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The growth determinants and transport properties of tunneling nanotube networks between B lymphocytes

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Abstract

Tunneling nanotubes (TNTs) are long intercellular connecting structures providing a special transport route between two neighboring cells. To date TNTs have been reported in different cell types including immune cells such as T-, NK, dendritic cells, or macrophages. Here we report that mature, but not immature, B cells spontaneously form extensive TNT networks under conditions resembling the physiological environment. Live-cell fluorescence, structured illumination, and atomic force microscopic imaging provide new insights into the structure and dynamics of B cell TNTs. Importantly, the selective interaction of cell surface integrins with fibronectin or laminin extracellular matrix proteins proved to be essential for initiating TNT growth in B cells. These TNTs display diversity in length and thickness and contain not only F-actin, but their majority also contain microtubules, which were found, however, not essential for TNT formation. Furthermore, we demonstrate that Ca2+-dependent cortical actin dynamics exert a fundamental control over TNT growth-retraction equilibrium, suggesting that actin filaments form the TNT skeleton. Non-muscle myosin 2 motor activity was shown to provide a negative control limiting the uncontrolled outgrowth of membranous protrusions. Moreover, we also show that spontaneous growth of TNTs is either reduced or increased by B cell receptor- or LPS-mediated activation signals, respectively, thus supporting the critical role of cytoplasmic Ca2+ in regulation of TNT formation. Finally, we observed transport of various GM1/GM3 + vesicles, lysosomes, and mitochondria inside TNTs, as well as intercellular exchange of MHC-II and B7-2 (CD86) molecules which may represent novel pathways of intercellular communication and immunoregulation.

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Acknowledgments

This work was supported by grants T 104971 (to JM), NN 107776 and K112794 (to MN), K108437 (to LN) and K109480 (to MK) sponsored by the Hungarian National Science Fund (OTKA) and partly by MedinProt Project (Hungarian Academy of Sciences) to JM. We thank National Development Agency (NFU) and the European Social Fund for partly supporting this project by Grant Agreement TÁMOP 4.2.1./B-09/1/KMR-2010-0003. The authors are grateful to Drs. András Málnási-Csizmadia, Boglárka Várkuti, Miklós Képíró, Mihály Kovács, Judit Ovádi, Andrea Balogh, Glória László, and Imre Derényi for valuable advice and discussions throughout this work and to Ms. Márta Pásztor and Árpád Mikesy for their skillful technical assistance. The authors are very grateful to Xabier Osteikoetxea for careful reading and for the English language revisions of the manuscript, as well as, for the valuable discussions.

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Authors and Affiliations

  1. Department of Immunology, Eötvös Loránd University, Budapest, Hungary

    Anikó Osteikoetxea-Molnár, Eszter Angéla Tóth, Ádám Oszvald, Emese Izsépi, Mariann Kremlitzka & Janos Matko

  2. Department of Biophysics, Medical Faculty, University of Pécs, Pecs, Hungary

    Edina Szabó-Meleg & Miklós Nyitrai

  3. MTA-PTE Nuclear-Mitochondrial Interactions Research Group, Pecs, Hungary

    Edina Szabó-Meleg & Miklós Nyitrai

  4. Department of Biochemistry, Eötvös Loránd University, Budapest, Hungary

    Beáta Biri & László Nyitray

  5. Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary

    Tamás Bozó & Miklós Kellermayer

  6. Environmental Chemistry Research Group, Research Centre for Natural Sciences, Budapest, Hungary

    Péter Németh

  7. MTA-SE Molecular Biophysics Research Group, Budapest, Hungary

    Miklós Kellermayer

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  1. Anikó Osteikoetxea-Molnár
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Correspondence to Janos Matko.

Electronic supplementary material

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18_2016_2233_MOESM1_ESM.eps

Figure S1: a Flow cytometric histograms show the presence of α6 (black: isotype control antibody; grey: anti-α6 antibody), b and the lack of β4 (black: isotype control antibody; grey: anti- β4 antibody) integrin chains on A20 mature murine B cells. c Representative fluorescent live cell confocal image of A20 B cells stained with DiI dye and incubated for 1 h on laminin. d A20 B cell NT growth frequency was found dependent on the interaction between laminin and both of its integrin receptor subunits (α6β1); significantly reduced number of NT growing cells was found upon blocking of β1 chains with the respective antibody. Blocking α6 chain alone or the two chains simultaneously almost fully suppressed NT formation. Mean and SD values for TNT forming cell  % were derived from three independent experiments, from approximately 500 cells/sample (*: p ≤ 0.05, **: p ≤ 0.01) (EPS 3495 kb)

Movie S1: Movement of vesicles within thick membrane nanotubes of A488-CTX-B stained A20 mature murine B cells as visualized by structured illumination microscopy (SIM) in real time for 280 s (MP4 209 kb)

Movie S2: Prototypical trajectories of vesicles (see blue and green lines) tracked from a section within a nanotube for 280 s illustrating bidirectional traffic of microvesicles within thick membrane nanotubes of A20 B cells. Position of the tracked microvesicles at the beginning of the process is indicated by purple circles. ImageJ/TrackMate plugin was used to identify and then track vesicles on a frame-by-frame basis. (http://fiji.sc/TrackMate) Time-lapse movie was acquired by SIM using 5 grid rotations. (MP4 461 kb)

18_2016_2233_MOESM4_ESM.mp4

Movie S3: Movement of mitochondria stained with MitoTracker between two adjacent A20 mature B cells via nanotube as shown by a time lapse movie generated from LC-CLSM image series recorded in real time for 96 s (MP4 334 kb)

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Movie S4: Retraction of an already existing nanotube (see white arrow) upon ionomycin administration (Ca2+-influx) within 11 s following addition of ionomycin as shown by a time lapse movie of DIC images of A20 B cells recorded in real time (MP4 2119 kb)

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Figure S2: Flow cytometric dot plots show that addition of ionomycin to A20 B cells did not have in itself cytotoxic effects. Annexin V (early apoptosis marker) and propidium iodide (PI) (late apoptosis and necrosis marker) were used to verifying the viability of cells. The percentage of Annexin V positive and Annexin V + PI double positive cells in control, untreated sample (a) and in control cells cultured for 1 h without ionomycin (c) were low. These percentages did not change significantly in the sample treated with 1 µg/ml ionomycin for 5 min (b) or in samples treated with 1 µg/ml ionomycin for 5 min, followed by a wash and culturing for 1 h (d) (EPS 1248 kb)

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Osteikoetxea-Molnár, A., Szabó-Meleg, E., Tóth, E.A. et al. The growth determinants and transport properties of tunneling nanotube networks between B lymphocytes. Cell. Mol. Life Sci. 73, 4531–4545 (2016). https://doi.org/10.1007/s00018-016-2233-y

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  • DOI: https://doi.org/10.1007/s00018-016-2233-y

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