BRC-BIO: Investigating the molecular mechanisms of fungal cell fusion

BRC-BIO：研究真菌细胞融合的分子机制

• 批准号: 2233325
• 负责人: Jean Smith
• 金额: $ 50.3万
• 依托单位: Stetson University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2233325&HistoricalAwards=false
• 关键词: BRC; BIO; Investigating; molecular; mechanisms

The process of two cells combining into one, known as cell fusion, happens frequently throughout the development of mammals, including humans. During reproduction, sperm and egg cells must fuse for fertilization to occur, and later in fetal development muscle cells fuse into long skeletal muscle fibers that allow for movement. These are just two examples of the many fusion events that occur and highlight the importance of this general process. Cell fusion does not happen spontaneously and is highly regulated by proteins that ensure the process occurs at the correct time and place. Identifying these regulators and determining how they function is crucial for understanding cell fusion but has proven difficult to study in mammals. The yeast, Saccharomyces cerevisiae, undergoes a fusion event similar to fertilization during their life cycle making them an excellent model system to study fusion. Yeast are easy to grow and manipulate and contain many of the same genes and proteins as human cells. This project aims to study the mechanisms of cell fusion in S. cerevisiae by identifying novel regulators and analyzing known proteins required for the process. This work will be performed by undergraduates through course-based research experiences and year-long independent research opportunities. Students will gain experience genetically manipulating yeast and performing cutting edge microscopy techniques. This project will encourage the next generation of scientists by allowing them access to research experiences where they will identify mechanisms of yeast fusion that can aid in the understanding of cell fusion in mammals.Yeast cells have a cell wall surrounding their plasma membrane, meaning that once two cells come into contact, they must first degrade their cell walls specifically where they will fuse, similar to the degradation of extracellular matrix required in mammalian systems. Once this occurs, proteins that promote plasma membrane fusion can act, leading to cytoplasmic mixing. While some regulators and morphological events of yeast fusion have been identified, there are still unanswered questions about the process. Overall, this proposal aims to uncover how cell wall degradation and plasma membrane fusion are regulated through mutational analysis of an understudied, key regulator of cell wall degradation, visualization of enzyme secretion during fusion, and identification of novel plasma membrane fusion regulators. Fluorogen-activating tags will be used to develop a microscopic assay for fusion-specific secretion, addressing a long-standing hypothesis in the field. Additionally, suppressor analysis of a membrane fusion mutation recently identified by a student researcher will allow for identification of novel plasma membrane fusion regulators, which have long-eluded scientists. Along with significant advancements to the understanding of yeast fusion, this work involves proteins that are conserved from yeast to humans allowing the potential to provide insight into this fundamental process in higher eukaryotes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

两个细胞合二为一的过程，称为细胞融合，在哺乳动物（包括人类）的发育过程中经常发生。在繁殖过程中，精子和卵细胞必须融合才能受精，随后在胎儿发育过程中，肌肉细胞融合成允许运动的长骨骼肌纤维。这些只是发生的许多聚变事件的两个例子，并强调了这一一般过程的重要性。细胞融合不会自发发生，而是受到蛋白质的高度调控，以确保该过程在正确的时间和地点发生。识别这些调节因子并确定它们的功能对于理解细胞融合至关重要，但事实证明很难在哺乳动物中进行研究。酿酒酵母在其生命周期中经历类似于受精的融合事件，使其成为研究融合的优秀模型系统。酵母易于生长和操作，并且含有许多与人类细胞相同的基因和蛋白质。该项目旨在通过鉴定新的调节因子并分析该过程所需的已知蛋白质来研究酿酒酵母的细胞融合机制。这项工作将由本科生通过基于课程的研究经验和为期一年的独立研究机会来完成。学生将获得基因操纵酵母和执行尖端显微镜技术的经验。该项目将鼓励下一代科学家，让他们获得研究经验，他们将确定酵母融合机制，有助于理解哺乳动物的细胞融合。酵母细胞的质膜周围有细胞壁，这意味着一旦两个细胞接触时，它们必须首先降解它们的细胞壁，特别是它们将融合的地方，类似于哺乳动物系统中所需的细胞外基质的降解。一旦发生这种情况，促进质膜融合的蛋白质就会起作用，导致细胞质混合。虽然酵母融合的一些调节因子和形态事件已被确定，但有关该过程的问题仍有待解答。总体而言，该提案旨在通过对正在研究的细胞壁降解关键调节因子的突变分析、融合过程中酶分泌的可视化以及新型质膜融合调节因子的鉴定来揭示细胞壁降解和质膜融合是如何调节的。荧光激活标签将用于开发融合特异性分泌的显微检测方法，解决该领域长期存在的假设。此外，对一名学生研究人员最近发现的膜融合突变进行抑制分析将有助于识别新型质膜融合调节因子，而这正是科学家长期以来一直未能实现的。随着对酵母融合的理解取得重大进展，这项工作涉及从酵母到人类的保守蛋白质，从而有可能深入了解高等真核生物中的这一基本过程。该奖项反映了 NSF 的法定使命，并被认为值得通过以下方式获得支持：使用基金会的智力价值和更广泛的影响审查标准进行评估。