BPS2026 – Progress toward understanding endosomal membrane identity via single-molecule imaging of Rab GTP-ase binding events

Endosomal compartments face a fascinating physical challenge—they must correctly identify and sort potential cargo into transport intermediates directed to the proper cellular locations. Growing evidence in the field suggests this process involves a complex signaling cascade on the endosomal membrane, incorporating luminal cargo recognition adaptors, Rab GTPases, and downstream effector molecules such as sorting nexins. In this project, we present early work toward adapting our high-speed, multicolor single-molecule tracking system to simultaneously visualize the interplay of multiple members of this cascade on the endosomes of living cells. We show preliminary results indicating that we can quantify binding events of individual members of the Rab GTPase family on native cellular endosomes in real time, and that distinct signatures of specific family members correlate with their predicted biological functions and the proposed identity of the compartment. These results demonstrate the viability of direct, in-cell visualization tools for understanding the sorting events underlying cellular endocytosis and compartment maturation.