Molecular Genesis and Organization of Olfactory Transduction Components and Cilia

描述(申请人提供)：哺乳动物的嗅觉系统通过新的遗传机制、细胞特化以及随后由神经回路处理感觉信息而获得显著的敏感性。气味信息转化为传播到大脑的信号的最早步骤发生在嗅觉纤毛上。嗅觉神经元的十几根长而不动的纤毛是感觉细胞暴露在外界环境中的唯一部分，也是气味刺激的来源，它们的存在对于有效的气味检测是必不可少的。嗅觉神经元已经发展出有效的机制来丰富纤毛中的嗅觉传导成分(气味受体(OR)、G蛋白(Golf)、腺苷环化酶(AC3)和嗅觉环核苷酸通道)，但人们对此知之甚少。含有AC3酶的单一多肽在所有嗅觉神经元中都有丰富的表达，这使得它特别适合于阐明纤毛定位和丰富的途径和机制。在这项资助中，我们将使用分子和遗传学方法来研究一种假设，即一种专门的但被广泛使用的机制和途径负责将AC3定位到纤毛上，并且这种定位对纤毛的动力学和功能至关重要。在具体目标1中，我们通过有条件的遗传干扰方法在体内靶向AC3的纤毛定位信号，并确定AC3重新定位到不同的细胞室的后果。在特定的目标2中，我们将利用ROBUT表达系统来鉴定导致纤毛定位的AC3转位/浓缩途径的组成部分，并研究AC3在纤毛中的动态以及AC3定位在纤毛长度调节中的作用。总之，这些实验将扩大我们对一类重要的蛋白质如何富含纤毛以及它们对这个细胞器的选择性定位的重要性的理解。纤毛存在于几乎所有终末分化的哺乳动物细胞中，对生物体的发育至关重要。腺酰环化酶定位的途径和机制将对嗅觉、感觉交流以及广泛的人类健康和疾病的理解产生重要的影响。嗅觉神经元中AC3的丰富使其特别适合进行详细研究。AC3酶在大脑和肾脏的许多神经元纤毛中广泛表达和高度丰富，它可能介导神经内分泌/神经递质信号，其在这些细胞中的亚细胞定位似乎是由类似的机制介导的。这一途径的调节应该扰乱复杂的行为和代谢过程。此外，AC3利用的纤毛定位途径很可能与其他蛋白质共享，这些蛋白质在纤毛中的存在对细胞功能至关重要。对这些途径的阐明将提供对人类健康具有广泛后果的细胞过程的洞察。

DESCRIPTION (provided by applicant): The mammalian olfactory system achieves remarkable sensitivity through novel genetic mechanisms, cellular specializations, and subsequent processing of sensory information by neuronal circuitry. The earliest steps in the transduction of odorant information into signals that are propagated to the brain occur in olfactory cilia. The dozen long immotile cilia of each olfactory neuron are the only part of the sensory cell exposed to the outside environment and the sources of odorant stimuli and their existence is essential for efficient odor detection. The olfactory neuron has developed efficient but poorly understood mechanisms for enriching the components of olfactory transduction (odorant receptor (OR), G protein (Golf), adenylyl cyclase (AC3) and olfactory cyclic nucleotide channel) in cilia. The abundant expression of the single polypeptide comprising the AC3 enzyme in all olfactory neurons makes it particularly amenable to elucidate the pathways and mechanisms responsible for cilia localization and enrichment. In this grant we will use molecular and genetic approaches to investigate the hypothesis that a specialized but broadly utilized mechanism and pathway is responsible for AC3 localization to cilia and that this localization is critical for cilia dynamics and function. In specific aim 1, we target the cilia localization signa of AC3 in vivo by a conditional genetic disruption approach and determine the consequences of re- localization of AC3 into different cellular compartments. In specific aim 2, we will utilize a robut expression system to identify components of the AC3 translocation/enrichment pathway leading to cilia localization and examine the dynamics of AC3 in cilia and role of AC3 localization in modulation of cilia length. Together, these experiments will expand our understanding of how an important class of proteins are enriched in cilia and the importance of their selective localizatio to this organelle. Cilia are present in essentially all terminally differentiated mammalian cells ad critical for the development of the organism. The pathways and mechanisms underlying adenylyl cyclase localization will have important consequences for olfaction, sensory communication and broadly for human health and understanding disease. The abundance of AC3 in olfactory neurons makes it particularly amenable for detailed studies. The AC3 enzyme is broadly expressed and highly enriched in many neuronal cilia of the brain and kidney where it likely mediates neuroendocrine/neurotransmitter signaling and its subcellular localization in these cells appear to be mediated by similar mechanisms. Modulation of this pathway should perturb complex behaviors and metabolic processes. Additionally, it is likely that the cilia localization pathway utilized by AC3 is shared by other proteins whose presence in cilia is critical for cellular function. Elucidation of these pathways will provide insight into cellular processes that have widespread consequences for human health.