title: Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy.
abstract: Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.
authors.affiliation: Division of Cell and Molecular Biology, Imperial College London, London, United Kingdom.
authors.forename: Alice C N
authors.initials: AC
authors.lastname: Brown
authors.forename: Stephane
authors.initials: S
authors.lastname: Oddos
authors.forename: Ian M
authors.initials: IM
authors.lastname: Dobbie
authors.forename: Juha-Matti
authors.initials: JM
authors.lastname: Alakoskela
authors.forename: Richard M
authors.initials: RM
authors.lastname: Parton
authors.forename: Philipp
authors.initials: P
authors.lastname: Eissmann
authors.forename: Mark A A
authors.initials: MA
authors.lastname: Neil
authors.forename: Christopher
authors.initials: C
authors.lastname: Dunsby
authors.forename: Paul M W
authors.initials: PM
authors.lastname: French
authors.forename: Ilan
authors.initials: I
authors.lastname: Davis
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Davis
mesh_terms: D000199:Actins / Q000378:metabolism*; D015550:Cell Degranulation; D002460:Cell Line; D051380:GRB2 Adaptor Protein / Q000378:metabolism; D006801:Humans; D007089:Image Enhancement / Q000379:methods; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D054992:Immunological Synapses / Q000378:metabolism*; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D007694:Killer Cells, Natural / Q000378:metabolism*; D008213:Lymphocyte Activation; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods*; D022101:Microtubule-Organizing Center / Q000378:metabolism; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000378:metabolism; D052898:Optical Tweezers; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D061251:Primary Cell Culture; D055571:Secretory Pathway; D014162:Transfection
chemical_list: D000199:Actins; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C527085:KLRK1 protein, human; D016169:Lymphocyte Function-Associated Antigen-1; D055655:NK Cell Lectin-Like Receptor Subfamily K; D018799:Intercellular Adhesion Molecule-1