Highlights:
• A3R5.7 and TF228.1.16 are capable of CD4-mediated membrane fusion.
• Cytoplasmic and nuclear material can be removed simultaneously from A3R5.7 cells.
• Resultant cell membrane remains intact, and retains presentation of CD4 and CXCR4.
• Products are still capable of CD4-mediated membrane fusion with a target cell.
• Useful for host-pathogen studies, and developing targeted delivery vehicles.
Authors:
Cherng-Wen Darren Tan, Andreas Forsthuber, Eva-Kathrin Ehmoser
Abstract:
Structurally-reduced cells and cell-derived structures are powerful tools for membrane studies. Using this approach, we probed whether a cell, without its nucleus and cytoplasm, is still capable of undergoing CD4-mediated membrane fusion. For this, we needed a cell-derived structure, akin to a giant liposome functionalised with CD4 and chemokine receptors. We present a method for the simultaneous removal of cytoplasmic and nuclear material from cells presenting CD4, CCR5, and CXCR4, using Colcemid treatment followed by hypotonic cytolysis, and then enriched using preparative flow cytometry. We show that the resultant cell membrane remains intact, retains presentation of CD4, CCR5, and CXCR4, and is still capable of CD4-mediated membrane fusion with a target cell. Finally, we detail how this protocol was developed, as well as how such samples should be handled for storage and assays. We envision the use of such systems for host-pathogen interaction studies, and the development of targeted delivery vehicles.
Accepted for publication in Experimental Cell Research on 10th January 2021.