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

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.