Microclusters of inhibitory killer immunoglobulin–like receptor signaling at natural killer cell immunological synapses

Treanor, Bebhinn; Lanigan, Peter M. P.; Kumar, Sunil; Dunsby, Chris; Munro, Ian; Auksorius, Egidijus; Culley, Fiona J.; Purbhoo, Marco A.; Phillips, David; Neil, Mark A. A.; Burshtyn, Deborah N.; French, Paul M. W.; Davis, Daniel M.

doi:10.1083/jcb.200601108

Cited by 101 publications

(69 citation statements)

References 52 publications

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“…histogram (Figs. 3(c) and 3(e), respectively) show that the fluorescence lifetimes are fairly uniform across the cells with a peak in the histogram of the GFP lifetimes at τ f = 2.38 ns, consistent with previous measurements of intracellular GFP [35,36,67,68] and our previous FLIM measurements of CAR-GFP alone [57]. The steady-state anisotropy image ( Fig.…”

Section: Combined Frap Flim Tr-faim Microscopy Of Car-gfp In Livingsupporting

confidence: 74%

Simultaneous FRAP, FLIM and FAIM for measurements of protein mobility and interaction in living cells

Levitt

Morton

Fruhwirth

et al. 2015

Biomed. Opt. Express

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We present a novel integrated multimodal fluorescence microscopy technique for simultaneous fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging (FLIM) and fluorescence anisotropy imaging (FAIM). This approach captures a series of polarization-resolved fluorescence lifetime images during a FRAP recovery, maximizing the information available from a limited photon budget. We have applied this method to analyse the behaviour of GFP-labelled coxsackievirus and adenovirus receptor (CAR) in living human epithelial cells. Our data reveal that CAR exists in oligomeric states throughout the cell, and that these complexes occur in conjunction with high immobile fractions of the receptor at cell-cell junctions. These findings shed light on previously unknown molecular associations between CAR receptors in intact cells and demonstrate the power of combined FRAP, FLIM and FAIM microscopy as a robust method to analyse complex multi-component dynamics in living cells. 28-36 (2009

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Section: Combined Frap Flim Tr-faim Microscopy Of Car-gfp In Livingsupporting

confidence: 74%

Simultaneous FRAP, FLIM and FAIM for measurements of protein mobility and interaction in living cells

Levitt

Morton

Fruhwirth

et al. 2015

Biomed. Opt. Express

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“…1, A and B, left) and in the other immunological synapse of the second conjugate (IS #1), the Cdc42 activity was more heterogeneous. This heterogeneous activity was reminiscent of images of microclusters containing phosphorylated killer cell immunoglobulinlike receptor (KIR) and the adaptor protein Lck that were previously observed at the inhibitory immunological synapse of NK cells (31). When our analysis was performed at the level of each individual immunological synapse rather than at the level of the whole cell (Fig.…”

Section: Cdc42 Activity First Increases and Then Oscillates During Nkmentioning

confidence: 99%

A Targeted siRNA Screen Identifies Regulators of Cdc42 Activity at the Natural Killer Cell Immunological Synapse

et al. 2011

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This information is current as of 30 November 2011.The following resources related to this article are available online at http://stke.sciencemag.org. The Rho guanosine triphosphatase (GTPase) Cdc42 coordinates cytoskeletal reorganization downstream of many receptors. The Rho-related GTPase from plants 1 (ROP1) exhibits oscillatory activation behavior at the apical plasma membrane of growing pollen tubes; however, a similar oscillation in Rho GTPase activity has so far not been demonstrated in mammalian cells. We hypothesized that oscillations in Cdc42 activity might occur within NK cells as they interact with target cells. Through fluorescence lifetime imaging of a Cdc42 biosensor, we observed that in live NK cells forming immunological synapses with target cells, Cdc42 activity oscillated after exhibiting an initial increase. We used protein-protein interaction networks and structural databases to identify candidate proteins that controlled Cdc42 activity, leading to the design of a targeted short interfering RNA screen. The guanine nucleotide exchange factors RhoGEF6 and RhoGEF7 were necessary for Cdc42 activation within the NK cell immunological synapse. In addition, the kinase Akt and the p85a subunit of phosphoinositide 3-kinase (PI3K) were required for Cdc42 activation, the periodicity of the oscillation in Cdc42 activity, and the subsequent polarization of cytotoxic vesicles toward target cells. Given that PI3Ks are targets of tumor therapies, our findings suggest the need to monitor innate immune function during the course of targeted therapy against these enzymes. Article Toolshttp

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“…Specifically, we assessed a "quantity-based" inhibition model, in which the number of bound inhibitory receptors is simply subtracted from the number of bound activating receptors, and a "distance-based" inhibition model, in which every bound inhibitory receptor inhibits signals from all bound activating receptors within a certain radius around it. The latter hypothesis was suggested by the observation that inhibitory receptors can stop the response while being gradually integrated into the synapse as the cell spreads (35) and that phosphorylation of KIR occurs in microclusters (36).…”

mentioning

confidence: 99%

Simulations of the NK Cell Immune Synapse Reveal that Activation Thresholds Can Be Established by Inhibitory Receptors Acting Locally

Kaplan

Kotzer

Almeida

et al. 2011

The Journal of Immunology

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NK cell activation is regulated by a balance between activating and inhibitory signals. To address the question of how these signals are spatially integrated, we created a computer simulation of activating and inhibitory NK cell immunological synapse (NKIS) assembly, implementing either a “quantity-based” inhibition model or a “distance-based” inhibition model. The simulations mimicked the observed molecule distributions in inhibitory and activating NKIS and yielded several new insights. First, the total signal is highly influenced by activating complex dissociation rates but not by adhesion and inhibitory complex dissociation rates. Second, concerted motion of receptors in clusters significantly accelerates NKIS maturation. Third, when the potential of a cis interaction between Ly49 receptors and MHC class I on murine NK cells was added to the model, the integrated signal as a function of receptor and ligand numbers was only slightly increased, at least up to the level of 50% cis-bound Ly49 receptors reached in the model. Fourth, and perhaps most importantly, the integrated signal behavior obtained when using the distance-based inhibition signal model was closer to the experimentally observed behavior, with an inhibition radius of the order 3–10 molecules. Microscopy to visualize Vav activation in NK cells on micropatterned surfaces of activating and inhibitory strips revealed that Vav is only locally activated where activating receptors are ligated within a single NK cell contact. Taken together, these data are consistent with a model in which inhibitory receptors act locally; that is, that every bound inhibitory receptor acts on activating receptors within a certain radius around it.

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Microclusters of inhibitory killer immunoglobulin–like receptor signaling at natural killer cell immunological synapses

Cited by 101 publications

References 52 publications

Simultaneous FRAP, FLIM and FAIM for measurements of protein mobility and interaction in living cells

Simultaneous FRAP, FLIM and FAIM for measurements of protein mobility and interaction in living cells

A Targeted siRNA Screen Identifies Regulators of Cdc42 Activity at the Natural Killer Cell Immunological Synapse

Simulations of the NK Cell Immune Synapse Reveal that Activation Thresholds Can Be Established by Inhibitory Receptors Acting Locally

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