Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Monday Speaker Abstracts

MULTIVESICULAR ENDOSOME FUSION KINETICS DEPEND ON EXOSOME TETHERING AND TEMPERATURE

Michelle K. Knowles 1 ; Zdenek Otruba 1 ; Broderick Bills 1 ; Melodie Nguyen 1 ; 1 University of Denver, Molecular and Cellular Biophysics, Denver, CO, USA

Multivesicular endosomes (MVEs) fuse with the plasma membrane to secrete exosomes into the extracellular space. From here, exosomes can be taken up by cells locally or flow through the body to affect cells further from the secretion site. It is not clear what controls whether exosomes are fully released from the secreting cell or remain nearby but temperature greatly affects the release kinetics. In this work, single MVE fusion events are visualized in a TIRF microscope using a pH-sensitive fluorescent protein tagged to CD63, a tetraspanin that is enriched in exosomes. Single events were measured in A549 cells at 23-37C where lower temperatures correlate to a reduced frequency of fusion and a reduction in the rate of content release. Content release is often incomplete at all temperatures but more so at lower temperatures. To interpret the complex kinetics observed, a simple diffusion-based model was used to simulate the fluorescence decays. Three components were required: freely diffusing exosomes, CD63 diffusion from the limiting membrane into the plasma membrane and tethered exosomes. Our data suggest that temperature affects release and the post-fusion attachment of exosomes at the fusion site.

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