Syncytin-mediated open-ended membrane tubular connections facilitate the intercellular transfer of cargos including Cas9 protein
Much attention has been focused on the possibility that cytoplasmic proteins and RNA may be conveyed between cells in extracellular vesicles (EVs) and tunneling nanotube (TNT) structures. Here, we set up two quantitative delivery reporters to study cargo transfer between cells. We found that EVs are internalized by reporter cells but do not efficiently deliver functional Cas9 protein to the nucleus. In contrast, donor and acceptor cells co-cultured to permit cell contact resulted in a highly effective transfer. Among our tested donor and acceptor cell pairs, HEK293T and MDA-MB-231 recorded optimal intercellular transfer. Depolymerization of F-actin greatly decreased Cas9 transfer whereas inhibitors of endocytosis or knock-down of genes implicated in this process had little effect on transfer. Imaging results suggest that intercellular transfer of cargos occurred through open-ended membrane tubular connections. In contrast, cultures consisting only of HEK293T cells form close-ended tubular connections ineffective in cargo transfer. Depletion of human endogenous fusogens, syncytins, especially syncytin-2 in MDA-MB-231 cells, significantly reduced Cas9 transfer. Full-length mouse syncytin, but not truncated mutants, rescued the effect of depletion of human syncytins on Cas9 transfer. Mouse syncytin overexpression in HEK293T cells partially facilitated Cas9 transfer among HEK293T cells. These findings suggest that syncytin may serve as the fusogen responsible for the formation of an open-ended connection between cells.
All data generated or analysed during this study are included in the manuscript and supporting file. Source data files have been provided for Figure 1B; Figure 1- figure supplement 1A and 1B; Figure 3- figure supplement 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I, and 2J; Figure 7- figure supplement 1A, 1B, 1D, 1F, 1G, and 1H; Figure 8- figure supplement 1D; Figure 9B and 9D.
Article and author information
Howard Hughes Medical Institute
- Randy Schekman
Innovative Genomics Institute, UC Berkeley
- Randy Schekman
Sergey Brin Family Foundation
- Randy Schekman
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Robert H Singer, Albert Einstein College of Medicine, United States
- Received: October 22, 2022
- Accepted: February 26, 2023
- Accepted Manuscript published: March 10, 2023 (version 1)
© 2023, Zhang & Schekman
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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