Journal of Structural Biology 168 (2009) 161–167

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Journal of Structural Biology

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Roles for SH2 and SH3 domains in Lyn kinase association with activated FceRIin RBL mast cells revealed by patterned surface analysis

Stephanie Hammond, Alice Wagenknecht-Wiesner, Sarah L. Veatch, David Holowka, Barbara Baird *

Department of Chemistry and Chemical Biology, Cornell University, Baker Laboratory, Ithaca, NY 14853-1301, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 17 December 2008Received in revised form 27 April 2009Accepted 28 April 2009Available online 7 May 2009

Keywords:IgE receptorsCross-correlation analysis

1047-8477/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.jsb.2009.04.012

* Corresponding author. Fax: +1 607 255 4137.E-mail address: bab13@cornell.edu (B. Baird).

In mast cells, antigen-mediated cross-linking of IgE bound to its high-affinity surface receptor, FceRI, ini-tiates a signaling cascade that culminates in degranulation and release of allergic mediators. Antigen-pat-terned surfaces, in which the antigen is deposited in micron-sized features on a silicon substrate, wereused to examine the spatial relationship between clustered IgE–FceRI complexes and Lyn, the signal-ini-tiating tyrosine kinase. RBL mast cells expressing wild-type Lyn-EGFP showed co-redistribution of thisprotein with clustered IgE receptors on antigen-patterned surfaces, whereas Lyn-EGFP containing aninhibitory point mutation in its SH2 domain did not significantly accumulate with the patterned antigen,and Lyn-EGFP with an inhibitory point mutation in its SH3 domain exhibited reduced interactions. Ourresults using antigen-patterned surfaces and quantitative cross-correlation image analysis reveal thatboth the SH2 and SH3 domains contribute to interactions between Lyn kinase and cross-linked IgE recep-tors in stimulated mast cells.

� 2009 Elsevier Inc. All rights reserved.

1. Introduction domains in unstimulated cells. Net phosphorylation of monomeric

Mast cells are the primary cellular mediators of allergic reac-tions (Metcalfe et al., 1997), and they play important roles in bothinnate (Marshall, 2004) and adaptive (Galli et al., 2005) immuneresponses. The high-affinity receptor for IgE, FceRI, is expressedat the plasma membrane of mast cells where it binds IgE to sensi-tize the cells (Gould et al., 2003). Antigen-mediated cross-linkingof IgE–FceRI complexes results in receptor phosphorylation, anevent that links receptor recognition of extracellular signal to theintracellular signaling cascade that culminates in degranulationand the release of allergic mediators, such as histamine (Metcalfeet al., 1997; Turner and Kinet, 1999).

Intracellular signaling requires recruitment of cytosolic proteinsto the plasma membrane and redistribution of membrane compo-nents relative to cross-linked IgE receptors. The first biochemicallydetectable step within this signaling cascade is phosphorylation ofintracellular receptor segments by Lyn, a member of the Src kinasefamily (Sheets et al., 1999). Accumulating evidence supports theview that antigen-mediated cross-linking promotes stable associa-tion of clustered IgE receptors with ordered regions of the plasmamembrane, often called lipid rafts, that are enriched in cholesterol,sphingomyelin, and phospholipids with saturated acyl chains(Dykstra et al., 2003; Holowka et al., 2005). Lyn is anchored tothe membrane by saturated acyl chains, and active, phosphory-lated Lyn is predominately associated with dynamic, ordered lipid

ll rights reserved.

FceRI in unstimulated cells is prevented by transmembrane phos-phatases that are more readily accommodated in regions of themembrane composed of lipids with unsaturated, disordered acylchains. Co-localization of active Lyn with antigen-cross-linked IgEreceptors in ordered lipid rafts results in FceRI phosphorylationand consequent downstream signaling events (Young et al., 2005).

To initiate signaling, Lyn phosphorylates FceRI b and c subunits(Jouvin et al., 1994; Kihara and Siraganian, 1994), leading torecruitment and activation of Syk kinase (Kihara and Siraganian,1994; Reth and Brummer, 2004; Sada et al., 2001; Shiue et al.,1995). Syk then phosphorylates several proteins, including the lin-ker for the activation of T cells (LAT), which mediates recruitmentof phospholipase C c (PLCc) to the plasma membrane (Saitoh et al.,2003). Upon tyrosine phosphorylation, PLCc hydrolyzes phosphati-dylinositol-4,5-bisphosphate to produce two second messengers,inositol-1,4,5-trisphosphate and diacylglycerol (Gilfillan and Tkac-zyk, 2006), leading to Ca2+ mobilization and protein kinase C acti-vation, which are necessary for degranulation (Beaven andMetzger, 1993).

Src-family kinases have common structural components,including an N-terminal unique domain, SH3 and SH2 domains,and a C-terminal kinase domain (Boggon and Eck, 2004; Ingley,2008). At the N-terminus, all family members are myristoylatedand most, including Lyn, are also palmitoylated (Koegl et al.,1994; Wolven et al., 1997) causing their association with the plas-ma membrane (Liang et al., 2001; Resh, 1996). The SH3 and SH2domains mediate interactions with proline-rich motifs (Rickleset al., 1994; Yu et al., 1994) and with phosphotyrosine-containing

162 S. Hammond et al. / Journal of Structural Biology 168 (2009) 161–167

sequences (Malek and Desiderio, 1993; Waksman et al., 1993),respectively. Phosphorylation of a C-terminal regulatory tyrosineby Csk maintains Src-family kinases in an inactive conformationinvolving an intramolecular interaction between this phosphotyro-sine residue and the SH2 domain. This ‘‘closed” conformation isfurther stabilized by an additional interaction between the SH3 do-main and a proline-rich sequence between the SH2 and kinase do-mains (Panchamoorthy et al., 1994; Superti-Furga et al., 1993; Xuet al., 1999). Upon dephosphorylation of the regulatory C-terminaltyrosine, Src-family kinases adopt an ‘‘open” conformation inwhich the activation loop tyrosine is phosphorylated and the ki-nase is fully activated (Ingley, 2008). Lyn activity is down-regu-lated by dephosphorylation of the active site tyrosine kinase bytransmembrane phosphatases such as PTPa (Young et al., 2005).

Lyn SH2 and SH3 domains also mediate inter-protein interac-tions that influence signal transduction in mast cells. The SH2 do-main interacts with phosphorylated FceRI b to amplify thephosphotyrosine signaling cascade (Kihara and Siraganian, 1994),and its deletion results in inhibition of stimulated Syk phosphory-lation and activation in an RBL cell reconstitution system (Hondaet al., 2000). Studies of the SH3 domain showed that it also contrib-utes to Lyn kinase activity (Abrams and Zhao, 1995). Peptides thatbind competitively to this domain were found to inhibit stimulatedIgE receptor phosphorylation and Ca2+ signaling (Stauffer et al.,1997). In contrast, deletion of this domain did not significantly al-ter Lyn-dependent signaling leading to Ca2+ mobilization (Hondaet al., 2000).

Based on this previous work demonstrating functional roles forthe SH2 and SH3 domains in Lyn kinase regulation and Lyn-depen-dent signaling, we investigated how specific mutations of these do-mains affects physical association of Lyn with FceRI duringsignaling. Co-localization of Lyn with cross-linked IgE receptorshas been observed with electron microscopy (Wilson et al.,2000), fluorescence imaging of cells on patterned antigen surfaces(Torres et al., 2008b; Wu et al., 2004), fluorescence correlationspectroscopy (Larson et al., 2005), and cross-correlation fluores-cence imaging (Das et al., 2008). To directly assess the respectiveroles of the SH2 and SH3 domains in mediating Lyn co-localizationwith FceRI, we generated Lyn mutants in which the SH2 or SH3 do-main is inactivated by a point mutation. We took advantage ofantigen-patterned surfaces to cluster the IgE receptors in well de-fined features, which enables co-redistributing fluorescently la-beled species to be reliably visualized (Torres et al., 2008a). Inaddition to visual inspection, we used cross-correlation analysisof Lyn co-localization with the antigen patterns to quantify the ef-fects of these mutations on stimulated Lyn redistribution. Wefound that mutation of the SH2 domain blocks co-redistributionof Lyn with clustered IgE receptors, and mutation of the SH3 do-main substantially reduces this antigen-dependent accumulation.Our results provide new information about binding interactionsbetween Lyn kinase and FceRI that relate to regulation of thisreceptor-mediated signaling.

2. Materials and methods

2.1. Reagents

Mouse monoclonal IgE specific for 2,4-dinitrophenyl (DNP) waspurified as described previously (Subramanian et al., 1996). Thisanti-DNP IgE in borate-buffered saline (BBS, 200 mM boric acid,33 mM NaOH, and 160 mM NaCl, pH 8.5) was fluorescently labeledwith Alexa488 as previously described (Gosse et al., 2005) and dia-lyzed extensively in PBS with EDTA (0.15 M NaCl, 10 mM sodiumphosphate, and 1 mM EDTA, pH 7.4) at 4 �C. The fluorescently mod-ified IgE had�7–10 dye molecules per protein. BSA was conjugated

with an average of 15 DNP groups per protein (Posner et al., 1992).DNP–BSA in BBS was fluorescently labeled with Alexa555 dye kit(Molecular Probes) at the recommended dye:protein ratio for24 h at room temperature (RT � 22 �C) in the dark. Reaction mix-tures were dialyzed extensively in PBS with EDTA at 4 �C. Fluores-cently modified Alexa555–DNP–BSA had �2 dye molecules perprotein.

2.2. Lyn constructs

The wt Lyn-EGFP construct was previously described (Hesset al., 2003). The Lyn constructs with single point mutations,Lyn-EGFP-SH2mut and Lyn-EGFP-SH3mut, were generated fromthe corresponding Lyn-mRFP (monomeric RFP) mutants by ex-change of mRFP for EGFP at the BamHI and NotI restriction sites.Lyn-mRFP was generated by amplifying mRFP using the sense pri-mer 50-TTAAAGGATCCA ATGGCCTCCTCCGAGGACG-30 and the anti-sense primer 50-GCGCAAACGGCCGCTTAGGCGCCGGTGGAGTGG-30

and then exchanging EGFP for the subcloned mRFP at the BamHIand NotI restriction sites in the Lyn-EGFP plasmid. The mRFP-pRSETb plasmid (Campbell et al., 2002) was a gift from Dr. EdwardCox, Princeton University. Lyn-mRFP-SH2mut was generated bythe Arg ? Ala mutation at position 135 using the sense primer50-GGGCTTTCCTGATCGCAGAAAGTGAAACTTTAAAGG-30 and theantisense primer 50-CCTTTAAAGTTTCACTTTCTGCGATCAG-GAAAGCCC-30. Lyn-mRFP-SH3mut was generated by the Trp ? Alamutation at position 78 using the sense primer 50-GGAAGAG-CACGGGGAAGCGTGGAAAGCTAAGTCCC-30 and the antisense pri-mer 50-GGGACTTAGCTTTCCACGCTTCCCCGTGCTCTTCC-30.

2.3. Clustering of IgE receptors on antigen-patterned surfaces

Preparation of parylene-patterned silicon wafers was describedpreviously (Ilic and Craighead, 2000). Antigen was immobilized onthese surfaces as previously described (Torres et al., 2008b). Briefly,the silicon surface, coated with parylene and patterned with theuse of a fabricated mask, was first derivatized with 2% v/v 3-(mer-captopropyl) trimethoxysilane (Sigma–Aldrich) in toluene for20 min at RT. This surface was reacted with 2 mM N-c-maleimido-butyryloxy succinimide ester (Sigma–Aldrich) in absolute ethanolfor 1 h at RT. Antigen was then immobilized on the surface by add-ing a 2:1 mixture of DNP–BSA:Alexa555–DNP–BSA (50 lg/mLDNP–BSA:25 lg/mL Alexa555–DNP–BSA) to the silicon chips for2 h at RT. Chips were rinsed with PBS containing 2 mg/mL BSA be-fore and after the patterned parylene layer was mechanicallypeeled away in solution.

Transient transfection was performed using a Gene Pulser Xcellelectroporation system (Bio-Rad Laboratories). RBL cells wereresuspended in cold electroporation buffer (137 mM NaCl,2.7 mM KCl, 1 MgCl2, 1 mg/mL glucose, 20 mM Hepes, pH 7.4) at30 � 106 cells/mL. For each construct, �10 lg of wt Lyn-EGFP,Lyn-EGFP-SH2mut, or Lyn-EGFP-SH3mut cDNA was added to0.3 mL of suspended RBL cells in a 4 mm cuvette, and cells wereelectroporated with one exponential pulse at 280 V and 950 lF.Transfected cells were sensitized overnight with 0.5 lg/mL anti-DNP IgE. Experiments were performed 24 h after transfection.

Suspended cells at a concentration of �0.5–1 � 106 cells/mLwere gently pipetted on top of the antigen-patterned silicon chipand incubated for 30 min at 37 �C. Then cells were fixed with 4%w/v paraformaldehyde for 10 min at RT and quenched with PBScontaining 0.01% sodium azide and 10 mg/mL BSA for 10 min atRT. Confocal images were acquired on a Leica TCS SP2 laser scan-ning confocal system with a 63�, 0.9 NA, HCX APO L U-V-I waterimmersion objective. Images were collected sequentially to mini-mize bleedthrough by alternatively exciting and collecting emis-sion from the green probe (EGFP: kex = 488 nm, kem = 500–

Fig. 1. Illustration of cross-correlation methodology. (A) Cell and (B) pattern imagesare acquired in parallel. Pixel intensity, I, is displayed in the false color scale shown,where higher numbers correspond to brighter fluorescence. A mask is created fromthe cell image by tracing by hand the cell outline that defines a region of interest(white trace). Scale bar represents 5 lm. (C) Cell and (D) pattern images areprepared for further processing by applying the mask and by subtracting meanpixel values (hIi) from each image. (E) Radially averaged autocorrelation functionsare calculated from each processed image as described in Section 2. Autocorrelationvalues at r = 0 contain additional contributions from photomultiplier (shot) noise;therefore the extrapolated values at zero shift (r = 0, denoted with an ‘x’ symbols)are used to normalize cross-correlation values. (F) The value of the radiallyaveraged cross-correlation function at zero shift (r = 0; dotted line) is the cross-correlation coefficient. At long distances (>1.5 lm), correlation function values canfall below zero, indicating that images become anti-correlated at large shifts. Inthese experiments, the decay of the correlation functions is a measure of patternedfeature size.

S. Hammond et al. / Journal of Structural Biology 168 (2009) 161–167 163

550 nm), and then exciting and collecting emission from the redprobe (Alexa555–DNP–BSA: kex = 543 nm, kem = 560–630 nm).These images were evaluated both by visual inspection and bycross-correlation analysis. For each of wt and mutant Lyn con-structs, 150–180 transfected cells from 5 to 6 experiments wereevaluated.

2.4. Cross-correlation analysis of Lyn construct co-localization withpatterned antigen

Cross-correlation coefficients are calculated for individual cellsas follows: first, the cell boundary is traced by hand to create themask applied to paired images of cells and underlying patternedfeatures. The mean pixel intensity is then subtracted from eachmasked image, respectively. Auto-correlation and cross-correlationfunctions are defined as:

autocorrð~rÞ ¼ hIð~Rþ~rÞ � Ið~RÞi hMð~Rþ~rÞ �Mð~RÞi.

crosscorrð~rÞ ¼ 1NhIð~Rþ~rÞ � Jð~RÞi hMð~Rþ~rÞ �Mð~RÞi

.

where Ið~rÞ and Jð~rÞ are the pixel values at position~r of the maskedimages, Mð~rÞ is the binary mask (ones on the cell and zeros offthe cell), N is a normalization factor, and the averages are over allpixels (values of ~R). Correlation functions are divided by the auto-correlation of the mask to account for the finite nature of the image,because the autocorrelation of the mask represents that maximumautocorrelation values possible in the measurement. In practice,auto-correlation and cross-correlation values are evaluated usingFourier transforms (FT),

hIð~Rþ~rÞ � Ið~RÞi ¼ FT�1ðjFTðIð~rÞÞj2Þ

hIð~Rþ~rÞ � Jð~RÞi ¼ real FT�1ðFTðIð~rÞÞ � FTðJð~rÞÞ�Þn o

where FT�1 is the inverse Fourier Transform, and * denotes a com-plex conjugate, and M refers to the binary mask (on or off tracedcell). Autocorrelations calculated using Fourier transforms are lesscomputationally intensive and are mathematically equivalent tothose calculated using brute force computations (Weisstein). Thecross-correlation function normalization factor is defined as:

N ¼ lim~r!0

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffihIð~R�~rÞIð~RÞihJð~R�~rÞJð~RÞi

q=hMð~RÞMð~RÞi

For acquired microscope images, the autocorrelation values at zeroshift ð~r ¼ 0Þ, which are used to calculate this normalization factor,contain contributions from camera or photomultiplier noise, as wellas autocorrelations from the cell itself. To correct for noise, autocor-relation functions obtained from individual images are extrapolatedto r = 0. Cross-correlation coefficients are defined as the magnitudeof the cross-correlation function at this zero shift. In this scheme, across-correlation value of 1 indicates perfect correlation of thepaired images, whereas a value of �1 indicates perfect anti-correla-tion. Fig. 1 illustrates the application of this method to a sampleimage.

For each of the three wt or mutant Lyn-EGFP constructs, cross-correlation coefficients were evaluated for 150–180 transfectedcells. Each experimental condition resulted in a broad distributionof cross-correlation values, which are well fit by a Gaussian line-shape, centered around the most probable cross-correlation value.

3. Results

3.1. Lyn SH2 and SH3 domain point mutations

Our previous studies with sensitized RBL mast cells demon-strated that Lyn-EGFP co-redistributes with IgE receptors that clus-

ter over patterns of specific antigen presented in micron-sizefeatures. To investigate the structural basis for this accumulation,we generated Lyn-EGFP constructs containing point mutations thattarget critical residues in Lyn’s SH2 and SH3 domains. For Lyn-EGFP-SH2mut, we mutated a conserved arginine residue (R135 inLynB) to alanine within the phosphotyrosine-binding pocket (Ing-ley, 2008). The analogous mutation in Src (R175A in Src) eliminatesphosphotyrosine binding to this domain (Shvartsman et al., 2007;Waksman et al., 1993). For Lyn-EGFP-SH3mut, we mutated a con-served tryptophan residue (W78 in LynB) to alanine in the ligandbinding surface (Bauer et al., 2005; Kuga et al., 2008). The analo-gous mutation in Src (W118A in Src) blocks interaction betweenSrc and PI-3-Kinase via this SH3 domain (Erpel et al., 1995; Shv-artsman et al., 2007). As previously shown for Lyn-EGFP (Gosseet al., 2005), we determined that wt Lyn-EGFP, Lyn-EGFP-SH2mut,

164 S. Hammond et al. / Journal of Structural Biology 168 (2009) 161–167

and Lyn-EGFP-SH3mut expressed in RBL cells show strong plasmamembrane localization in cells on unpatterned surfaces (Fig. 2).

3.2. Redistribution of Lyn on Antigen-Patterned Surfaces

Previously we used antigen presented in patterned lipid bilay-ers with feature dimensions of �1–4 lm to study membrane traf-ficking and the reorganization of membrane associatedcomponents that occurs at the cell-surface interface (Torres et al.,2008b; Wu et al., 2004, 2007). We demonstrated that for RBL cellssensitized with Alexa488–IgE, these labeled IgE receptors clusterspecifically over antigen patterns on the time scale of several min-utes as the cells contact the substrate, and no clustering occurswhen antigen is omitted from the patterned features (Orth et al.,2003). Our subsequent experiments with transfected cells showedthat micron-scale co-redistribution of Lyn-EGFP with IgE receptorsoccurs on patterned bilayer surfaces, becoming detectably clus-tered after 5–10 min at 37 �C and maximally clustered after 15–20 min. No accumulation of Lyn-EGFP is detectable in the presenceof cytochalasin D, which inhibits actin polymerization (Wu et al.,2004). Our present experiments used an alternative means of pat-terning the antigen with fluorescently labeled DNP–BSA immobi-lized on the silicon chips. With this method also, anti-DNP IgEbound to receptors on RBL cells accumulate over the patternedantigen, similar to that observed with patterned antigen presenta-

Fig. 2. Wild-type Lyn-EGFP and constructs with point mutations in SH2 or SH3 domainimages of RBL cells expressing wt Lyn-EGFP, Lyn-EGFP-SH2mut, and Lyn-EGFP-SH3mut

Fig. 3. Distribution of Lyn-EGFP constructs in transfected cells adhering to surfaces withgreen), Lyn-EGFP-SH2mut (middle image, green), and Lyn-EGFP-SH3mut (right image,5 lm.

tion with lipid bilayers (Torres et al., 2008b). To evaluate the effectsof functionally inhibitory point mutations in Lyn-EGFP on bindinginteractions, RBL cells transiently expressing Lyn-EGFP, Lyn-EGFP-SH2mut and Lyn-EGFP-SH3mut were incubated for 30 min withthese antigen-patterned surfaces at 37 �C. We found these condi-tions to be optimal for maximal Lyn-EGFP co-redistribution withpatterned antigen, as represented in Fig. 3, left panel. Visualinspection showed that Lyn-EGFP-SH3mut co-clusters to a reducedextent over antigen patterns (Fig. 3, right panel), whereas Lyn-EGFP-SH2mut does not detectably co-cluster on these surfaces(Fig. 3, middle panel).

As in our previous studies with patterned antigen, we assessedthe tendency of the Lyn-EGFP constructs to co-redistribute withantigen-clustered receptors by scoring transfected cells that set-tled on surfaces and showed EGFP accumulation that matchedthe patterned features. By this visual assessment of 150–180 trans-fected cells of each type in 5–6 experiments, we found that 60% ofLyn-EGFP, 0% of Lyn-EGFP-SH2mut, and 20% of Lyn-EGFP-SH3mutclustered over antigen patterns (Fig. 4A). The spatial definition ofpatterns provides reliability to this visual assessment, as describedpreviously (Torres et al., 2008a). We also applied cross-correlationanalysis to quantify the extent of co-clustering for the transfectedcells in the same experimental samples. In this approach, a mask isused to trace the outline of adherent cells, and quantitative imageanalysis calculates the extent of overlapping fluorescent labels cor-

s localize to the plasma membranes of transfected RBL mast cells. Representative. Scale bar represents 5 lm.

patterned antigen. Representative images of cells expressing Lyn-EGFP (left image,green) on surfaces patterned with immobilized antigen (red). Scale bars represent

% o

f cel

ls w

ith E

GFP

clu

ster

ing

Lyn-EGFP Lyn-EGFP- SH2mut

Lyn-EGFP- SH3mut

20

40

60

80

0

Clustering on Patterned SurfacesA

00.80.40-0.4 1.2

10

20

30

Lyn-EGFP 0.37 ± 0.03Lyn-EGFP-SH2mut 0.06 ± 0.02Lyn-EGFP SH3mut 0.14 ± 0.01

Rel

ativ

e Fr

eque

ncy

Cross-Correlation AnalysisB

peak cross-correlation value

Fig. 4. Quantification of variant Lyn-EGFP distribution in transfected cells adhering to surfaces with patterned antigen. Approaches represented in (A) and (B) were carriedout on the same samples: For each of wt Lyn-EGFP, Lyn-EGFP-SH2mut, and Lyn-EGFP-SH3mut, 150–180 cells from 5 to 6 independent experiments were evaluated. (A) Visualinspection: Bar graph shows average percent of transfected cells on patterned antigen surfaces exhibiting Lyn-EGFP co-localization with patterned features. Error barsindicate the standard error for independent experiments. (B) Cross-correlation analysis: histogram shows the distribution of calculated cross-correlation coefficients thatquantify overlap of Lyn-EGFP constructs and patterned fluorescent antigen. The fitted Gaussian line-shapes are centered around the value corresponding to the most probable(peak) cross-correlation coefficient value for each construct. Standard errors are included to demonstrate the fit to the mean.

S. Hammond et al. / Journal of Structural Biology 168 (2009) 161–167 165

responding to the Lyn construct and the patterned antigen (Fig. 1).A cross-correlation coefficient was determined for each transfectedcell that had settled on the patterned surface. We found that eachof the constructs displays a broad distribution of values corre-sponding to different cells, and these collectively are well fit by aGaussian distribution with a peak (most probable) value(Fig. 4B). Lyn-EGFP showed a wide range of cross-correlation coef-ficients, with a peak value of 0.37, compared to 1.0 for theoreticallyperfect overlap of the fluorescence labels. Values less than 1.0 andvariation within a single population arises from fluorescence label-ing of internal structures as well as typical cell-to-cell variation.Lyn-EGFP-SH2mut showed very low cross-correlation coefficients,with a peak value of 0.06. Lyn-EGFP-SH3mut showed cross-corre-lation coefficients intermediate to these two constructs, with apeak value of 0.14. Although the statistical basis is different for vi-sual inspection and cross-correlation analysis, the trends in theseresults are consistent (Fig. 4).

4. Discussion

Our study used surfaces that present patterned antigen toexamine Lyn co-redistribution with IgE receptors (FceRI) afterthese receptors are clustered to initiate transmembrane signaling.Previous studies showed that Lyn-mediated phosphorylation ofFceRI becomes maximal within 2–5 min of soluble antigen stimu-lation at 22 �C, and this is followed by a reduction in Lyn-EGFP dif-fusion that temporally correlates with interactions between Lyn-EGFP and IgE receptors, as measured with fluorescence correlationspectroscopy (Larson et al., 2005). Additionally, micron-scale accu-mulation of Lyn-EGFP in cells stimulated on antigen-patterned sur-faces can be visualized about ten minutes after both IgE receptorclustering and co-localized tyrosine phosphorylation is detectedat earlier times in the same cells (Wu et al., 2004). Accumulationof Lyn-EGFP over antigen patterns is prevented by pretreatmentwith cytochalasin D, an inhibitor of actin polymerization. In con-trast, this treatment enhances antigen-stimulated tyrosine phos-phorylation of FceRI (Holowka et al., 2000), further supportingthe view that micron-scale accumulation of Lyn-EGFP with clus-tered IgE receptors can be uncoupled from its role in initial phos-phorylation events. The stabilized, cytoskeleton-dependentinteractions between Lyn-EGFP and IgE receptors we observe withpatterned antigen appear to be a consequence of the initial phos-

phorylation events that occur in ordered lipid domains in the plas-ma membrane (Holowka et al., 2005).

We evaluated antigen-patterned surfaces for co-redistributionof wt and mutant Lyn constructs with cross-linked IgE receptors;the same samples were assessed both by visual inspection andby cross-correlation analysis (Fig. 4). The latter is a rigorous math-ematical approach and yields a distribution of cross-correlationcoefficients, which quantify the extent of overlapping fluorescentlabels in imaged cells. The most probable cross-correlation coeffi-cient, i.e., the peak of the distribution, can be determined for eachconstruct. We observed �60% of cells expressing wt Lyn-EGFP withvisible concentration of this probe over the patterned antigen, andwe determined a peak cross-correlation value of 0.37. Mutation ofthe SH2 domain (Lyn-EGFP-SH2mut) blocked this stimulated accu-mulation almost completely, such that negligible positive cellswere scored, and correspondingly peak cross-correlation for Lyn-EGFP-SH2mut had a very low value of 0.06. These consistent re-sults indicate that the SH2 domain participates in stabilized, F-ac-tin-dependent, co-localization of Lyn with clustered IgE receptors.Mutation of the SH3 domain of Lyn substantially reduces, but doesnot eliminate, Lyn accumulation in the regions of patterned anti-gens. Only �20% of cells expressing Lyn-EGFP-SH3mut exhibitedco-clustering, and an intermediate peak cross-correlation value of0.14 was determined. Evidently, the SH3 domain of Lyn contributesto, but is not essential for, its interaction with clustered IgEreceptors.

Our results suggest that both the SH2 and SH3 domains play apositive role in IgE-mediated signaling by stabilizing interactionsbetween Lyn, FceRI, and focal adhesion proteins such as paxillin(Stauffer et al., 1997; Torres et al., 2008b). We previously observedthat the saturated acyl anchor of Lyn (PM-EGFP) is sufficient tomediate its accumulation with patterned antigen in an IgE-depen-dent manner (Wu et al., 2004). These palmitate and myristoylatechains may facilitate Lyn’s localization with cross-linked IgE recep-tors that preferentially partition into ordered membrane domains(Field et al., 1997), allowing the SH2 domain of recruited Lyn tointeract with focal adhesion proteins in a dynamic complex. In-deed, our previous fluorescence photobleaching recovery measure-ments showed that, whereas clustered IgE receptors areimmobilized, Lyn-EGFP and PM-EGFP show diffusive exchange,and the latter exhibits a larger mobile fraction than the former(Torres et al., 2008a; Wu et al., 2004). Lyn may mediate receptor-

166 S. Hammond et al. / Journal of Structural Biology 168 (2009) 161–167

actin interactions by binding to the phosphorylated b subunit ofFceRI via its SH2 domain (Kihara and Siraganian, 1994) and fromthat position recruit other focal adhesion proteins, including paxil-lin (Minoguchi et al., 1994), to form an F-actin-stabilized network,similar to a focal adhesion complex.

These interactions may also negatively regulate IgE-mediatedsignaling, as implicated in gene knockout studies of bone mar-row-derived mast cells from mice (Odom et al., 2004). Paxillinwas shown participate in down-regulation of Src family kinaseactivity by recruiting the C-terminal Src kinase, Csk (Rathoreet al., 2007). The knock-down of paxillin in RBL cells reveals evi-dence for both negative and positive effects of this protein: it en-hances stimulated tyrosine phosphorylation of FceRI b whilereducing Ca2+ responses in these cells (Torres et al., 2008b). Thus,Lyn interactions with both phosphotyrosine and proline-rich se-quences of adaptor proteins linked to the actin cytoskeleton maymediate both positive and negative regulation of FceRI signalingin these cells. Further studies will be necessary to distinguish thespecific protein–protein interactions that participate in this pro-cess. In the current work, we over-expressed fluorescently labeledLyn constructs, which may compete with endogenous Lyn for bind-ing partners. Expression of these constructs in a Lyn-deficientbackground, generated by using siRNA that targets endogenousLyn in RBL mast cells, may provide more insight into the role ofthese domains early in the signaling pathway.

A striking advantage of patterned substrates is their reliabilityfor visual evaluation of co-localized fluorescent labels (Torreset al., 2008a). Our mathematical approach for quantifying the co-redistribution of Lyn constructs with patterned antigen, and there-by clustered IgE receptors, yields a distribution of cross-correlationcoefficients for a large number of cells. These distributions reflectvariations within the sample that likely arise from backgroundfluorescence labeling as well as typical cell-to-cell variation. None-theless, significant differences in the peaks of these distributionsdemonstrate distinction in the capacities of these constructs tointeract with clustered IgE receptors. Complementary and improv-ing upon visual inspection, this cross-correlation approach pro-vides an objective and quantitative method for evaluating co-localization in experiments utilizing patterned substrates.

Acknowledgments

We are grateful to Alexis Torres for preparing the silicon chipswith patterned parylene and for assistance in early stages of theseexperiments. This research was supported by the Nanobiotechnol-ogy Center (NSF: ECS9876771) and by NIH Grants: R01-AI018306,and T32-GM08210. S.L.V. was supported in part through the Irving-ton Institute Fellowship Program in Cancer Immunology.

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