Bipartite regulation of cellular osmosensing in C. elegans

描述（由申请人提供）：维持细胞溶质和水分含量的生理过程被称为渗透稳态或渗透调节，对所有形式的细胞生命都是必不可少的。在人类中，渗透稳态在多种情况下起着至关重要的作用，包括调节肾脏的尿浓缩机制、控制血压和激活免疫反应。渗透性平衡失调与许多与年龄有关的疾病有关，包括慢性肾病、肾功能衰竭、高血压和周围神经病变。尽管渗透调节在生理和病理生理疾病状态中都具有明显的重要性，但动物细胞感知和响应渗透应激的机制尚不清楚。更好地了解这些机制可能有助于及早发现和干预与年龄有关的疾病。大多数关于渗透调节的研究都是用培养的细胞进行的，这种细胞不能模拟大多数细胞所处的复杂环境。这些研究导致了许多假设来解释细胞渗透传感的机制，例如膜或细胞骨架的机械“拉伸”，大分子拥挤，细胞质离子含量的改变，等等。然而，几乎没有数据支持这些模型。为了获得动物体内细胞渗透机制的观点，我们正在模式生物秀丽隐杆线虫中研究这一过程，其中保存了复杂的细胞-细胞和细胞-细胞外基质（ECM）相互作用。使用无偏正向和反向遗传方法，我们发现了ECM （Rohlfing等人，PLoS Genetics, 2011）和蛋白质错误折叠（Moronetti Mazzeo等人，PNAS, 2012）在秀丽隐杆线虫细胞渗透调节中的关键作用。基于这些发现，我们假设动物细胞使用机械转导和蛋白质损伤检测机制来感知渗透干扰并激活渗透敏感基因表达。在Aim 1中，我们将确定秀丽隐杆线虫表皮ECM是否作为一种结构“渗透传感器”，通过黏液样蛋白OSM-8和跨膜蛋白PTR-23之间的相互作用来传递信息。在目标2中，我们将确定蛋白质和化学伴侣如何在渗透胁迫的背景下防止蛋白质损伤。在Aim 3中，我们将研究ECM和蛋白质损伤检测途径如何相互作用以控制渗透调节生理。我们的研究最大限度地利用秀丽隐杆线虫系统来填补我们对后生动物细胞生理学知识的重要空白。这些发现将为渗透调节的保守过程提供革命性的见解，使我们能够更好地理解、检测和管理与年龄相关的渗透平衡失调疾病。

DESCRIPTION (provided by applicant): The physiological process of maintaining cellular solute and water content is termed osmotic homeostasis, or osmoregulation, and is essential for all forms of cellular life. In humans, osmotic homeostasis plays vital roles in several contexs, including regulation of the kidney's urinary concentrating mechanism, control of blood pressure, and activation of immune responses. Osmotic dyshomeostasis is associated with several age- related diseases, including chronic kidney disease, renal failure, hypertension, and peripheral neuropathy. Despite the obvious importance of osmoregulation in both physiological and pathophysiological disease states, little is known about the mechanisms by which animal cells sense and respond to osmotic stress. A better understanding of these mechanisms may allow earlier detection and intervention in age-related diseases. Most studies of osmoregulation have been carried out using cultured cells, which fail to mimic the complex environments in which most cells are found. These studies have led to many hypotheses to explain the mechanism(s) of cellular osmosensing, such as mechanical 'stretching' of the membrane or cytoskeleton, macromolecular crowding, and alterations in cytoplasmic ionic content, to name a few. However, there is little data supporting any of these models. To gain an in vivo perspective on mechanisms of cellular osmosensing in animals, we are studying this process in the model organism C. elegans, in which complex cell-cell and cell- extracellular matrix (ECM) interactions are preserved. Using unbiased forward and reverse genetic approaches, we discovered critical roles for the ECM (Rohlfing et al, PLoS Genetics, 2011) and protein misfolding (Moronetti Mazzeo et al, PNAS, 2012) in the regulation of cellular osmosensing in C. elegans. Based on these findings we hypothesize that animal cells use both mechanotransduction and protein damage detection mechanisms to sense osmotic disturbances and activate osmosensitive gene expression. In Aim 1, we will determine if the C. elegans cuticular ECM acts as a structural 'osmosensor' to transduce information via interactions between the mucin-like protein OSM-8 and a transmembrane protein PTR-23. In Aim 2, we will determine how protein and chemical chaperones prevent protein damage in the context of osmotic stress. In Aim 3, we will examine how ECM and protein damage detection pathways interact with each other to control osmoregulatory physiology. Our studies take maximal advantage of the C. elegans system to fill an important gap in our knowledge of metazoan cell physiology. These findings will provide transformative insights into the conserved process of osmoregulation that will allow us to better understand, detect, and manage age-related diseases of osmotic dyshomeostasis.