Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking

Saïda Abounit1, Elise Delage1, Chiara Zurzolo2

1 These authors contributed equally to this work, 2 Corresponding author
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 12.10
DOI:  10.1002/0471143030.cb1210s67
Online Posting Date:  June, 2015
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Abstract

Tunneling nanotubes (TNTs) are thin membranous channels providing direct cytoplasmic connection between remote cells. They are commonly observed in different cell cultures and increasing evidence supports their role in intercellular communication and pathogen transfer. However, the study of TNTs presents several pitfalls (e.g., difficulty in preserving such delicate structures, possible confusion with other protrusions, structural and functional heterogeneity, etc.) and therefore requires thoroughly designed approaches. The methods described in this unit represent a guideline for the characterization of TNTs (or TNT‐like structures) in cell culture. Specifically, optimized protocols to (1) identify TNTs and the cytoskeletal elements present inside them; (2) evaluate TNT frequency in cell culture; (3) unambiguously distinguish them from other cellular connections or protrusions; and (4) monitor their formation in living cells are provided. Finally, this unit describes how to assess TNT‐mediated cell‐to‐cell transfer of cellular components, which is a fundamental criterion for identifying functional TNTs. © 2015 by John Wiley & Sons, Inc.

Keywords: tunneling nanotubes (TNTs); intercellular communication; membrane protrusion; cytoskeleton

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Identification of Tunneling Nanotubes
  • Alternate Protocol 1: Identifying the Cytoskeletal Elements Present in Tunneling Nanotubes
  • Alternate Protocol 2: Distinguishing Tunneling Nanotubes from Intercellular Bridges Formed During Cell Division
  • Basic Protocol 2: Deciphering Tunneling Nanotube Formation by Live Fluorescent Microscopy
  • Alternate Protocol 3: Deciphering Tunneling Nanotube Formation Using a Live‐Compatible Dye
  • Basic Protocol 3: Tunneling Nanotubes Functionality in Intercellular Transfer
  • Alternate Protocol 4: Flow Cytometry Approach to Quantify the Rate of Vesicle Transfer
  • Support Protocol 1: Controls to Support TNT‐Mediated Transfer
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Identification of Tunneling Nanotubes

  Materials
  • CAD cells (Sigma, cat. no. 08100805)
  • CAD cell growth medium (see recipe)
  • Fixative 1 (see recipe)
  • Fixative 2 (see recipe)
  • 1× PBS
  • Alexa Fluor conjugate of wheat germ agglutinin (Molecular Probes)
  • HCS CellMask Blue stain (Molecular Probes)
  • Aqua‐Poly/Mount (Polysciences)
  • 25‐cm2 culture flasks
  • 37°C incubator
  • Hemacytometer or automated cell counter
  • Ibidi 35‐mm μ‐dishes, high, tissue‐culture‐treated (ibidi)
  • 24‐mm round coverslips
  • Inverted confocal fluorescence microscope
  • Imaging software

Alternate Protocol 1: Identifying the Cytoskeletal Elements Present in Tunneling Nanotubes

  Additional Materials (also see protocol 1)
  • Fixative 3 (see recipe)
  • 100 mM NH 4Cl in PBS
  • 1% (v/v) Triton‐X 100
  • 2% (w/v) bovine serum albumin (BSA) in PBS
  • Anti‐α‐tubulin antibody
  • Alexa Fluor–conjugated secondary antibody
  • 6.6 μM fluorescent phallotoxin (Molecular Probes) in methanol

Alternate Protocol 2: Distinguishing Tunneling Nanotubes from Intercellular Bridges Formed During Cell Division

  Additional Materials (also see protocol 1)
  • Serum‐free Opti‐MEM (Life Technologies)
  • Lipofectamine 2000 (Life Technologies)
  • DNA preparations of vectors encoding chosen fusion‐proteins
  • Fluorescent protein markers to distinguish between two cell populations (see Critical Parameters)
  • 1.5‐ml microcentrifuge tubes

Basic Protocol 2: Deciphering Tunneling Nanotube Formation by Live Fluorescent Microscopy

  Materials
  • CAD cells
  • CAD cell growth medium (complete medium)
  • Serum‐free Opti‐MEM I reduced serum medium (Life Technologies)
  • Lipofectamine 2000 (Life Technologies)
  • DNA preparations of vectors encoding chosen fusion‐proteins
  • 25‐cm2 culture flasks
  • 37°C incubator
  • Hemacytometer or automated cell counter
  • 35‐mm μ‐dishes, high, tissue culture treated (ibidi)
  • Inverted spinning disk confocal fluorescence microscope equipped with live observation chamber

Alternate Protocol 3: Deciphering Tunneling Nanotube Formation Using a Live‐Compatible Dye

  Additional Materials (also see protocol 4)
  • CellMask Orange Plasma Membrane Stain (Molecular Probes), also available in green and far red

Basic Protocol 3: Tunneling Nanotubes Functionality in Intercellular Transfer

  Materials
  • CAD cells
  • CAD cell growth medium (see recipe)
  • Serum‐free Opti‐MEM I reduced serum medium (Life Technologies)
  • Lipofectamine 2000 (Life Technologies)
  • DNA preparation of GFP plasmid
  • DiD lipophilic tracer (Life Technologies)
  • 1× PBS
  • Fixative 1 (see recipe)
  • Fixative 2 (see recipe)
  • HCS CellMask Blue stain (Molecular Probes)
  • Aqua‐Poly/Mount (Polysciences)
  • 37°C incubator
  • 25‐cm2 flask
  • 5‐ or 15‐ml centrifuge tubes
  • 1.5‐ml microcentrifuge tubes
  • 35‐mm μ‐dishes, high, tissue culture–treated (ibidi)
  • 24‐mm round coverslips
  • Inverted spinning disk confocal fluorescence microscope equipped with live observation chamber
  • Confocal microscope

Alternate Protocol 4: Flow Cytometry Approach to Quantify the Rate of Vesicle Transfer

  Additional Materials (also see protocol 6)
  • 1× PBS, ice cold
  • 4% PFA (w/v), ice cold
  • 37°C incubator
  • 24‐well plates
  • 12 × 75–mm tubes with cell strainer caps (Falcon)
  • Flow cytometer and flow cytometry analysis software (e.g., Kaluza, FlowJo)
  • Additional reagents and equipment for transfection of acceptor CAD cells (see protocol 3)

Support Protocol 1: Controls to Support TNT‐Mediated Transfer

  Additional Materials (also see protocol 6)
  • Transwell clear polyester membrane inserts for 24‐well plates (6.5 mm with 0.4‐μm pores; Costar)
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Figures

Videos

Literature Cited

Literature Cited
  Abounit, S. and Zurzolo, C. 2012. Wiring through tunneling nanotubes‐from electrical signals to organelle transfer. J. Cell Sci. 125:1089‐1098.
  Andresen, V., Wang, X., Ghimire, S., Omsland, M., Gjertsen, B.T., and Gerdes, H.H. 2013. Tunneling nanotube (TNT) formation is independent of p53 expression. Cell Death Differ. 20:1124‐1124.
  Bukoreshtliev, N.V., Wang, X., Hodneland, E., Gurke, S., Barroso, J.F.V., and Gerdes, H.‐H. 2009. Selective block of tunneling nanotube (TNT) formation inhibits intercellular organelle transfer between PC12 cells. FEBS Lett. 583:1481‐1488.
  Chinnery, H.R., Pearlman, E., and McMenamin, P.G. 2008. Cutting edge: Membrane nanotubes in vivo: A feature of MHC class II+ cells in the mouse cornea. J. Immunol. 180:5779‐5783.
  Costanzo, M. and Zurzolo, C. 2013. The cell biology of prion‐like spread of protein aggregates: Mechanisms and implication in neurodegeneration. Biochem. J. 452:1‐17.
  Costanzo, M., Abounit, S., Marzo, L., Danckaert, A., Chamoun, Z., Roux, P., and Zurzolo, C. 2013. Transfer of polyglutamine aggregates in neuronal cells occurs in tunneling nanotubes. J. Cell Sci. 126:3678‐3685.
  Crowell, E.F., Gaffuri, A.‐L., Gayraud‐Morel, B., Tajbakhsh, S., and Echard, A. 2014. Engulfment of the midbody remnant after cytokinesis in mammalian cells. J. Cell Sci. 127:3840‐3851.
  Dambournet, D., Machicoane, M., Chesneau, L., Sachse, M., Rocancourt, M., El Marjou, A., Formstecher, E., Salomon, R., Goud, B., and Echard, A. 2011. Rab35 GTPase and OCRL phosphatase remodel lipids and F‐actin for successful cytokinesis. Nat. Cell Biol. 13:981‐988.
  Delage, E. and Zurzolo, C. 2013. Exploring the role of lipids in intercellular conduits: Breakthroughs in the pipeline. Front Plant Sci. 4:504.
  Elia, N., Ott, C., and Lippincott‐Schwartz, J. 2013. Incisive imaging and computation for cellular mysteries: Lessons from abscission. Cell 155:1220‐1231.
  Gousset, K. and Zurzolo, C. 2009. Tunnelling nanotubes: A highway for prion spreading? Prion 3:94‐98.
  Gousset, K., Marzo, L., Commere, P.‐H., and Zurzolo, C. 2013. Myo10 is a key regulator of TNT formation in neuronal cells. J. Cell Sci. 126:4424‐4435.
  Gousset, K., Schiff, E., Langevin, C., Marijanovic, Z., Caputo, A., Browman, D.T., Chenouard, N., de Chaumont, F., Martino, A., Enninga, J., Olivo‐Marin, J.C., Männel, D., and Zurzolo, C. 2009. Prions hijack tunnelling nanotubes for intercellular spread. Nat. Cell Biol. 11:328‐336.
  Gurke, S., Barroso, J.F.V., Hodneland, E., Bukoreshtliev, N.V., Schlicker, O., and Gerdes, H.‐H. 2008. Tunneling nanotube (TNT)‐like structures facilitate a constitutive, actomyosin‐dependent exchange of endocytic organelles between normal rat kidney cells. Exp. Cell Res. 314:3669‐3683.
  Hase, K., Kimura, S., Takatsu, H., Ohmae, M., Kawano, S., Kitamura, H., Ito, M., Watarai, H., Hazelett, C.C., Yeaman, C., and Ohno, H. 2009. M‐Sec promotes membrane nanotube formation by interacting with Ral and the exocyst complex. Nat. Cell Biol. 11:1427‐1432.
  He, K., Luo, W., Zhang, Y., Liu, F., Liu, D., Xu, L., Qin, L., Xiong, C., Lu, Z., Fang, X., and Zhang, Y. 2010. Intercellular transportation of quantum dots mediated by membrane nanotubes. ACS Nano 4:3015‐3022.
  He, K., Shi, X., Zhang, X., Dang, S., Ma, X., Liu, F., Xu, M., Lv, Z., Han, D., Fang, X., and Zhang, Y. 2011. Long‐distance intercellular connectivity between cardiomyocytes and cardiofibroblasts mediated by membrane nanotubes. Cardiovasc. Res. 92:39‐47.
  Kadiu, I. and Gendelman, H.E. 2011a. Human immunodeficiency virus type 1 endocytic trafficking through macrophage bridging conduits facilitates spread of infection. J. Neuroimmune Pharmacol. 6:658‐675.
  Kadiu, I. and Gendelman, H.E. 2011b. Macrophage bridging conduit trafficking of HIV‐1 through the endoplasmic reticulum and Golgi network. J. Proteome Res. 10:3225‐3238.
  Kimura, S., Hase, K., and Ohno, H. 2012. Tunneling nanotubes: Emerging view of their molecular components and formation mechanisms. Exp. Cell Res. 318:1699‐1706.
  Lou, E., Fujisawa, S., Morozov, A., Barlas, A., Romin, Y., Dogan, Y., Gholami, S., Moreira, A.L., Manova‐Todorova, K., and Moore, M.A.S. 2012a. Tunneling nanotubes provide a unique conduit for intercellular transfer of cellular contents in human malignant pleural mesothelioma. PloS One 7:e33093.
  Lou, E., Fujisawa, S., Barlas, A., Romin, Y., Manova‐Todorova, K., Moore, M.A.S., and Subramanian, S. 2012b. Tunneling Nanotubes: A new paradigm for studying intercellular communication and therapeutics in cancer. Commun. Integr. Biol. 5:399‐403.
  Marzo, L., Gousset, K., and Zurzolo, C. 2012. Multifaceted roles of tunneling nanotubes in intercellular communication. Front. Physiol. 3:72.
  Onfelt, B., Nedvetzki, S., Benninger, R.K.P., Purbhoo, M.A., Sowinski, S., Hume, A.N., Seabra, M.C., Neil, M.A.A., French, P.M.W., and Davis, D.M. 2006. Structurally distinct membrane nanotubes between human macrophages support long‐distance vesicular traffic or surfing of bacteria. J. Immunol. 177:8476‐8483.
  Pasquier, J., Guerrouahen, B.S., Al Thawadi, H., Ghiabi, P., Maleki, M., Abu‐Kaoud, N., Jacob, A., Mirshahi, M., Galas, L., Rafii, S., Le Foll, F., and Rafii, A. 2013. Preferential transfer of mitochondria from endothelial to cancer cells through tunneling nanotubes modulates chemoresistance. J. Transl. Med. 11:94.
  Qi, Y., Wang, J.K., McMillian, M., and Chikaraishi, D.M. 1997. Characterization of a CNS cell line, CAD, in which morphological differentiation is initiated by serum deprivation. J. Neurosci. 17:1217‐1225.
  Rustom, A., Saffrich, R., Markovic, I., Walther, P., and Gerdes, H.‐H. 2004. Nanotubular highways for intercellular organelle transport. Science 303:1007‐1010.
  Segura‐Aguilar, J. 2011. Catecholaminergic cell lines for the study of dopamine metabolism and neurotoxicity. In Cell Culture Techniques. (M. Aschner, C. Suñol, and A. Bal‐Price, eds.) pp. 383‐402. Humana Press, Totowa, N.J. Available at: http://link.springer.com/10.1007/978‐1‐61779‐077‐5_19.
  Seyed‐Razavi, Y., Hickey, M.J., Kuffová, L., McMenamin, P.G., and Chinnery, H.R. 2013. Membrane nanotubes in myeloid cells in the adult mouse cornea represent a novel mode of immune cell interaction. Immunol. Cell Biol. 91:89‐95.
  Smith, I.F., Shuai, J., and Parker, I. 2011. Active generation and propagation of Ca2+ signals within tunneling membrane nanotubes. Biophys. J. 100:L37‐L39.
  Sowinski, S., Alakoskela, J.‐M., Jolly, C., and Davis, D.M. 2011. Optimized methods for imaging membrane nanotubes between T cells and trafficking of HIV‐1. Methods 53:27‐33.
  Sowinski, S., Jolly, C., Berninghausen, O., Purbhoo, M.A., Chauveau, A., Köhler, K., Oddos, S., Eissmann, P., Brodsky, F.M., Hopkins, C., Onfelt, B., Sattentau, Q., and Davis, D.M. 2008. Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV‐1 transmission. Nat. Cell Biol. 10:211‐219.
  Veranič, P., Lokar, M., Schütz, G.J., Weghuber, J., Wieser, S., Hägerstrand, H., Kralj‐Iglič, V., and Iglič, A. 2008. Different types of cell‐to‐cell connections mediated by nanotubular structures. Biophys. J. 95:4416‐4425.
  Wang, X., Bukoreshtliev, N.V., and Gerdes, H.‐H. 2012. Developing neurons form transient nanotubes facilitating electrical coupling and calcium signaling with distant astrocytes. PloS One 7:e47429.
  Wang, Y., Cui, J., Sun, X., and Zhang, Y. 2011. Tunneling‐nanotube development in astrocytes depends on p53 activation. Cell Death Differ. 18:732‐742.
  Watkins, S.C. and Salter, R.D. 2005. Functional connectivity between immune cells mediated by tunneling nanotubules. Immunity 23:309‐318.
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