MCA - Application of quantitative imaging methods to identify molecular components of multi-vesicular body fusion sites

MCA - 应用定量成像方法识别多囊泡体融合位点的分子成分

• 批准号: 2122289
• 负责人: Michelle Knowles
• 金额: $ 33.57万
• 依托单位: University of Denver
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2122289&HistoricalAwards=false
• 关键词: MCA; Application; quantitative; imaging; methods

All cells communicate with one another by sending out packets of molecules that relay information to other cells, both near and far. The regulation of how these packets are sent is currently unknown but this mechanism of communication can affect a wide range of biological processes, such as aging, immunity, neuronal health, and cell migration. This project focuses on identifying how cells control the secretion of these packets. The packets, called “exosomes”, are formed by sorting molecules into membrane envelopes, and then sending many exosomes to the cell surface to be released simultaneously. Many exosomes are packed together in a multi-vesicular body (MVB), a larger membrane envelope, that is transported to the cell surface and releases exosomes upon fusing with the cell membrane. To make this happen, cells organize molecules on their membranes to ensure that the necessary proteins are present at the right time. When all the needed molecules are present, membranes fuse and the packets of molecules are released from the donor cell into the extracellular space. In this research project, the proteins that regulate the exosome secretion process will be identified and mapped in time to determine when and where essential proteins are needed. Recently designed fluorescent probes allow for the direct visualization of membrane fusion between MVBs and the plasma membrane using total internal reflection fluorescence microscopy. The low pH of the MVB quenches an exosome enriched, pH-sensitive fluorescent protein and the unquenching of these probes during fusion leads to a flash of fluorescence that can be spatially and temporally localized. SNARE proteins, well-established regulators of membrane fusion, will be screened for their role in the MVB fusion process. SNARE proteins on the cell membrane are predicted to cluster beneath MVBs to aid in both docking and fusion. Microscopy will be used in conjunction with computational modeling approaches to identify the molecular components necessary for SNARE cluster formation. Beyond the research activities, this work will also lead to collaboration, training, and mentorship for female faculty performing research in the biomolecular sciences at the University of Denver.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.

所有的细胞都通过发送分子包来相互交流，这些分子包将信息传递给远近的其他细胞。这些信息包如何发送的调控目前尚不清楚，但这种通信机制可以影响广泛的生物过程，如衰老、免疫、神经元健康和细胞迁移。这个项目的重点是确定细胞如何控制这些信息包的分泌。这些被称为“外泌体”的包，是通过将分子分类到膜包膜中，然后将许多外泌体送到细胞表面同时释放而形成的。许多外泌体被包裹在一个多泡体（MVB）中，这是一个更大的膜包膜，被运输到细胞表面，并在与细胞膜融合时释放外泌体。为了实现这一点，细胞在膜上组织分子，以确保必要的蛋白质在正确的时间出现。当所有需要的分子都存在时，细胞膜融合，分子包从供体细胞释放到细胞外空间。在本研究项目中，将及时识别和绘制调节外泌体分泌过程的蛋白质，以确定何时何地需要必需蛋白质。最近设计的荧光探针允许使用全内反射荧光显微镜直接可视化MVBs和质膜之间的膜融合。MVB的低pH值会淬灭外泌体富集的pH敏感荧光蛋白，而这些探针在融合过程中不淬灭会导致荧光闪光，这种荧光可以在空间和时间上定位。SNARE蛋白是一种成熟的膜融合调节因子，将对其在MVB融合过程中的作用进行筛选。预计细胞膜上的SNARE蛋白会聚集在MVBs下，以帮助对接和融合。显微镜将与计算建模方法结合使用，以确定SNARE簇形成所需的分子成分。除了研究活动之外，这项工作还将为丹佛大学从事生物分子科学研究的女教师提供合作、培训和指导。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Exosome secretion kinetics are controlled by temperature

• DOI: 10.1016/j.bpj.2023.02.025
• 发表时间: 2023-04-04
• 期刊: BIOPHYSICAL JOURNAL
• 影响因子: 3.4
• 作者: Mahmood，Anarkali;Otruba，Zdenek;Knowles，Michelle K.
• 通讯作者: Knowles，Michelle K.