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

本研究的长期目标是了解Amphora属硅藻的粘附、运动和趋化性的调控。该项目将扩展钙在这些过程的调节中起作用的初步观察。初步结果表明，一定水平的细胞外钙是粘连和运动所必需的，并且双耳菌对单糖和谷氨酸有化学反应。钙作为第二信使在控制许多生物过程中起着关键作用。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，该离子负责信号-反应耦合。钙敏感荧光探针，如Fura-2、Indo-1、Fluo-3的可用性，使得使用共聚焦激光扫描显微镜直接可视化单个细胞中胞质游离钙的时空变化成为可能，并且可以使用适当的荧光光谱仪检测同步细胞悬浮液中细胞内钙的变化。这项小型探索性研究资助的研究将建立实验条件，以便将这些重要的实验工具应用于钙在调节硅藻细胞粘附、运动和趋化性中的作用的研究。这些工具将用于研究促进细胞内游离钙水平趋化的外部钙和糖的影响，作为钙在这些相当原始的真核生物中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氧气。作为本研究对象的硅藻生长在近岸底栖区被光照的表面上。趋化性是否是这些生物的共同特性尚不清楚。虽然硅藻是绿藻，可以进行光合作用，但这个生态位中的许多硅藻也可以使用一些单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，它们可以感知沉积物孔隙水中这些化合物的化学梯度，并向更高浓度移动，这些硅藻可能对光照下沉积物中溶解有机碳的转换很重要。然而，尽管这些生物具有潜在的重要性，但人们对它们对刺激（如有机化合物）的反应机制知之甚少。这笔小额探索性研究基金将用于为调节硅藻粘附、运动性和趋化性的信号转导途径的研究奠定实验基础。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反应机制的知识空白，而且还将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. ***