Modelling the effect of tethering on exosome secretion.

Abstract

Exosomes are small membrane vesicles that originate from multivesicular bodies (MVBs). Exosomes contain cargo molecules that are transported between cells. This type of transport is used for intercellular communication, but exosome secretion is upregulated in certain diseases, such as cancer. For exosomes to leave cells, MVBs fuse with the cellular membrane and exosomes are subsequently externalized. Recent research uncovered an interesting property of this secretion: exosomes take a significantly longer time to leave the site of the MVB fusion than they would if they could diffuse freely after the fusion event. Others have also shown that exosomes remain attached to cell surface with EM data. It appears that there is something that holds the exosomes attached. Our research addressed this question by observing single MVB fusion events in time to determine the kinetics of exosome release. Exosomes and MVB membranes accumulate CD63-pHluorin and the loss of fluorescence from the fusion site is related to the loss of CD63-pHluorin containing exosomes and diffusion of CD63-pHluorin into the plasma membranes. We developed a simulation that models the movement of exosomes post-fusion to interpret the fluorescence loss. The simulation treats this as an exponential decay composed of the number of exosomes left at the fusion site, and the diffusion of the probe in the plasma membrane. Experimentally determined parameters, like the diffusion coefficient of CD63-pHluroin in the cell membrane, were used to simulate the decays. The simulation accurately replicated the experimental decays and determined properties of these exosomal tethers such as their lifetime or the fraction of exosomes that start tethered. The simulated properties can be used to better describe how exosomes leave cells, which will provide insight into the mechanisms used by some diseases to propagate and a better understanding of cell communication.