Limits...
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.

Show MeSH

Related in: MedlinePlus

KIR2DL2 photostimulation induces actin remodeling. (A–D) NKL cells expressing KIR2DL2 and Lifeact-RFP were imaged using TIRF microscopy and UV irradiated on bilayers containing ULBP3, ICAM, and either HLA-Cw3(cage) (A, C, and D) or HLA-Cw3(Tyr) (B). Photostimulation was performed using cells left untreated (A and B) or treated with NSC87877 (C) or Mn2+ (D). For each panel, a time-lapse montage (∼75-s intervals) is shown (top) along with an associated kymograph. UV irradiation is indicated by magenta text in the time lapse and by a magenta line in the kymograph. Kymographs were generated using the yellow line in the first image of each time lapse. Shown on the bottom in each panel, normalized mean fluorescence intensity of Lifeact-RFP in the center of the contact is graphed as a function of time together with cell area. The contact center is indicated by a cyan ellipse in each time lapse. Data are representative of at least two independent experiments. ΔF/F, normalized fluorescence intensity. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC3044118&req=5

fig7: KIR2DL2 photostimulation induces actin remodeling. (A–D) NKL cells expressing KIR2DL2 and Lifeact-RFP were imaged using TIRF microscopy and UV irradiated on bilayers containing ULBP3, ICAM, and either HLA-Cw3(cage) (A, C, and D) or HLA-Cw3(Tyr) (B). Photostimulation was performed using cells left untreated (A and B) or treated with NSC87877 (C) or Mn2+ (D). For each panel, a time-lapse montage (∼75-s intervals) is shown (top) along with an associated kymograph. UV irradiation is indicated by magenta text in the time lapse and by a magenta line in the kymograph. Kymographs were generated using the yellow line in the first image of each time lapse. Shown on the bottom in each panel, normalized mean fluorescence intensity of Lifeact-RFP in the center of the contact is graphed as a function of time together with cell area. The contact center is indicated by a cyan ellipse in each time lapse. Data are representative of at least two independent experiments. ΔF/F, normalized fluorescence intensity. Bars, 5 µm.

Mentions: To investigate the effects of ITIM receptor signaling on actin structure, we transduced NKL cells with the Lifeact peptide (Riedl et al., 2008), which binds specifically to filamentous actin. When cells expressing KIR2DL2-GFP and Lifeact-RFP contacted bilayers containing ULBP3, ICAM, and HLA-Cw3(cage), the Lifeact-RFP probe accumulated in a ring at the periphery of the synapse, which is characteristic of an activated lymphocyte. Subsequent UV irradiation dramatically altered this configuration (Fig. 7 A and Video 5). As the contact retracted, the actin ring “filled in” so that the intensity of the Lifeact-RFP probe became uniform over the entire interface. UV-induced dissolution of the actin ring was far less frequent on bilayers containing HLA-Cw3(Tyr) instead of HLA-Cw3(cage) (Fig. 7 B). This remodeling response was also impaired by NSC87877 (Fig. 7 C). Hence, the reorganization of actin induced by KIR2DL2 requires both ITIM signaling and SHP-1/2 activity.


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 photostimulation induces actin remodeling. (A–D) NKL cells expressing KIR2DL2 and Lifeact-RFP were imaged using TIRF microscopy and UV irradiated on bilayers containing ULBP3, ICAM, and either HLA-Cw3(cage) (A, C, and D) or HLA-Cw3(Tyr) (B). Photostimulation was performed using cells left untreated (A and B) or treated with NSC87877 (C) or Mn2+ (D). For each panel, a time-lapse montage (∼75-s intervals) is shown (top) along with an associated kymograph. UV irradiation is indicated by magenta text in the time lapse and by a magenta line in the kymograph. Kymographs were generated using the yellow line in the first image of each time lapse. Shown on the bottom in each panel, normalized mean fluorescence intensity of Lifeact-RFP in the center of the contact is graphed as a function of time together with cell area. The contact center is indicated by a cyan ellipse in each time lapse. Data are representative of at least two independent experiments. ΔF/F, normalized fluorescence intensity. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3044118&req=5

fig7: KIR2DL2 photostimulation induces actin remodeling. (A–D) NKL cells expressing KIR2DL2 and Lifeact-RFP were imaged using TIRF microscopy and UV irradiated on bilayers containing ULBP3, ICAM, and either HLA-Cw3(cage) (A, C, and D) or HLA-Cw3(Tyr) (B). Photostimulation was performed using cells left untreated (A and B) or treated with NSC87877 (C) or Mn2+ (D). For each panel, a time-lapse montage (∼75-s intervals) is shown (top) along with an associated kymograph. UV irradiation is indicated by magenta text in the time lapse and by a magenta line in the kymograph. Kymographs were generated using the yellow line in the first image of each time lapse. Shown on the bottom in each panel, normalized mean fluorescence intensity of Lifeact-RFP in the center of the contact is graphed as a function of time together with cell area. The contact center is indicated by a cyan ellipse in each time lapse. Data are representative of at least two independent experiments. ΔF/F, normalized fluorescence intensity. Bars, 5 µm.
Mentions: To investigate the effects of ITIM receptor signaling on actin structure, we transduced NKL cells with the Lifeact peptide (Riedl et al., 2008), which binds specifically to filamentous actin. When cells expressing KIR2DL2-GFP and Lifeact-RFP contacted bilayers containing ULBP3, ICAM, and HLA-Cw3(cage), the Lifeact-RFP probe accumulated in a ring at the periphery of the synapse, which is characteristic of an activated lymphocyte. Subsequent UV irradiation dramatically altered this configuration (Fig. 7 A and Video 5). As the contact retracted, the actin ring “filled in” so that the intensity of the Lifeact-RFP probe became uniform over the entire interface. UV-induced dissolution of the actin ring was far less frequent on bilayers containing HLA-Cw3(Tyr) instead of HLA-Cw3(cage) (Fig. 7 B). This remodeling response was also impaired by NSC87877 (Fig. 7 C). Hence, the reorganization of actin induced by KIR2DL2 requires both ITIM signaling and SHP-1/2 activity.

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