemotactic Effectors: A First Step in the Dissection of the Signal Transduction Pathway

9320335 Cooksey这项研究的长期目标是了解文氏菌属硅藻的粘附性、运动性和趋化性的调节。这个项目将对钙在这些过程的调节中起作用的初步观察结果进行扩展。初步结果表明，一定水平的细胞外钙是黏附和运动所必需的，而Amphora对单糖和谷氨酸有化学反应。钙在许多生物过程中起着第二信使的关键作用。这种离子在动物、植物和其他细胞的分泌和细胞极性形成等生理过程中负责信号-反应耦合。随着Fura-2、Indo-1、Fluo-3等钙敏感荧光探针的问世，激光共聚焦扫描显微镜可以直接显示单个细胞胞浆内游离钙的时空变化，而细胞内钙的变化也可以通过合适的荧光分光光度计检测到。这项由探索性研究小额拨款支持的研究将为将这些重要的实验工具应用于研究钙在调节硅藻细胞黏附、运动和趋化性中的作用奠定实验条件。然后，这些工具将被用于研究促进趋化作用的外部钙和糖对细胞内游离钙水平的影响，这是对这些相当原始的真核生物中钙控制生理过程的长期研究的一部分。海洋硅藻提供了世界上一半以上的替代氧气。作为这项研究的主题的硅藻附着在近岸底栖带的照亮表面上。趋化性是否是这些生物的共同属性尚不清楚。虽然硅藻是绿藻，可以进行光合作用，但来自这个生态位的大多数硅藻也可以利用一些简单的糖和氨基酸来支持它们的生长。如果正如初步观察所表明的那样，它们可以感觉到沉积物孔隙水中这些化合物的化学梯度，并向更高的浓度移动，这些硅藻可能对照明沉积物中溶解的有机碳的周转起到重要作用。然而，尽管这些生物具有潜在的重要性，但人们对它们对有机化合物等刺激的反应机制知之甚少。这一小笔探索性研究拨款将用于为研究调控硅藻黏附、移动和趋化的信号转导通路奠定实验基础。这项研究的长期结果不仅将填补这些鲜为人知的生物对环境信号做出反应的机制方面的知识空白，还应该为它们在近岸食物网中的生态作用提供新的见解。***

9320335 Cooksey The long term goal of this research is to understand the regulation of adhesion, motility and chemotaxis in diatoms of the genus Amphora. This project will expand on preliminary observations that calcium plays a role in the regulation of these processes. Preliminary results indicate that a certain level of extracellular calcium is necessary for adhesion and motility and that Amphora responds chemotactically to simple sugars and glutamate. Calcium has a pivotal role as a second messenger in control of numerous biological processes. This ion is responsible for signal-response coupling in animal, plant, and other cells during such physiological processes as secretion and the development of cellular polarity. The availability of calcium-sensitive fluorescent probes such as Fura-2, Indo-1, Fluo-3 has made possible the direct visualization of spatial and temporal changes in cytosolic free calcium in single cells using confocal laser scanning microscopy, and changes in intracellular calcium can be detected in suspensions of synchronized cells using an appropriate spectrofluorimeter. Research supported by this Small Grant for Exploratory Research will establish the experimental conditions for applying these important experimental tools in studies of the role of calcium in regulation of cell adhesion, motility, and chemotaxis in diatoms. These tools will then be used to study the effects of external calcium and sugars that promote chemotaxis on intracellular free calcium levels as part of a longer term study of the control of physiological processes by calcium in these, rather primitive, eukaryotes. %%% Marine diatoms are responsible for more than half of the world's replacement oxygen. The diatoms that are the subject of this research grow attached to illuminated surfaces in the nearshore benthic zone. Whether chemotaxis is a common property of these organisms is not known. Although diatoms are green algae and can carry out photosynthesis, ma ny diatoms from this niche also can use some simple sugars and amino acids to support their growth. If, as suggested by preliminary observations, they can sense chemical gradients of these compounds in the sediment pore water and move toward higher concentrations, these diatoms could be important in turnover of dissolved organic carbon in illuminated sediments. Yet, despite the potential importance of these organisms, very little is known about the mechanisms of their responses to stimuli such as organic compounds. This Small Grant for Exploratory Research will be used to lay the experimental groundwork for studies of the signal transduction pathways regulating diatom adhesion, motility, and chemotaxis. The longer term results of this research will not only fill a gap in knowledge of the mechanisms by which these little-understood organisms respond to environmental signals, but should also provide new insights into their ecological role in the nearshore food web. ***