Abstract
SummaryNatural killer (NK) cells are innate immune effectors that lyse virally infected and tumorigenic cells through the formation of an immunological synapse. Actin remodeling at the lytic immunological synapse is a critical requirement for multiple facets of cytotoxic function. Activating receptor and integrin signaling leads to the regulated turnover and remodeling of actin, which is required for adhesion, sustained receptor signaling, and ultimately exocytosis. NK cells undergo lytic granule exocytosis in hypodense regions of a pervasive actin network. Although these requirements have been well demonstrated, neither the dynamic regulation of synaptic actin nor its specific function, however, has been determined at a nanoscale level. Here, live-cell super-resolution microscopy demonstrates nanoscale filamentous actin dynamism in NK cell lytic granule secretion. Following cell spreading, the overall content of the branched actin network at an immune synapse is stable over time and contains branched actin fibers and discrete actin foci. Similar actin architecture is generated in cytolytic T cells, although the timescale differs from that of NK cells. Individual filament displacement leads to stochastic clearance formation and disappearance, which are independent of lytic granule positioning. Actin dynamism is dependent upon branched network formation mediated by Arp2/3 and contractility generated by myosin IIA. Importantly, the use of small-molecule inhibitors demonstrates that actin dynamism is ultimately needed for granule secretion. Thus, we describe a requirement for nanoscale actin fiber rearrangement in generating the complex actin architecture that enables lytic granule secretion.
Highlights
Natural killer (NK) cells access cytotoxicity through a series of discrete steps [1] that begin with actin accumulation, continue to polarization of the microtubule organizing center (MTOC) and specialized lysosome-related organelles toward the immune synapse (IS), and culminate with degranulation onto diseased cells [2,3,4,5]
A pervasive synaptic actin network containing lytic granule permissive clearances has been previously defined in NK cells after their activation, but this work was limited to the observation of fixed samples using several super-resolution microscopy techniques [14,15,16]
Actin Organization at the NK Cell Synapse Begins with a Dual Architecture To interrogate the organization of actin during the formation of the lytic synapse in living NK cells, we imaged NK92 cells expressing the filamentous actin (F-actin) reporter (LifeAct-mEmerald) activated by immobilized anti-CD18 and/or anti-NKp30 antibodies [16]
Summary
Natural killer (NK) cells access cytotoxicity through a series of discrete steps [1] that begin with actin accumulation, continue to polarization of the microtubule organizing center (MTOC) and specialized lysosome-related organelles (lytic granules) toward the immune synapse (IS), and culminate with degranulation onto diseased cells [2,3,4,5]. Commitment to target cell lysis follows dominant activation signals in the absence of sufficient inhibitory signaling, after which filamentous actin (F-actin) accumulates at the synaptic interface Both major cytotoxic lymphocytes, NK and T cells, require actin polymerization and remodeling for lytic synapse formation and function, and human diseases arise from aberrations in this process [6,7,8,9,10,11,12,13]. Regions of actin at the NK and T cell ISs are structured differently [14,15,16, 29,30,31], indirect evidence in both cell types implies that the malleability of synaptic actin contributes to secretory function It is unclear, whether this is because of large-scale eradication of filamentous actin from the synaptic interface occurring during the lamellipodium stage, or a more complex mechanism taking place within the late and denser actin mesh
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