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

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附近和远处的其他细胞。这些数据包如何发送的调节目前尚不清楚，但这种通信机制可以影响广泛的生物过程，如衰老，免疫力，神经元健康和细胞迁移。该项目的重点是确定细胞如何控制这些包的分泌。这些被称为“外泌体”的包裹是通过将分子分选到膜包膜中，然后将许多外泌体发送到细胞表面以同时释放而形成的。许多外泌体被包装在一个多囊泡体（MVB）中，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。为了做到这一点，细胞在其膜上组织分子，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。在这个研究项目中，将及时识别和映射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观察MVB和质膜之间的膜融合。MVB的低pH淬灭富含外来体的pH敏感性荧光蛋白，并且融合期间这些探针的未淬灭导致可以在空间和时间上定位的荧光闪光。SNARE蛋白，膜融合的公认调节剂，将筛选其在MVB融合过程中的作用。预测细胞膜上的SNARE蛋白在MVB下方聚集以帮助对接和融合。显微镜将与计算建模方法结合使用，以确定SNARE簇形成所需的分子组分。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NSF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。

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.