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.

所有细胞通过发送分子数据包来相互通信，这些分子数据包将信息传递给附近和远处的其他细胞。目前尚不清楚这些数据包如何发送的调节，但这种通信机制可以影响广泛的生物过程，例如。该项目的重点是确定细胞如何控制这些被称为“外泌体”的包的分泌，它们是通过将分子分类到膜包膜中，然后将许多外泌体发送到细胞而形成的。细胞表面为许多外泌体被包装在一个更大的膜泡体（MVB）中，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，细胞在其上组织分子。当所有需要的分子都存在时，膜会融合，分子包从供体细胞释放到细胞外空间。外泌体的分泌过程将及时识别并绘制图谱，以确定何时何地需要必需的蛋白质。最近设计的荧光探针允许使用全内反射荧光显微镜直接观察 MVB 和膜血浆之间的膜融合。MVB 的低 pH 值会淬灭外泌体。富集的、pH 敏感的荧光蛋白以及这些探针在融合过程中的不猝灭会产生可以在空间和时间上定位的 SNARE 蛋白（成熟的膜调节剂）的荧光闪光。将筛选细胞膜上的 SNARE 蛋白在 MVB 融合过程中的作用，以帮助对接和融合，并将与计算建模方法结合使用来识别所需的分子成分。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性教师带来合作、培训和指导。该奖项反映了 NSF 的法定使命和通过使用基金会的智力优点和更广泛的影响审查标准进行评估，该项目被认为值得支持。

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.