Sensory Transduction in Bacterial Chemotaxis

描述（由申请人提供）：细胞感知其环境的变化并作出反应，通常以全有或全无的方式调节运动性、生长、发育命运或突触功效。细菌趋化性是研究感觉传导的一个杰出的模型系统，其中机制可以在原子细节上理解。E.大肠杆菌是奈米科技的奇迹，只有一微米大小的细胞由数条螺旋状细丝推动，每一条细丝的底部由一个直径为50奈米的旋转马达驱动，马达的动力来自质子流。当细丝逆时针旋转时，细胞稳定地向前移动-它“运行”。当一个或多个细丝顺时针（CW）旋转时，细胞改变路线。细胞计数其环境中感兴趣的分子并延长被认为有利的运行。计数是由调节激酶活性的受体完成的，激酶磷酸化反应调节因子，当磷酸化时，反应调节因子扩散穿过细胞质并结合到鞭毛马达的基部，增加它们顺时针旋转的可能性。使用荧光融合蛋白在体内之间的荧光共振能量转移（FRET），我们将研究负责高系统增益的受体-受体相互作用，遵循单细胞内反应调节剂的扩散，评估电机开关的机制，并试图了解是否有反馈连接电机到受体。我们将通过计算机建模来测试我们的理解。将开发新的方法来探测运动功能：研究质子转移的高速限制，内部运动组件的相互作用，并在体外运动试验的行为。视频分析将被用来了解更多关于荧光鞭毛细丝的运动：多态转换如何重新定位细胞体，以及不同细胞上的鞭毛如何相互作用以产生合作运动。虽然这项工作与微生物毒力直接相关，但它意味着研究基本的生物过程：化学感受，细胞内信号传导和化学渗透能转化为机械功。

DESCRIPTION (provided by applicant): Cells sense changes in their environment and respond, often in an all-or-none fashion, modulating motility, growth, developmental fate, or synaptic efficacy. Bacterial chemotaxis is a pre-eminent model system for studies of sensory transduction, where mechanisms can be understood in atomic detail. E. coli is a nanotechnological marvel, with cells only a micron in size propelled by several helical filaments, each driven at its base by a rotary motor 50 nm in diameter powered by a proton flux. When the filaments spin counterclockwise (CCW), a cell moves steadily forward - it "runs". When one or more filaments spin clockwise (CW), the cell changes course. A cell counts molecules of interest in its environment and extends runs deemed favorable. The counting is done by receptors that regulate the activity of a kinase that phosphorylates a response regulator that, when phosphorylated, diffuses across the cytoplasm and binds to the base of the flagellar motors, increasing the probability that they spin CW. Using fluorescence resonance energy transfer (FRET) between fluorescent fusion proteins in vivo, we will study receptor-receptor interactions responsible for high system gain, follow the diffusion of the response regulator within single cells, assess mechanisms for motor switching, and to try to learn whether there is feedback linking motors to receptors. We will test our understanding by computer modeling. New methods will be developed to probe motor function: to study proton-transfer in the high-speed limit, interactions of internal motor components, and behavior in an in vitro motor assay. Video analysis will be used to learn more about the motion of fluorescent flagellar filaments: how polymorphic transformations reorient the cell body, and how flagella on different cells interact to generate cooperative movement. While this effort is directly relevant to microbial virulence, it is meant as a study of fundamental biological processes: chemoreception, intracellular signaling, and conversion of chemiosmotic energy to mechanical work.