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Inhibitory signaling blocks activating receptor clustering and induces cytoskeletal retraction in natural killer cells.

Abeyweera TP, Merino E, Huse M - J. Cell Biol. (2011)

Bottom Line: Photostimulation of KIR2DL2 induces the rapid formation of inhibitory receptor microclusters in the plasma membrane and the simultaneous suppression of microclusters containing activating receptors.This is followed by the collapse of the peripheral actin cytoskeleton and retraction of the NK cell from the source of inhibitory stimulation.These results suggest a cell biological basis for ITIM receptor signaling and establish an experimental framework for analyzing it.

View Article: PubMed Central - HTML - PubMed

Affiliation: Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

ABSTRACT
Natural killer (NK) lymphocytes use a variety of activating receptors to recognize and kill infected or tumorigenic cells during an innate immune response. To prevent targeting healthy tissue, NK cells also express numerous inhibitory receptors that signal through immunotyrosine-based inhibitory motifs (ITIMs). Precisely how signals from competing activating and inhibitory receptors are integrated and resolved is not understood. To investigate how ITIM receptor signaling impinges on activating pathways, we developed a photochemical approach for stimulating the inhibitory receptor KIR2DL2 during ongoing NK cell-activating responses in high-resolution imaging experiments. Photostimulation of KIR2DL2 induces the rapid formation of inhibitory receptor microclusters in the plasma membrane and the simultaneous suppression of microclusters containing activating receptors. This is followed by the collapse of the peripheral actin cytoskeleton and retraction of the NK cell from the source of inhibitory stimulation. These results suggest a cell biological basis for ITIM receptor signaling and establish an experimental framework for analyzing it.

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KIR2DL2 signaling inhibits cell spreading and the initiation of Ca2+ flux. (A and B) NKL cells expressing KIR2DL2 were stained with PKH26 and imaged using TIRF microscopy on lipid bilayers containing the indicated proteins. (A) Representative time-lapse montages (∼90-s intervals) under both activating (top) and inhibitory (bottom) conditions. (B) Bar graph representing the distribution of cell behavior on surfaces containing the indicated ligands. Only cells visible in the imaging field for ≥5 min were analyzed. Cells were described as spread if they formed a stationary footprint at least 10 µm in diameter (yellow arrow in A), collapsed if they engaged in minimal dynamic interactions with the membrane (magenta arrow in A), or motile if they exhibited directional migration (cyan arrow in A). Occasionally, cells would display two phenotypes during the imaging period. (C) NKL cells expressing KIR2DL2 (KIR-WT) or KIR2DL2(mut) (KIR-Mut) were loaded with Fura-2AM and imaged on lipid bilayers containing ULBP3, ICAM, and the indicated HLA-Cw3 proteins. (right) Representative time-lapse montages (4-min intervals) showing a pseudocolored Fura-2AM ratio (warmer colors indicate higher intracellular Ca2+ concentrations). (left) Background-corrected mean Fura-2AM ratios for all imaging fields are plotted versus time for each condition. Error bars show SEM. All data are representative of at least two independent experiments. Bars, 10 µm.
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fig2: KIR2DL2 signaling inhibits cell spreading and the initiation of Ca2+ flux. (A and B) NKL cells expressing KIR2DL2 were stained with PKH26 and imaged using TIRF microscopy on lipid bilayers containing the indicated proteins. (A) Representative time-lapse montages (∼90-s intervals) under both activating (top) and inhibitory (bottom) conditions. (B) Bar graph representing the distribution of cell behavior on surfaces containing the indicated ligands. Only cells visible in the imaging field for ≥5 min were analyzed. Cells were described as spread if they formed a stationary footprint at least 10 µm in diameter (yellow arrow in A), collapsed if they engaged in minimal dynamic interactions with the membrane (magenta arrow in A), or motile if they exhibited directional migration (cyan arrow in A). Occasionally, cells would display two phenotypes during the imaging period. (C) NKL cells expressing KIR2DL2 (KIR-WT) or KIR2DL2(mut) (KIR-Mut) were loaded with Fura-2AM and imaged on lipid bilayers containing ULBP3, ICAM, and the indicated HLA-Cw3 proteins. (right) Representative time-lapse montages (4-min intervals) showing a pseudocolored Fura-2AM ratio (warmer colors indicate higher intracellular Ca2+ concentrations). (left) Background-corrected mean Fura-2AM ratios for all imaging fields are plotted versus time for each condition. Error bars show SEM. All data are representative of at least two independent experiments. Bars, 10 µm.

Mentions: To examine the cell biological basis for this inhibition, we stained KIR2DL2-expressing NKL cells with the vital membrane dye PKH26 and imaged them using total internal reflection fluorescence (TIRF) microscopy as they interacted with supported lipid bilayers containing activating and inhibitory ligands. Consistent with previous work (Culley et al., 2009; Liu et al., 2009), NKL cells formed stable, synapselike contacts on bilayers containing ULBP3 (an NKG2D ligand), the intercellular adhesion molecule (ICAM), and the nonfunctional HLA-Cw3(Tyr) (Fig. 2, A and B; and Video 1). Substitution of HLA-Cw3(Tyr) with the functional HLA-Cw3(Ser) dramatically altered this spreading behavior; most cells did not spread at all, instead forming small, dynamic contacts (Fig. 2, A and B; and Video 2). A subset of cells exhibited a highly mobile crawling phenotype (Fig. 2 A, cyan arrow), which was consistent with the notion that inhibitory receptors block the ability of activating signals to arrest cell motility (Culley et al., 2009).


Inhibitory signaling blocks activating receptor clustering and induces cytoskeletal retraction in natural killer cells.

Abeyweera TP, Merino E, Huse M - J. Cell Biol. (2011)

KIR2DL2 signaling inhibits cell spreading and the initiation of Ca2+ flux. (A and B) NKL cells expressing KIR2DL2 were stained with PKH26 and imaged using TIRF microscopy on lipid bilayers containing the indicated proteins. (A) Representative time-lapse montages (∼90-s intervals) under both activating (top) and inhibitory (bottom) conditions. (B) Bar graph representing the distribution of cell behavior on surfaces containing the indicated ligands. Only cells visible in the imaging field for ≥5 min were analyzed. Cells were described as spread if they formed a stationary footprint at least 10 µm in diameter (yellow arrow in A), collapsed if they engaged in minimal dynamic interactions with the membrane (magenta arrow in A), or motile if they exhibited directional migration (cyan arrow in A). Occasionally, cells would display two phenotypes during the imaging period. (C) NKL cells expressing KIR2DL2 (KIR-WT) or KIR2DL2(mut) (KIR-Mut) were loaded with Fura-2AM and imaged on lipid bilayers containing ULBP3, ICAM, and the indicated HLA-Cw3 proteins. (right) Representative time-lapse montages (4-min intervals) showing a pseudocolored Fura-2AM ratio (warmer colors indicate higher intracellular Ca2+ concentrations). (left) Background-corrected mean Fura-2AM ratios for all imaging fields are plotted versus time for each condition. Error bars show SEM. All data are representative of at least two independent experiments. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3044118&req=5

fig2: KIR2DL2 signaling inhibits cell spreading and the initiation of Ca2+ flux. (A and B) NKL cells expressing KIR2DL2 were stained with PKH26 and imaged using TIRF microscopy on lipid bilayers containing the indicated proteins. (A) Representative time-lapse montages (∼90-s intervals) under both activating (top) and inhibitory (bottom) conditions. (B) Bar graph representing the distribution of cell behavior on surfaces containing the indicated ligands. Only cells visible in the imaging field for ≥5 min were analyzed. Cells were described as spread if they formed a stationary footprint at least 10 µm in diameter (yellow arrow in A), collapsed if they engaged in minimal dynamic interactions with the membrane (magenta arrow in A), or motile if they exhibited directional migration (cyan arrow in A). Occasionally, cells would display two phenotypes during the imaging period. (C) NKL cells expressing KIR2DL2 (KIR-WT) or KIR2DL2(mut) (KIR-Mut) were loaded with Fura-2AM and imaged on lipid bilayers containing ULBP3, ICAM, and the indicated HLA-Cw3 proteins. (right) Representative time-lapse montages (4-min intervals) showing a pseudocolored Fura-2AM ratio (warmer colors indicate higher intracellular Ca2+ concentrations). (left) Background-corrected mean Fura-2AM ratios for all imaging fields are plotted versus time for each condition. Error bars show SEM. All data are representative of at least two independent experiments. Bars, 10 µm.
Mentions: To examine the cell biological basis for this inhibition, we stained KIR2DL2-expressing NKL cells with the vital membrane dye PKH26 and imaged them using total internal reflection fluorescence (TIRF) microscopy as they interacted with supported lipid bilayers containing activating and inhibitory ligands. Consistent with previous work (Culley et al., 2009; Liu et al., 2009), NKL cells formed stable, synapselike contacts on bilayers containing ULBP3 (an NKG2D ligand), the intercellular adhesion molecule (ICAM), and the nonfunctional HLA-Cw3(Tyr) (Fig. 2, A and B; and Video 1). Substitution of HLA-Cw3(Tyr) with the functional HLA-Cw3(Ser) dramatically altered this spreading behavior; most cells did not spread at all, instead forming small, dynamic contacts (Fig. 2, A and B; and Video 2). A subset of cells exhibited a highly mobile crawling phenotype (Fig. 2 A, cyan arrow), which was consistent with the notion that inhibitory receptors block the ability of activating signals to arrest cell motility (Culley et al., 2009).

Bottom Line: Photostimulation of KIR2DL2 induces the rapid formation of inhibitory receptor microclusters in the plasma membrane and the simultaneous suppression of microclusters containing activating receptors.This is followed by the collapse of the peripheral actin cytoskeleton and retraction of the NK cell from the source of inhibitory stimulation.These results suggest a cell biological basis for ITIM receptor signaling and establish an experimental framework for analyzing it.

View Article: PubMed Central - HTML - PubMed

Affiliation: Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

ABSTRACT
Natural killer (NK) lymphocytes use a variety of activating receptors to recognize and kill infected or tumorigenic cells during an innate immune response. To prevent targeting healthy tissue, NK cells also express numerous inhibitory receptors that signal through immunotyrosine-based inhibitory motifs (ITIMs). Precisely how signals from competing activating and inhibitory receptors are integrated and resolved is not understood. To investigate how ITIM receptor signaling impinges on activating pathways, we developed a photochemical approach for stimulating the inhibitory receptor KIR2DL2 during ongoing NK cell-activating responses in high-resolution imaging experiments. Photostimulation of KIR2DL2 induces the rapid formation of inhibitory receptor microclusters in the plasma membrane and the simultaneous suppression of microclusters containing activating receptors. This is followed by the collapse of the peripheral actin cytoskeleton and retraction of the NK cell from the source of inhibitory stimulation. These results suggest a cell biological basis for ITIM receptor signaling and establish an experimental framework for analyzing it.

Show MeSH
Related in: MedlinePlus