Focal adhesion proteins connect IgE receptors to the cytoskeleton as revealed by micropatterned ligand arrays - PubMed (original) (raw)

Comparative Study

Focal adhesion proteins connect IgE receptors to the cytoskeleton as revealed by micropatterned ligand arrays

Alexis J Torres et al. Proc Natl Acad Sci U S A. 2008.

Abstract

Patterned surfaces that present specific ligands in spatially defined arrays are used to examine structural linkages between clustered IgE receptors (IgE-Fc epsilonRI) and the cytoskeleton in rat basophilic leukemia (RBL) mast cells. We showed with fluorescence microscopy that cytoskeletal F-actin concentrates in the same regions as cell surface IgE-Fc epsilonRI that bind to the micrometer-size patterned ligands. However, the proteins mediating these cytoskeletal connections and their functional relevance were not known. We now show that whereas the adaptor proteins ezrin and moesin do not detectably concentrate with the array of clustered IgE-Fc epsilonRI, focal adhesion proteins vinculin, paxillin, and talin, which are known to link F-actin with integrins, accumulate in these regions on the same time scale as F-actin. Moreover, colocalization of these focal adhesion proteins with clustered IgE-Fc epsilonRI is enhanced after addition of fibronectin-RGD peptides. Significantly, the most prominent rat basophilic leukemia cell integrin (alpha5) avoids the patterned regions occupied by the ligands and associates preferentially with exposed regions of the silicon substrate. Thus, spatial separation provided by the patterned surface reveals that particular focal adhesion proteins, which connect to the actin cytoskeleton, associate with ligand-cross-linked IgE-Fc epsilonRI, independently of integrins. We investigated the functional role of one of these proteins, paxillin, in IgE-Fc epsilonRI-mediated signaling by using small interfering RNA. From these results, we determine that paxillin reduces stimulated phosphorylation of the Fc epsilonRI beta subunit but enhances stimulated Ca(2+) release from intracellular stores. The results suggest that paxillin associated with clustered IgE-Fc epsilonRI has a net positive effect on Fc epsilonRI signaling.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Vinculin and talin, but not ezrin and moesin, visibly coredistribute with clustered IgE-FcεRI. Confocal florescence micrographs show cells incubated at 37°C with patterned lipid bilayers containing DNP-cap-PE and stained for specific intracellular proteins (red). Clustered IgE-FcεRI on cell surfaces are visualized by Alexa Fluor 488-labeled IgE (green). (A) F-actin accumulates consistently with the clustered FcεRI-IgE, whereas ERM family members, ezrin and moesin, do not. (B) Focal adhesion proteins vinculin and talin redistribute with clustered IgE-FcεRI in the absence and presence of fibronectin-RGD peptides (RGDSP or RGDSPT). Intracellular proteins were labeled after ≈40-min incubation of cells with substrates, followed by cell fixation and treatment with Alexa Fluor 568 phalloidin (F-actin) or specific antibodies (ezrin, moesin, vinculin, talin). (Scale bars: 20 μm.)

Fig. 2.

Fig. 2.

Paxillin coredistributes with clustered IgE-FcεRI and patterned lipid bilayers, but integrins avoid these regions. Confocal micrographs show cells expressing paxillin-EGFP or integrin α5-EGFP (green) after incubation for 40 min at 37°C with patterned lipid bilayers (red) containing DNP-cap-PE and fixation before imaging. (A) Paxillin is recruited with IgE-FcεRI in the regions of patterned ligands, and α5-integrin is excluded. (Scale bars: 20 μm.) (B) Intensity line profiles from images in A confirm that paxillin accumulation and integrin exclusion correspond exactly to where the patterned bilayers are localized.

Fig. 3.

Fig. 3.

Paxillin knockdown affects phosphorylation stimulated by IgE-FcεRI. (A) Immunoblots show that paxillin-specific siRNA causes >90% decrease in paxillin expression levels. To confirm knockdown, the same PDVF membrane was stripped and reprobed with anti-tubulin as a loading control. (B) Antigen-stimulated phosphorylation was examined in lysates from suspended whole cells (2 × 106/ml) 48 h after siRNA delivery, sensitization with anti-DNP IgE, and stimulation with DNP-BSA (50 ng/ml) for the times indicated. After SDS/PAGE, gels were blotted with anti-phosphotyrosine antibody, 4G10. Boxes indicate 35-kDa (p35) and 50-kDa (p50) bands that are consistently increased after paxillin knockdown in this representative experiment; anti-tubulin bands serve as loading control. (C) Densitometric analysis of tyrosine phosphorylation (PY) for p35 band (FcεRI β) after 3-min stimulation. (D) Densitometric plot showing stimulation time course for relative tyrosine phosphorylation of the p50 band. (C and D) Data are averaged over five different experiments in which the phosphorylated band was normalized by tubulin band (loading control). The data were further normalized to control (sham knockdown) cells (C) or to the 15-min time point in the knockdown cells (D). **, P < 0.01; ***, P < 0.001. Error bars indicate S.E.M.

Fig. 4.

Fig. 4.

Paxillin knockdown affects Ca2+ mobilization stimulated by IgE-FcεRI. Intracellular Ca2+ changes in paxillin knockdown (gray) and sham knockdown (black) cells after stimulation by DNP-BSA (50–200 ng/ml) at 37°C, in representative (A and B) and summarized (C) experiments. (A) Cells were stimulated in buffer containing 2 mM Ca2+; release from intracellular stores and influx from the extracellular medium occurs under these conditions. (B) Cells were stimulated in the absence of extracellular Ca2+ such that only release from stores occurs; after 500 s the Ca2+ concentration in the buffer is increased to 2 mM (by addition of CaCl2) such that influx occurs. (C) Averages over five experiments similar to that shown in B. The first two sets of two bars correspond to curves before and after addition of extracellular Ca2+ (2 mM), respectively; the last set corresponds to both curves additively. In each case, changes in intracellular Ca2+ concentration were integrated >500 s and normalized to the control cell response. *, P < 0.05; **, P < 0.01. Error bars indicate S.E.M.

References

    1. Lock JG, Wehrle-Haller B, Stromblad S. Cell–matrix adhesion complexes: Master control machinery of cell migration. Semin Cancer Biol. 2008;18:65–76. - PubMed
    1. Kinet J-P. The high-affinity IgE receptor (FcεRI): From physiology to pathology. Annu Rev Immunol. 1999;17:931–972. - PubMed
    1. Holowka D, et al. Lipid segregation and IgE receptor signaling: A decade of progress. Biochim Biophys Acta. 2005;1746:252–259. - PubMed
    1. Wu M, Holowka D, Craighead HG, Baird B. Visualization of plasma membrane compartmentalization with patterned lipid bilayers. Proc Natl Acad Sci USA. 2004;101:13798–13803. - PMC - PubMed
    1. Critchley DR. Focal adhesions: The cytoskeletal connection. Curr Opin Cell Biol. 2000;12:133–139. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources