Sensory Transduction in Bacterial Chemotaxis

描述（由申请人提供）：细胞感知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、生长、发育命运或突触功效。细菌趋化性是研究感觉转导的卓越模型系统，其机制可以在原子细节上理解。大肠杆菌是纳米技术的奇迹，它的细胞只有一微米大小，由几根螺旋状细丝推动，每根细丝的底部由直径50纳米的旋转马达驱动，马达由质子通量提供动力。当细丝逆时针旋转（CCW）时，细胞稳定地向前移动——它“奔跑”。当一根或多根细丝顺时针旋转时，细胞改变方向。细胞对其环境中感兴趣的分子进行计数，并延长被认为有利的运行时间。计数是由调节激酶活性的受体完成的，该激酶磷酸化了一种反应调节因子，当被磷酸化时，这种反应调节因子扩散到细胞质中，并与鞭毛马达的基部结合，增加了它们旋转CW的可能性。利用荧光融合蛋白之间的荧光共振能量转移（FRET），我们将研究导致高系统增益的受体-受体相互作用，跟踪响应调节器在单个细胞内的扩散，评估运动开关的机制，并试图了解是否存在将马达与受体连接起来的反馈。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：在高速极限下研究质子转移，内部运动组件的相互作用，以及在体外运动实验中的行为。视频分析将用于了解更多关于荧光鞭毛丝的运动：多态转化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，但它意味着对基本生物过程的研究：化学接受、细胞内信号传导和化学渗透能向机械功的转化。

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.