Disruption of SLP-76 interaction with Gads inhibits dynamic clustering of SLP-76 and FcepsilonRI signaling in mast cells - PubMed (original) (raw)
Disruption of SLP-76 interaction with Gads inhibits dynamic clustering of SLP-76 and FcepsilonRI signaling in mast cells
Michael A Silverman et al. Mol Cell Biol. 2006 Mar.
Abstract
We developed a confocal real-time imaging approach that allows direct observation of the subcellular localization pattern of proteins involved in proximal FcepsilonRI signaling in RBL cells and primary bone marrow-derived mast cells. The adaptor protein Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is critical for FcepsilonRI-induced calcium flux, degranulation, and cytokine secretion. In this study, we imaged SLP-76 and found it in the cytosol of unstimulated cells. Upon FcepsilonRI cross-linking, SLP-76 translocates to the cell membrane, forming clusters that colocalize with the FcepsilonRI, the tyrosine kinase Syk, the adaptor LAT, and phosphotyrosine. The disruption of the SLP-76 interaction with its constitutive binding partner, Gads, through the mutation of SLP-76 or the expression of the Gads-binding region of SLP-76, inhibits the translocation and clustering of SLP-76, suggesting that the interaction of SLP-76 with Gads is critical for appropriate subcellular localization of SLP-76. We further demonstrated that the expression of the Gads-binding region of SLP-76 in bone marrow-derived mast cells inhibits FcepsilonRI-induced calcium flux, degranulation, and cytokine secretion. These studies revealed, for the first time, that SLP-76 forms signaling clusters following FcepsilonRI stimulation and demonstrated that the Gads-binding region of SLP-76 regulates clustering of SLP-76 and FcepsilonRI-induced mast cell responses.
Figures
FIG. 1.
FcɛRI cross-linking induces dynamic SLP-76 clustering at the membrane. (A and B) Anti-DNP IgE-sensitized RBL cells expressing GFP-SLP-76 were dropped onto a coverslip coated with either PLL or DNP-HSA. Upon contact with the coverslip, multiple _z_-stack images of live cells were acquired using the UltraVIEW spinning disk confocal system. Images shown are from the plane of interaction between the cell and the coverslip.
FIG. 2.
SLP-76 colocalizes with FcɛRI, Syk, LAT, and phosphotyrosine. (A) RBL cells expressing GFP-SLP-76 (green) were sensitized with Alexa 568-conjugated anti-DNP IgE (red) and then dropped onto a coverslip coated with either PLL or DNP-HSA. Live cells were imaged from 0 to 30 min using the UltraVIEW spinning disk confocal system. Multiple vertical _z_-stack images were acquired. Images shown are from the plane of interaction between the cell and the coverslip. (B and C) RBL cells were cotransfected with mRFP-SLP-76 and Syk-GFP (B) or mRFP-SLP-76 and LAT-GFP (C), sensitized with anti-DNP IgE, and then dropped onto coverslips with either PLL or PLL with DNP-HSA. Cells were then fixed with 4% paraformaldehyde, and multiple confocal _z_-stack images were acquired in the GFP and RFP channels that were merged using IP Labs software. Images shown are from the plane of interaction between the cell and the coverslip. Inset images are from the same cell ∼1 μm away from the plane of interface between the cell and the coverslip, allowing visualization of the cell membrane and cytosol. (D) Anti-DNP IgE-sensitized RBL cells expressing GFP-SLP-76 were dropped onto a coverslip coated with either PLL or DNP-HSA. Cells were then fixed, permeabilized, and stained with rhodamine-conjugated PY20 antiphosphotyrosine (red). Multiple confocal _z_-stack images were obtained in the GFP and rhodamine channels that were merged using IP Labs software. Images shown are from the plane of interaction between the cell and the coverslip. pY, phosphotyrosine.
FIG. 3.
Anti-DNP-IgE-sensitized GFP-SLP-76 expressing BMMCs were dropped onto a coverslip coated with either PLL or DNP-HSA. Cells were then fixed, permeabilized, and stained with rhodamine-conjugated PY20 antiphosphotyrosine (red). Multiple confocal _z_-stack images were obtained from the GFP and rhodamine channels and were merged with IP labs software. Images shown are from the plane of interaction between the cell and the coverslip. pY, phosphotyrosine.
FIG. 4.
FcɛRI-induced calcium flux and SLP-76 clustering requires an interaction between Gads and SLP-76. (A) Anti-DNP-IgE-sensitized SLP-76-deficient BMMCs reconstituted with GFP-SLP-76, GFP-SLP-76 G2, or GFP alone were loaded with the calcium-sensitive dye Indo-1. Calcium flux was assessed by flow cytometry with DNP-HSA added at 30 s and ionomycin added at 225 s. (B) Anti-DNP IgE-sensitized RBL cells expressing GFP-SLP-76 or GFP-SLP-76 G2 were dropped onto a coverslip coated with either PLL or DNP-HSA. Cells were then fixed following a 10-min incubation. Multiple confocal _z_-stack images were obtained in the GFP channel. Images shown are from the plane of interaction between the cell and the coverslip.
FIG. 5.
GBF inhibits SLP-76 clustering at the membrane. (A) Schematic of the GBF of SLP-76. (B) RBL cells stably expressing GFP-SLP-76 were transiently transfected with either DsRed2 (vector) or GBF-DsRed2 (GBF) and then sorted by FACS for coexpression of GFP and DsRed2. These cells were rested overnight, incubated for 1 h with IgE specific for DNP-HSA, and then dropped onto a coverslip coated with either PLL or DNP-HSA. Live cell images for GFP and DsRed2 were recorded from 0 to 30 min by confocal microscopy. Two representative images of cells expressing DsRed2 or GBF-DsRed2 are shown. (C) SLP-76 clustering was quantitated by a blinded observer in cells coexpressing GFP-SLP-76 and GBF-DsRed2 (GBF) or GFP-SLP-76 and DsRed2 (vector). More than 75 cells were scored for both groups. Percentages of cells with GFP-SLP-76 clustering were compared between GBF-DsRed2 and the DsRed2 vector by Student's t test (P < 0.01).
FIG. 6.
Generation of GBF-DsRed2-expressing RBL cells. (A) RBL cells were transfected with DsRed2 (vector) or GBF-DsRed2 (GBF) plasmids, selected by G418, and sorted by FACS for equal levels of DsRed2 expression. Expression of DsRed2 protein was assessed by Western blotting (WB) and FACS. (B) Histogram shows FcɛRI levels. Anti-DNP IgE-sensitized RBL cells were stained with anti-IgE-biotin and streptavidin-APC.
FIG. 7.
GBF expression blocks FcɛRI signaling in RBL cells. (A) Anti-DNP IgE-sensitized RBL cells sorted for equal expression of GBF-DsRed2 or the DsRed2 vector were loaded with the calcium-sensitive dye Indo-1. Calcium flux was assessed by flow cytometry with DNP-HSA added at 30 s. (B) Anti-DNP IgE-sensitized RBL cells expressing either GBF-DsRed2 or the DsRed2 vector were stimulated with DNP-HSA for 1 h, and degranulation was assessed by measuring the activity of hexosaminidase released from the cells into the supernatant divided by the total hexosaminidase activity in the cells. Error bars represent the standard deviations. (C) Schematic of GBF peptide consisting of 17 amino acids of the antennapedia protein fused to the GBF. (D) Anti-DNP IgE-sensitized RBL cells were incubated with GBF peptide or dimethyl sulfoxide (DMSO) for 150 min and then loaded with the calcium-sensitive dye Indo-1. Calcium flux was assessed by flow cytometry with DNP-HSA added at 30 s, and ionomycin was added at 225 s.
FIG. 8.
Generation of BMMCs expressing GBF-DsRed2. (A) Bone marrow was infected with an MSCV-based retroviral plasmid encoding DsRed2 vector alone or fused to the GBF, differentiated into bone marrow-derived mast cells, and then FACS sorted for expression of DsRed2. Expression of DsRed2 protein was assessed by Western blotting (WB) and FACS. (B) Following sorting, all cells were assessed for FcɛRI levels by flow cytometry.
FIG. 9.
GBF expression blocks FcɛRI signaling in BMMCs. (A) Anti-DNP IgE-sensitized unsorted BMMCs expressing GBF-DsRed2 or DsRed2 vector alone were loaded with the calcium-sensitive dye Indo-1 and assayed for calcium flux by flow cytometry. Cells were gated for high, low, and negative expression levels of DsRed2 as shown in the histogram, and calcium fluxes were analyzed and compared for each population as shown in the overlay. (B) Anti-DNP IgE-sensitized BMMCs sorted for similar expression levels of GBF-DsRed2 or DsRed2 vector alone were stimulated with DNP-HSA for 1 h. Degranulation was assessed by measuring the activity of hexosaminidase released from the cells into the supernatant divided by the hexosaminidase activity released from PMA-ionomycin-stimulated cells. (C) Anti-DNP IgE-sensitized BMMCs sorted for similar expression levels of GBF-DsRed2 or DsRed2 alone were stimulated with DNP-HSA for 24 h, and IL-6 secretion was measured from culture supernatant by ELISA. Error bars represent the standard deviations.
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