Abstract
Natural killer (NK) cells contain specialized lysosome-related organelles termed lytic granules allowing them to mediate cytotoxicity against tumorigenic or virally infected target cells. NK cells polarize their lytic granules toward a target cell via the microtubule-organizing center (MTOC). Prior to that, however, lytic granules converge to the MTOC along microtubules utilizing minus-end-directed microtubule motors. Herein we describe how to visualize and quantify lytic granule convergence using confocal microscopy to gain quantitative insight into NK cell cytotoxicity and its regulation.
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References
Orange JS (2008) Formation and function of the lytic NK-cell immunological synapse. Nat Rev Immunol 8:713–725
Mace EM, Dongre P, Hsu HT, Sinha P, James AM, Mann SS, Forbes LR, Watkin LB, Orange JS (2014) Cell biological steps and checkpoints in accessing NK cell cytotoxicity. Immunol Cell Biol 92:245–255
Mentlik AN, Sanborn KB, Holzbaur EL, Orange JS (2010) Rapid lytic granule convergence to the MTOC in natural killer cells is dependent on dynein but not cytolytic commitment. Mol Biol Cell 21:2241–2256
James AM, Hsu HT, Dongre P, Uzel G, Mace EM, Banerjee PP, Orange JS (2013) Rapid activation receptor- or IL-2-induced lytic granule convergence in human natural killer cells requires Src, but not downstream signaling. Blood 121:2627–2637
Hsu, H.T., Orange, J.S. (2014) Distinct integrin-dependent signals define requirements for lytic granule convergence and polarization in natural killer cells. Sci Signal 7:pe24.
Ritter AT, Asano Y, Stinchcombe JC, Dieckmann NM, Chen BC, Gawden-Bone C, van Engelenburg S, Legant W, Gao L, Davidson MW, Betzig E, Lippincott-Schwartz J, Griffiths GM (2015) Actin depletion initiates events leading to granule secretion at the immunological synapse. Immunity 42:864–876
Zhang M, March ME, Lane WS, Long EO (2014) A signaling network stimulated by beta2 integrin promotes the polarization of lytic granules in cytotoxic cells. Sci Signal 7:ra96
Mace EM, Wu WW, Ho T, Mann SS, Hsu HT, Orange JS (2012) NK cell lytic granules are highly motile at the immunological synapse and require F-actin for post-degranulation persistence. J Immunol 189:4870–4880
Briercheck EL, Trotta R, Chen L, Hartlage AS, Cole JP, Cole TD, Mao C, Banerjee PP, Hsu HT, Mace EM, Ciarlariello D, Mundy-Bosse BL, Garcia-Cao I, Scoville SD, Yu L, Pilarski R, Carson WE 3rd, Leone G, Pandolfi PP, Yu J, Orange JS, Caligiuri MA (2015) PTEN is a negative regulator of NK cell cytolytic function. J Immunol 194:1832–1840
Ham H, Huynh W, Schoon RA, Vale RD, Billadeau DD (2015) HkRP3 is a microtubule-binding protein regulating lytic granule clustering and NK cell killing. J Immunol 194:3984–3996
Tuli A, Thiery J, James AM, Michelet X, Sharma M, Garg S, Sanborn KB, Orange JS, Lieberman J, Brenner MB (2013) Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell-mediated cytotoxicity. Mol Biol Cell 24:3721–3735
Bertolet G, Liu D (2016) The planar lipid bilayer system serves as a reductionist approach for studying NK cell immunological synapses and their functions. Methods Mol Biol 1441:151–165
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682
Rak GD, Mace EM, Banerjee PP, Svitkina T, Orange JS (2011) Natural killer cell lytic granule secretion occurs through a pervasive actin network at the immune synapse. PLoS Biol 9:e1001151
Acknowledgments
We would like to thank Dr. Ashley Mentlik-James for her efforts in developing this protocol in part as part of her Ph.D. thesis. This work was supported by NIH grants R01AI069746 and R01AI120989 to J.S.O.
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Hsu, HT., Carisey, A.F., Orange, J.S. (2017). Measurement of Lytic Granule Convergence After Formation of an NK Cell Immunological Synapse. In: Baldari, C., Dustin, M. (eds) The Immune Synapse. Methods in Molecular Biology, vol 1584. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6881-7_31
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DOI: https://doi.org/10.1007/978-1-4939-6881-7_31
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