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

两个细胞结合成一个细胞的过程，称为细胞融合，在包括人类在内的哺乳动物的整个发育过程中经常发生。在生殖过程中，精子和卵细胞必须融合才能受精，然后在胎儿发育过程中，肌肉细胞融合成长的骨骼肌纤维，从而允许运动。这些只是发生的许多聚变事件中的两个例子，并突出了这一一般过程的重要性。细胞融合不是自发发生的，受到蛋白质的高度调控，确保这一过程在正确的时间和地点发生。识别这些调控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，在哺乳动物中研究这些调控因子是困难的。酵母，酿酒酵母，在它们的生命周期中经历了类似于受精的融合事件，使它们成为研究融合的优秀模型系统。酵母易于培养和操纵，含有许多与人类细胞相同的基因和蛋白质。本项目旨在通过识别新的调控因子和分析该过程所需的已知蛋白质来研究酿酒酵母细胞融合的机制。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。学生将获得遗传操作酵母和执行尖端显微镜技术的经验。这个项目将鼓励下一代科学家，让他们获得研究经验，在那里他们将识别酵母融合的机制，这有助于了解哺乳动物的细胞融合。哺乳动物细胞的质膜周围有细胞壁，这意味着一旦两个细胞接触，它们必须首先降解细胞壁，特别是在它们将融合的地方，类似于哺乳动物系统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可以起作用，导致细胞质混合。虽然酵母融合的一些调控因子和形态事件已经确定，但关于这个过程仍然有一些悬而未决的问题。总之，本提案旨在通过对一个未被研究的细胞壁降解的关键调控因子的突变分析、融合过程中酶分泌的可视化以及新的质膜融合调控因子的鉴定来揭示细胞壁降解和质膜融合是如何受到调控的。荧光激活标签将被用来开发一种融合特异性分泌的显微分析，解决该领域长期存在的一个假说。此外，对一名学生研究人员最近发现的膜融合突变的抑制子分析将有助于识别新的质膜融合调节因子，这是科学家长期以来一直难以理解的。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。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.