Behavior and Properties of Mature Lytic Granules at the Immunological Synapse of Human Cytotoxic T Lymphocytes.

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  • Additional Information
    • Source:
      Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: eCollection Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE
    • Publication Information:
      Original Publication: San Francisco, CA : Public Library of Science
    • Subject Terms:
      Cell Membrane/*metabolism ; Cytoplasmic Granules/*metabolism ; Immunological Synapses/*metabolism ; Membrane Fusion/*immunology ; T-Lymphocytes, Cytotoxic/*metabolism; Calcium/immunology ; Calcium/metabolism ; Cell Membrane/immunology ; Cell Membrane/ultrastructure ; Cytoplasmic Granules/immunology ; Cytoplasmic Granules/ultrastructure ; Cytotoxicity, Immunologic ; Electric Capacitance ; Electroporation ; Exocytosis ; Gene Expression ; Granzymes/genetics ; Granzymes/immunology ; Granzymes/metabolism ; Humans ; Immunological Synapses/immunology ; Immunological Synapses/ultrastructure ; Kinetics ; Luminescent Proteins/genetics ; Luminescent Proteins/immunology ; Luminescent Proteins/metabolism ; Microscopy, Fluorescence ; Primary Cell Culture ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/immunology ; Recombinant Fusion Proteins/metabolism ; T-Lymphocytes, Cytotoxic/immunology ; T-Lymphocytes, Cytotoxic/ultrastructure ; Time Factors ; Red Fluorescent Protein
    • Abstract:
      Killing of virally infected cells or tumor cells by cytotoxic T lymphocytes requires targeting of lytic granules to the junction between the CTL and its target. We used whole-cell patch clamp to measure the cell capacitance at fixed intracellular [Ca2+] to study fusion of lytic granules in human CTLs. Expression of a fluorescently labeled human granzyme B construct allowed identification of lytic granule fusion using total internal reflection fluorescence microscopy. In this way capacitance steps due to lytic granule fusion were identified. Our goal was to determine the size of fusing lytic granules and to describe their behavior at the plasma membrane. On average, 5.02 ± 3.09 (mean ± s.d.) lytic granules were released per CTL. The amplitude of lytic granule fusion events was ~ 3.3 fF consistent with a diameter of about 325 nm. Fusion latency was biphasic with time constants of 15.9 and 106 seconds. The dwell time of fusing lytic granules was exponentially distributed with a mean dwell time of 28.5 seconds. Fusion ended in spite of the continued presence of granules at the immune synapse. The mobility of fusing granules at the membrane was indistinguishable from that of lytic granules which failed to fuse. While dwelling at the plasma membrane lytic granules exhibit mobility consistent with docking interspersed with short periods of greater mobility. The failure of lytic granules to fuse when visible in TIRF at the membrane may indicate that a membrane-confined reaction is rate limiting.
    • References:
      Traffic. 2013 Jul;14(7):798-809. (PMID: 23590328)
      Immunity. 2011 Aug 26;35(2):161-8. (PMID: 21867926)
      Nat Rev Mol Cell Biol. 2006 Sep;7(9):631-43. (PMID: 16912714)
      J Exp Med. 2013 Oct 21;210(11):2415-33. (PMID: 24101378)
      Blood. 2004 Nov 1;104(9):2840-8. (PMID: 15238416)
      PLoS Biol. 2011 Sep;9(9):e1001152. (PMID: 21931537)
      Cell. 2009 Sep 4;138(5):935-46. (PMID: 19716167)
      Eur J Immunol. 2014 Feb;44(2):573-84. (PMID: 24227526)
      EMBO J. 2006 Aug 23;25(16):3725-37. (PMID: 16902411)
      Nat Genet. 2000 Jun;25(2):173-6. (PMID: 10835631)
      J Cell Biol. 1993 Feb;120(4):885-96. (PMID: 8432729)
      J Neurosci. 2007 Feb 7;27(6):1386-95. (PMID: 17287513)
      Eur Biophys J. 2000;29(2):67-89. (PMID: 10877017)
      Proc Natl Acad Sci U S A. 2003 Jul 22;100(15):8975-80. (PMID: 12815098)
      J Immunol. 2011 Jun 15;186(12):6894-904. (PMID: 21562157)
      Nat Methods. 2005 Dec;2(12):905-9. (PMID: 16299475)
      Cell Immunol. 2011;271(2):267-79. (PMID: 21843881)
      Immunol Rev. 2009 Sep;231(1):160-73. (PMID: 19754896)
      Biochem J. 2000 Jul 1;349(Pt 1):247-53. (PMID: 10861235)
      PLoS One. 2011;6(11):e27057. (PMID: 22073254)
      Proc Natl Acad Sci U S A. 2013 Apr 9;110(15):6073-8. (PMID: 23536289)
      FEBS Lett. 2010 Dec 15;584(24):4823-31. (PMID: 20965175)
      Annu Rev Immunol. 2010;28:491-533. (PMID: 20307213)
      Curr Opin Cell Biol. 2013 Aug;25(4):495-505. (PMID: 23726022)
      J Physiol. 1995 Feb 15;483 ( Pt 1):201-9. (PMID: 7776232)
      Curr Opin Neurobiol. 1996 Jun;6(3):350-7. (PMID: 8794084)
      Traffic. 2011 Jul;12(7):890-901. (PMID: 21438968)
      J Cell Biol. 2015 Jul 6;210(1):135-51. (PMID: 26124288)
      Cell. 1988 Feb 12;52(3):329-41. (PMID: 2964276)
      Cell Tissue Res. 2006 Nov;326(2):393-407. (PMID: 16819626)
      Nat Rev Immunol. 2010 Aug;10(8):568-79. (PMID: 20634814)
      Immunol Rev. 2002 Nov;189:152-60. (PMID: 12445272)
      Traffic. 2000 May;1(5):435-44. (PMID: 11208129)
      Nat Commun. 2013;4:1439. (PMID: 23385584)
      Nat Rev Mol Cell Biol. 2002 Feb;3(2):122-31. (PMID: 11836514)
      Histochem J. 1991 Oct;23(10):474-82. (PMID: 1743996)
      Eur J Immunol. 1989 Aug;19(8):1469-75. (PMID: 2789142)
      PLoS One. 2012;7(5):e36416. (PMID: 22590540)
      Proc Natl Acad Sci U S A. 1982 Nov;79(21):6712-6. (PMID: 6959149)
      Proc Natl Acad Sci U S A. 1997 Jun 24;94(13):6735-40. (PMID: 9192634)
      Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56. (PMID: 9531970)
      J Exp Med. 1991 May 1;173(5):1099-109. (PMID: 2022921)
      Eur Biophys J. 1998;27(2):83-98. (PMID: 9530824)
      Sci Signal. 2010;3(121):pe16. (PMID: 20460646)
      Immunity. 2001 Nov;15(5):751-61. (PMID: 11728337)
      Front Immunol. 2012 Nov 09;3:335. (PMID: 23162553)
      Immunol Rev. 2010 May;235(1):24-34. (PMID: 20536553)
      J Cell Biol. 1990 Dec;111(6 Pt 1):2327-40. (PMID: 2277062)
      Rev Physiol Biochem Pharmacol. 1978;82:96-190. (PMID: 356157)
      J Physiol. 2005 Sep 15;567(Pt 3):891-903. (PMID: 16020463)
      PLoS One. 2006;1:e126. (PMID: 17205130)
      Cell Calcium. 2012 Sep-Oct;52(3-4):303-12. (PMID: 22560267)
      Eur J Immunol. 1990 Oct;20(10):2161-70. (PMID: 2173661)
      Nat Immunol. 2010 Jun;11(6):495-502. (PMID: 20473299)
      Cell. 2003 Nov 14;115(4):461-73. (PMID: 14622600)
      Eur J Immunol. 2007 Oct;37(10):2723-33. (PMID: 17899547)
    • Accession Number:
      0 (Luminescent Proteins)
      0 (Recombinant Fusion Proteins)
      EC 3.4.21.- (Granzymes)
      SY7Q814VUP (Calcium)
    • Publication Date:
      Date Created: 20150822 Date Completed: 20160513 Latest Revision: 20231213
    • Publication Date:
      20231215
    • Accession Number:
      PMC4546685
    • Accession Number:
      10.1371/journal.pone.0135994
    • Accession Number:
      26296096