title: H-Ras transfers from B to T cells via tunneling nanotubes.
abstract: Lymphocytes form cell-cell connections by various mechanisms, including intercellular networks through actin-supported long-range plasma membrane (PM) extensions, termed tunneling nanotubes (TNTs). In this study, we tested in vitro whether TNTs form between human antigen-presenting B cells and T cells following cell contact and whether they enable the transfer of PM-associated proteins, such as green fluorescent protein (GFP)-tagged H-Ras (GFP-H-Ras). To address this question, we employed advanced techniques, including cell trapping by optical tweezers and live-cell imaging by 4D spinning-disk confocal microscopy. First, we showed that TNTs can form after optically trapped conjugated B and T cells are being pulled apart. Next, we determined by measuring fluorescence recovery after photobleaching that GFP-H-Ras diffuses freely in the membrane of TNTs that form spontaneously between B and T cells during coculturing. Importantly, by 4D time-lapse imaging, we showed that GFP-H-Ras-enriched PM patches accumulate at the junction between TNTs and the T-cell body and subsequently transfer to the T-cell surface. Furthermore, the PM patches adopted by T cells were enriched for another B-cell-derived transmembrane receptor, CD86. As predicted, the capacity of GFP-H-Ras to transfer between B and T cells, during coculturing, was dependent on its normal post-transcriptional lipidation and consequent PM anchorage. In summary, our data indicate that TNTs connecting B and T cells provide a hitherto undescribed route for the transfer of PM patches containing, for example, H-Ras from B to T cells.
authors.affiliation: Department of Neurobiology, The George S Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: N
authors.initials: N
authors.lastname: Rainy
authors.forename: D
authors.initials: D
authors.lastname: Chetrit
authors.forename: V
authors.initials: V
authors.lastname: Rouger
authors.forename: H
authors.initials: H
authors.lastname: Vernitsky
authors.forename: O
authors.initials: O
authors.lastname: Rechavi
authors.forename: D
authors.initials: D
authors.lastname: Marguet
authors.forename: I
authors.initials: I
authors.lastname: Goldstein
authors.forename: M
authors.initials: M
authors.lastname: Ehrlich
authors.forename: Y
authors.initials: Y
authors.lastname: Kloog
mesh_terms: D001402:B-Lymphocytes / Q000201:enzymology* / Q000648:ultrastructure; D022081:Cell Surface Extensions / Q000201:enzymology*; D018920:Coculture Techniques; D004058:Diffusion; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D054878:Lipoylation; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D043942:Nanotubes; D017368:Protein Prenylation; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013601:T-Lymphocytes / Q000201:enzymology / Q000648:ultrastructure; D059008:Time-Lapse Imaging
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)