Identification of novel osmosensing receptors in C. elegans

项目摘要 感知渗透压变化和维持液体渗透压的能力是正常生理所必需的 每一个细胞的功能都是如此，因此对人类健康至关重要。高渗透压引起的液体失衡 (高渗透压)或低渗透压(低渗透压)，可对器官造成不可逆转的损害并引起 致命的神经创伤。在人类中，体液的渗透压在大脑和 保持在非常窄的范围内(275-299mOsm/kg)。血液渗透压只有1%的增加 足以引发口渴。这种强大的渗透调节的一个关键因素是分子渗透传感器 检测渗透压变化。然而，动物王国中渗透感受器的分子特性 仍然难以捉摸。在动物身上识别渗透感受器的困难部分是由于缺乏公正的 筛选系统。以前在动物中识别渗透感受器的努力仅限于Trp通道。 然而，渗透感受器并不一定属于Trp通道家族，因此可能会逃脱 侦测。线虫是研究渗透传感的理想模型。像哺乳动物一样，线虫有 渗透传感系统和保守的信号分子已经被识别出来。这一点，加上它的短小 世代时间(~3天)和简便丰富的遗传工具，使线虫成为鉴定的理想系统 新型渗透传感器。为了识别线虫的渗透感受器，我们设计并进行了一项神经活动- 基于基因的筛查。我们已经确定OSMS-1和OSMS-2是线虫的候选渗透敏感器。 尽管这一令人兴奋的发现，许多问题仍然没有得到回答。在目前的提案中，我们建议测试 Osms-1和Osms-2是真正的渗透敏感器的假设和分子特征 OSMS-1和OSMS-2感受渗透压的机制。我们将采取多学科的方法，通过 集成了分子遗传学、行为分析、钙成像、电生理学和冷冻-EM。去做 因此，我将接受钙成像、细胞培养、电生理记录和冷冻方面的广泛培训。 嗯。K99/R00大奖将允许我在导师徐绍恩博士的指导下获得这些技能 和Melanie Ohi，这将帮助我开始独立的研究生涯。拟议的工作将导致 动物界第一批渗透感受器的鉴定，获得了如何在分子上理解 渗透传感器感知渗透刺激，并提供渗透感受、渗透调节和 相关的人类疾病。

Project Summary The ability to sense osmolarity changes and maintain fluid osmolarity is required for normal physiological functions of every single cell and thus vital for human health. Fluid imbalance, caused by high osmolarity (hyperosmolarity) or low osmolarity (hypoosmolarity), can lead to irreversible damage to organs and cause lethal neurological trauma. In humans, the osmolarity of body fluids is continuously monitored in the brain and kept within a very narrow range (275-299 mOsm/kg). As little as a one percent increase in blood osmolarity is enough to trigger thirst. A key element for such robust osmoregulation is the molecular osmosensors that detect osmolarity changes. However, the molecular identities of osmosensors in the animal kingdom have remained elusive. The difficulty in identifying osmosensors in animals is partly due to the lack of an unbiased screening system. Previous efforts to identify osmosensors in animals were restricted to TRP channels. However, osmosensors do not necessarily fall into the TRP channel family and thus may have eluded detection. The nematode C. elegans is an ideal model to study osmosensing. Like mammals, C. elegans has osmosensing systems, and conserved signaling molecules have been identified. This, together with its short generation time (~3 days) and facile and rich genetic tools, makes C. elegans an ideal system for identifying novel osmosensors. To identify osmosensors in C. elegans, we designed and conducted a neural activity- based genetic screen. We have identified OSMS-1 and OSMS-2 as candidate osmosensors in C. elegans. Despite this exciting finding, many questions remain unanswered. In the current proposal, we propose to test the hypothesis that OSMS-1 and OSMS-2 are bona fide osmosensors and characterize the molecular mechanisms by which OSMS-1 and OSMS-2 sense osmolarity. We will take a multidisciplinary approach by integrating molecular genetics, behavioral analysis, calcium imaging, electrophysiology, and cryo-EM. To do so, I will receive extensive training on calcium imaging, cell culture, electrophysiological recording, and cryo- EM. The K99/R00 award will allow me to acquire these skills with guidance from my mentors Drs. Shawn Xu and Melanie Ohi, which will help me to launch an independent research career. The proposed work will lead to the identification of the first osmosensors in the animal kingdom, gain a molecular understanding of how osmosensors sense osmotic stimuli, and provide novel insights into osmosensation, osmoregulation and related human diseases.