Cln3 function in the model organism Dictyostelium discoideum

背景cln3 （ceroid lipofuscinosis neuronal 3）属于NCL蛋白家族。目前的知识表明，NCL蛋白参与吞噬和处理细胞外物质的途径，为细胞提供营养和消化不需要的物质。然而，它们在这些途径中的具体功能仍有待阐明。先前的研究已经将CLN3与多种细胞过程联系起来。然而，其在细胞中的确切功能仍不清楚，这在很大程度上是由于之前的工作无法确定蛋白质的精确定位。盘状盘基骨柱（Dictyostelium disideum）长期以来被认为是细胞和发育生物学的模型系统。这种土壤微生物的生命周期由单细胞和多细胞阶段组成，这使得各种细胞过程可以非常详细地进行检查。我们之前的研究表明，Dictyostelium中CLN3的缺失（简称CLN3）会导致异常生长和多细胞发育。我们还发现Cln3定位于收缩液泡（CV）系统，这是一个细胞内的隔室，帮助细胞响应和适应不同的环境条件（例如，维持水和离子平衡），以及在细胞内的隔室之间移动物质。然而，CV系统的重要性并不仅仅局限于盘基骨柱。它对淡水环境中多种生态重要生物的生存也至关重要，并已被研究为新型植物保护策略的潜在目标。目的：研究Cln3在盘基骨柱中的定位，以确定其在细胞中的精确功能。我们将使用多种细胞、分子和生化方法来揭示Cln3在盘基骨柱中的功能、与之相互作用的蛋白质以及调节其功能的信号通路。这将包括评估Cln3缺乏对与CV系统相关的过程的影响（例如，水和离子平衡），并研究Cln3与CV系统功能相关的蛋白质（例如，Rab蛋白，钙调蛋白）的相互作用。我们的长期目标是利用盘基ostelium作为模型系统来研究所有NCL蛋白的功能。意义本研究将有助于我们进一步了解Cln3在盘柄骨中的作用。细胞内物质的有效运输对细胞的整体健康和功能至关重要。在人类中，NCL蛋白的突变导致巴滕病，这是儿童神经变性最常见的形式。从长远来看，这项工作将对我们理解这些重要但特征不明确的蛋白质在细胞中所起的作用产生重大影响。最后，参与这项研究的人员将获得知识和发展技术和可转移技能，这将有助于支持创造高技能的加拿大劳动力。

BackgroundCLN3 (ceroid lipofuscinosis neuronal 3) belongs to the NCL family of proteins. Current knowledge suggests that NCL proteins participate in pathways that engulf and process extracellular material to provide the cell with nutrients and digest unwanted material. However their specific functions in these pathways remain to be clarified. Previous research has linked CLN3 to a diversity of cellular processes. However its precise function in the cell is still unclear, in large part due to previous work being unable to determine the precise localization of the protein. Dictyostelium discoideum has long served as a model system for cell and developmental biology. The life cycle of this soil microbe is comprised of both single-cell and multicellular phases, which allows for a variety of cellular processes to be examined in great detail. Our previous work showed that loss of CLN3 in Dictyostelium (denoted Cln3) causes abnormal growth and multicellular development. We also revealed that Cln3 localizes to the contractile vacuole (CV) system, which is an intracellular compartment that helps the cell respond and adapt to different environmental conditions (e.g., maintain water and ion balance), as well as move material between compartments within the cell. However, the importance of the CV system is not just limited to Dictyostelium. It is also essential to the survival of a diversity of ecologically important organisms in freshwater environments and has been investigated as a potential target for novel plant protection strategies.ObjectivesWe are using the defined localization of Cln3 in Dictyostelium to fuel research into determining its precise function in the cell. We will use a diversity of cellular, molecular, and biochemical approaches to reveal the function of Cln3 in Dictyostelium, the proteins it interacts with, and the signalling pathways that regulate its function. This will involve assessing the effect of Cln3-deficiency on processes linked to the CV system (e.g., water and ion balance) and studying the interaction of Cln3 with proteins involved in CV system function (e.g., Rab proteins, calmodulin). Our long-term objective is to use Dictyostelium as a model system for studying the functions of all NCL proteins.SignificanceThis research will significantly advance our understanding of Cln3 function in Dictyostelium. The efficient trafficking of material within a cell is critical to its overall health and function. In humans, mutations in NCL proteins cause Batten disease, the most common form of childhood neurodegeneration. In the long-term, this work will have a significant impact on our understanding of the roles these important, but poorly characterized proteins play in the cell. Finally, personnel participating in this research will acquire knowledge and develop technical and transferrable skills that will help support the creation of highly-skilled Canadian workforce.