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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.

SUBMITTER: Rainy N

PROVIDER: S-EPMC3730418 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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H-Ras transfers from B to T cells via tunneling nanotubes.

Rainy N N Chetrit D D Rouger V V Vernitsky H H Rechavi O O Marguet D D Goldstein I I Ehrlich M M Kloog Y Y

Cell death & disease 20130718

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 advanc ...[more]

PMID: 23868059

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Project description:BACKGROUND: Tunneling nanotubes (TNTs) may offer a very specific and effective way of intercellular communication. Here we investigated TNTs in the human retinal pigment epithelial (RPE) cell line ARPE-19. Morphology of TNTs was examined by immunostaining and scanning electron microscopy. To determine the function of TNTs between cells, we studied the TNT-dependent intercellular communication at different levels including electrical and calcium signalling, small molecular diffusion as well as mitochondrial re-localization. Further, intercellular organelles transfer was assayed by FACS analysis. METHODOLOGY AND PRINCIPAL FINDINGS: Microscopy showed that cultured ARPE-19 cells are frequently connected by TNTs, which are not attached to the substratum. The TNTs were straight connections between cells, had a typical diameter of 50 to 300 nm and a length of up to 120 µm. We observed de novo formation of TNTs by diverging from migrating cells after a short time of interaction. Scanning electron microscopy confirmed characteristic features of TNTs. Fluorescence microscopy revealed that TNTs between ARPE-19 cells contain F-actin but no microtubules. Depolymerisation of F-actin, induced by addition of latrunculin-B, led to disappearance of TNTs. Importantly, these TNTs could function as channels for the diffusion of small molecules such as Lucifer Yellow, but not for large molecules like Dextran Red. Further, organelle exchange between cells via TNTs was observed by microscopy. Using Ca²? imaging we show the intercellular transmission of calcium signals through TNTs. Mechanical stimulation led to membrane depolarisation, which expand through TNT connections between ARPE-19 cells. We further demonstrate that TNTs can mediate electrical coupling between distant cells. Immunolabelling for Cx43 showed that this gap junction protein is interposed at one end of 44% of TNTs between ARPE-19 cells. CONCLUSIONS AND SIGNIFICANCE: Our observations indicate that human RPE cell line ARPE-19 cells communicate by tunneling nanotubes and can support different types of intercellular traffic.

Project description:Despite advances in cancer therapies, nanomedicine approaches including the treatment of glioblastoma (GBM), the most common, aggressive brain tumor, remains inefficient. These failures are likely attributable to the complex and not yet completely known biology of this tumor, which is responsible for its strong invasiveness, high degree of metastasis, high proliferation potential, and resistance to radiation and chemotherapy. The intimate connection through which the cells communicate between them plays an important role in these biological processes. In this scenario, tunneling nanotubes (TnTs) are recently gaining importance as a key feature in tumor progression and in particular in the re-growth of GBM after surgery. In this context, we firstly identified structural differences of TnTs formed by U87-MG cells, as model of GBM cells, in comparison with those formed by normal human astrocytes (NHA), used as a model of healthy cells. Successively, we have studied the possibility to exploit U87-MG TnTs as drug-delivery channels in cancer therapy, using liposomes composed of cholesterol/sphingomyelin and surface functionalized with mApoE and chlorotoxin peptides (Mf-LIP) as nanovehicle model. The results showed that U87-MG cells formed almost exclusively thick and long protrusions, whereas NHA formed more thin and short TnTs. Considering that thick TnTs are more efficient in transport of vesicles and organelles, we showed that fluorescent-labeled Mf-LIP can be transported via TnTs between U87-MG cells and with less extent through the protrusions formed by NHA cells. Our results demonstrate that nanotubes are potentially useful as drug-delivery channels for cancer therapy, facilitating the intercellular redistribution of this drug in close and far away cells, thus reaching isolated tumor niches that are hardly targeted by simple drug diffusion in the brain parenchyma. Moreover, the differences identified in TnTs formed by GBM and NHA cells can be exploited to increase treatment precision and specificity.

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H-Ras transfers from B to T cells via tunneling nanotubes.

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H-Ras transfers from B to T cells via tunneling nanotubes.

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