Cytoskeletal Modulation of Lipid Interactions Regulates Lck Kinase Activity

Background: Rafts are important for phosphoregulation of Lck, but how they are formed and maintained in cell membranes is poorly understood.

Results: Disrupting the actomyosin cytoskeleton declusters raft lipophilic reporters and deregulates Lck.

Conclusion: The actomyosin cytoskeleton maintains lipid interactions that sustain rafts for Lck regulation.

Significance: These data provide new information regarding how rafts are maintained for Lck regulation.

Abstract

The actin cytoskeleton promotes clustering of proteins associated with cholesterol-dependent rafts, but its effect on lipid interactions that form and maintain rafts is not understood. We addressed this question by determining the effect of disrupting the cytoskeleton on co-clustering of dihexadecyl-(C₁₆)-anchored DiO and DiI, which co-enrich in ordered lipid environments such as rafts. Co-clustering was assayed by fluorescence resonance energy transfer (FRET) in labeled T cells, where rafts function in the phosphoregulation of the Src family kinase Lck. Our results show that probe co-clustering was sensitive to depolymerization of actin filaments with latrunculin B (Lat B), inhibition of myosin II with blebbistatin, and treatment with neomycin to sequester phosphatidylinositol 4,5-bisphosphate. Cytoskeletal effects on lipid interactions were not restricted to order-preferring label because co-clustering of C₁₆-anchored DiO with didodecyl (C₁₂)-anchored DiI, which favors disordered lipids, was also reduced by Lat B and blebbistatin. Furthermore, conditions that disrupted probe co-clustering resulted in activation of Lck. These data show that the cytoskeleton globally modulates lipid interactions in the plasma membrane, and this property maintains rafts that function in Lck regulation.

Introduction

Cell membranes are fluid structures, yet they retain discrete protein and lipid domains that functionally compartmentalize the bilayer. One example of this property is the cholesterol-dependent rafts, which function in signal transduction (1), membrane trafficking (2), and cell adhesion (3). In T cells, rafts are posited to modulate interactions between the Src family kinase (SFK)² Lck and its regulators CD45 and Csk (4, 5). Factors that regulate the formation of rafts therefore impact cell viability and survival, and, in T cells, regulate host response to antigen.

Structurally, rafts are posited to consist of lipids that are ordered through interactions with cholesterol. Cholesterol-dependent lipid ordering to form discrete domains is represented in model membranes by combinations of cholesterol and fluid phase lipids, notably sphingolipids, that form a cholesterol-dependent liquid-ordered (L_o) phase (6–8). Some bilayer compositions contain coexisting L_o and liquid-disordered (L_d) phases (8, 9), and this is suggested to be representative of coexisting raft and nonraft environments in cell membranes (10). Coexisting ordered and disordered phases can be produced in blebs that are generated by chemical treatment of cells (11), showing that the plasma membrane contains lipid mixtures that will undergo phase separations in certain conditions.

Consistent with the membrane raft model, proteins and lipids that favor L_o lipid environments exhibit a cholesterol-dependent clustering in the plasma membrane (12, 13). Furthermore, clustering of raft markers in the plasma membrane often correlate with the F-actin content of the cell (14, 15), suggesting that the cytoskeleton can promote, by poorly understood mechanisms, lipid interactions that transition them to a more ordered state (16). This interpretation is supported by data showing that lipids in model membranes undergo ordering as a result of attachment of actin filaments to the bilayer (17). However, cytoskeletal ordering of lipids in minimalist systems such as lipid vesicles may not be representative of cell membranes. Emission from the lipophilic probe Laurdan, which is sensitive to solvent polarity, is shifted by disrupting the cell cytoskeleton in a manner that is consistent with disordering of plasma membrane lipids (16), yet this may reflect changes in probe fluorescence by parameters unrelated to lipid ordering. Also unknown is whether the effect of the cytoskeleton on lipid interactions is specific to lipids that favor ordered lipid environments such as rafts.

Most rafts have a diameter less than ∼20 nm (18), thus challenging their characterization in intact cells because they are too small to visually resolve by light microscopy. Nonetheless, separate approaches exist for resolving nanoscale complexes, one being fluorescence resonance energy transfer (FRET) (13, 19–21). Measuring FRET between dialkyl forms of the carbocyanine dyes DiO and DiI, Baird and co-workers (21) showed evidence of cholesterol-dependent lipid heterogeneities in the plasma membrane of RBL cells. However, the role of the cytoskeleton in forming these lipid complexes was not reported.

Herein, we report experiments that measured the effect of altering the cytoskeleton on FRET between membrane-anchored forms of DiO and DiI. Our data show that conditions that disrupt either the structural or functional integrity of the cytoskeleton resulted in a decrease in probe clustering associated with lipid ordering. These conditions also altered phosphorylation of Lck on its regulatory tyrosines to increase the amount of active protein. Altogether, these data favor a model where the cytoskeleton regulates formation of lipid domains that are necessary for efficient regulation of Lck.

EXPERIMENTAL PROCEDURES

Cell Culture and Sample Preparation

Jurkat T cells (clone E6-1) were prepared for microscopy by seeding ∼10⁶ cells onto a poly-l-lysine-coated (Sigma) coverslip, followed by washing with RPMI containing 50 mm HEPES (pH 7.4) (RPMI-HEPES), and then adding RMPI-HEPES containing either 1% dimethyl sulfoxide (Sigma) alone, or drug diluted from a 100× stock solution in dimethyl sulfoxide. Final drug concentrations were 5 μm latrunculin B (Lat B) (Calbiochem, La Jolla, CA), 50 μm blebbistatin ((−) stereoisomer) (Calbiochem), and 5 μg/ml filipin (Cayman Chemicals, Ann Arbor, MI). Incubations with either drug or vehicle alone were for 30 min at 37 °C. Alternatively, cells were treated with 20 mm neomycin overnight at 37 °C in RPMI-HEPES containing 2% FBS. Following treatment, the samples were labeled with C₁₆-DiO and either C₁₆-DiI or C₁₂-DiI by incubating for 10 min at 4 °C in RPMI-HEPES containing 1 μm label. Samples were maintained at 4 °C until imaging, after which they were equilibrated to and maintained at 37 °C.

In experiments employing immunoblotting, cells were treated in suspension at a density of 10⁶/ml using the media and drug concentrations described above. Following incubation, the cells were sedimented, quickly resuspended in Laemmeli sample buffer containing 1% 2-mercaptoethanol, and then incubated at 100 °C for 5 min. Proteins were separated by gel electrophoresis (10% acrylamide) and then immunoblotted using monoclonal antibody to Lck (clone 2B, BD Biosciences), rabbit antibody to pTyr⁵⁰⁵ of Lck (Cell Signaling Technology; Danvers, MA), or rabbit antibody to pSrc⁴¹⁶ (Cell Signaling Technology). Immunoblots were developed by ECL (Amersham Biosciences, Rockford, IL), and detected using a G:Box imaging system (Syngene, Frederick, MD).

Ca²⁺ Flux Measurements

Ca²⁺ flux from T cell stimulation was measured using the Ca²⁺ indicator Indo-1 (Invitrogen) as described previously (22). Indo-1 fluorescence was measured by flow cytometry (MOFLO; Dako Cytomation; Fort Collins, CO) based on the fluorescence emission at 475 and 400 nm. Excitation for Indo-1 was at 390 nm. A base line was achieved by passing the cells for approximately 1 min prior to addition of αCD3 monoclonal antibody (OKT3) for cell stimulation. The temperature of the instrument was maintained at 37 °C, and the flow rate was maintained by a base sheath pressure of 60 psi ± 1 psi.

Preparation of GUVs

0.05 mg of lipid containing 0.1 mole % C₁₆-DiO and C₁₂-DiI was dried on to a indium tin oxide-coated slide (Delta Technologies, Stillwater, MN), first using a gentle stream of Ar gas, followed by incubation under vacuum for 1 h at room temperature. Giant unilamellar vesicles (GUVs) were generated by electroformation as described (23), using a sandwich composed of two indium tin oxide-coated slides and containing ∼0.5 ml of 300 mm sucrose in Milli-Q water (Millipore, Billerica, MA). GUVs were grown using a sinusoidal wave current (1 V, 10 Hz) for not <1 h at 60 °C. The GUV fraction was collected and diluted with an equal volume of 150 mm NaCl in Millipore water for imaging.

Flow Cytometry

Jurkat T cells were fixed in 2% (w/v) paraformaldehyde for 20 min at 37 °C and permeabilized in 0.1% (v/v) Triton X-100 in PBS-glycine for 10 min at room temperature. Staining with Texas Red-conjugated phalloidin was done as we have described (24). Cells were analyzed on an LSR II Flow Cytometer (BD Biosciences). 10,000 events were counted from each sample. Cells were gated on Forward Scatter and Side Scatter using untreated cells, and this gate was applied to the remaining samples for measurement of Texas Red fluorescence.

Cell Imaging and Analysis

Unless otherwise noted, imaging was performed using a Zeiss LSM 510 META confocal microscope (Oklahoma Medical Research Foundation imaging core facility) equipped with a 63× water objective (NA, 1.2) and a thermo-controlled chamber for maintaining the samples at 37 °C (19). Image processing and quantitation were performed using iVision imaging software (version 4.0, BioVision Technologies, Exton, PA).

FRET was measured by detecting sensitized emission of acceptor following donor excitation. Accordingly, images were acquired in three separate channels: donor channel (DiO, 488 nm excitation/505 to 550 nm emission), acceptor channel (DiI, 543 nm excitation/560 nm < emission), and FRET channel (488 nm excitation/560 nm < emission). FRET efficiency was calculated using images collected in the FRET channel. Images of cells labeled with either C₁₆-DiO or C₁₆-DiI alone were collected in the DiO and DiI channels to determine correction factors necessary to eliminate contributions from donor and acceptor bleed-through to the FRET channel (25–27). The parameter K_A for conversion of fluorescence ratios to FRET efficiency values (see Equation 1) (27) was determined by measuring quenching of donor fluorescence following addition of acceptor. Specifically, cells labeled with C₁₆-DiO alone were imaged in the donor channel (F^D) and then re-imaged in the donor channel following addition of an equal amount of C₁₆-DiO (F^DA). The decrease in donor fluorescence by labeling with acceptor represents the FRET efficiency, E, calculated using the equation:

K_A was calculated from E_quenching as described (see Equation 5 in Ref. 27).

RESULTS

Cell Labeling and Detection of FRET between Lipophilic Carbocyanine Dyes

We used dihexadecyl (C₁₆)- and didodecyl (C₁₂)-anchored forms of DiO and DiI to measure by FRET microscopy membrane heterogeneities associated with lipid ordering. The principal behind this approach is illustrated in Fig. 1A. Specifically, the C₁₆ tail targets the probes to domains composed of ordered lipids, whereas C₁₂-anchored probes favor less-ordered L_d phase lipid environments (28, 29). Accordingly, rafts composed of ordered lipids co-label with C₁₆-anchored DiO (C₁₆-DiO) and C₁₆-anchored DiI (C₁₆-DiI), minimizing their intermolecular distance to increase the FRET efficiency relative to that between C₁₆-DiO and C₁₂-anchored DiI (C₁₂-DiI) (21).

FIGURE 1.

Targeted labeling of separate lipid environments by dialkyl carbocyanine dyes. A, C₁₆-anchored dialkyl carbocyanine dyes incorporate into ordered phase lipids, such as the L_o phase rafts, whereas the C₁₂-anchored probes prefer L_d phase lipids (21). In membranes double-labeled with C₁₆-DiO and C₁₆-DiI, the probes will co-localize in ordered lipid microenvironments, thus elevating FRET efficiency relative to that of C₁₆-DiO and C₁₂-DiI. B, epifluorescence images of GUVs double-labeled with C₁₆-DiO and C₁₂-DiI. In the top row, the vesicle is composed of DPPC:DOPC:cholesterol (2:2:1), which forms co-existing L_d and L_o lipid phases at room temperature (8). The vesicle in the bottom row is composed of DOPC alone, which occurs as a single L_d phase at room temperature. C, FRET efficiency values measured between C₁₆-DiO and either C₁₆-DiI (C₁₆/C₁₆) or C₁₂-DiI (C₁₆/C₁₂) in GUVs composed of DOPC alone. D, confocal images of labeled Jurkat cells. The cells were double-labeled with C₁₆-anchored DiO and DiI (top), or labeled with either C₁₆-DiO alone (middle) or C₁₆-DiI alone (bottom). The FRET channel represents DiO excitation and detection of DiI emission. E, Ca²⁺ flux measured in Jurkat cells stimulated by cross-linking CD3 with the monoclonal antibody OKT3. The cells were either unlabeled or double-labeled with C₁₆-DiO and C₁₆-DiI.

We show examples of the distinct affinities of the C₁₆- and C₁₂-anchored probes for L_o phase lipids in Fig. 1B. Each row consists of images of a GUV double-labeled with C₁₆-DiO and C₁₂-DiI. In the top row is a GUV containing co-existing L_o and L_d phases; C₁₆-DiO and C₁₂-DiI segregate due to enrichment in the separate lipid phases. Conversely, in the bottom row is a GUV containing only L_d phase lipids, and C₁₆-DiO and C₁₂-DiI colocalize throughout the vesicle. Measurement of multiple (n = 31) mixed L_o/L_d phase GUVs showed that 20% of the C₁₆-DiO signal colocalized with regions of C₁₂-DiI enrichment. Thus, 80% of the C₁₆-DiO was restricted to the L_o phase.

We controlled for probe co-clustering absent lipid ordering by measuring FRET in GUVs composed of L_d phase lipids. Specifically, demixing of probes due to unexpected interactions between C₁₆-DiO and either C₁₆-DiI or C₁₂-DiI will show as an elevated FRET efficiency. However, we observed no significant difference in FRET efficiency between the separate donor-acceptor pairs (Fig. 1C), showing that unequal mixing of C₁₆-DiO with either C₁₆-DiI versus C₁₂-DiI was not detected even at nanoscale dimensions.

Lipid Ordering in T Cell Plasma Membrane by Cytoskeleton

We measured the plasma membrane of Jurkat T cells, where rafts are critical for cell regulation and stimulation through the T cell receptor (4, 30). The cells were double-labeled using conditions that minimized internalization of the probes (see “Experimental Procedures”), while also producing efficient labeling of the plasma membrane and a FRET signal that was specific to cells that contained both DiO and DiI (Fig. 1D). To control for perturbation of the plasma membrane by the labeling, we measured the increase in intracellular Ca²⁺, or Ca²⁺ flux, which occurs following cross-linking the T cell receptor as this is sensitive to changes in plasma membrane permeability (31). We observed that double-labeling cells with C₁₆-DiO and C₁₆-DiI had no affect on either the magnitude or duration of the Ca²⁺ flux (Fig. 1E), thus indicating no significant perturbation of the outer membrane by the probes.

Summarized in Fig. 2A are results from measuring FRET between C₁₆-DiO and either C₁₆-DiI or C₁₂-DiI in double-labeled Jurkat cells containing the indicated ratios of donor-to-acceptor (D:A). These data show co-clustering of C₁₆-DiO and C₁₆-DiI was significantly greater than that of C₁₆-DiO and C₁₂-DiI. For example, FRET efficiency for C₁₆-anchored DiO and DiI was ∼2-fold or greater than that of C₁₆-DiO and C₁₂-DiI at each D:A ratio. Furthermore, FRET between C₁₆-anchored DiO and DiI was sensitive to the D:A ratio, which is another signature of co-clustering of the donor and acceptor (32). The elevated co-clustering of C₁₆-DiO and C₁₆-DiI was cholesterol-dependent because sequestering cholesterol with filipin decreased their FRET efficiency to values similar to that of C₁₆-DiO and C₁₂-DiI, and the FRET became independent of the D:A ratio (Fig. 2B). Importantly, the decrease in FRET efficiency for the C₁₆-anchored probes was not due to anomalous quenching of the DiI by filipin because fluorescence intensities of cells labeled with C₁₆-DiI were unchanged upon addition of the drug (supplemental Fig. S1). Altogether, these data are consistent with the notion of cholesterol-dependent rafts in the plasma membrane that are composed of L_o phase lipids.

FIGURE 2.

C₁₆-anchored lipophilic probes exhibit elevated FRET that is sensitive to disruption of the actomyosin cytoskeleton. A, FRET efficiency values measured in Jurkat cells double-labeled with the indicated donor-acceptor pairs. Measurements were restricted to cells containing a relative fluorescence intensity of acceptor of 1500 (± 150), and a donor-to-acceptor (D:A) ratio, which represent the relative intensity of donor (D) and acceptor (A), of 1:1, 1:2, or 1:3. *, p < 0.05 by Dunnett's multiple comparison test. For C₁₆/C₁₂ FRET efficiency values, 0.05 < p for both the 1:2 and 1:3 samples relative to the 1:1 sample. B, FRET efficiency values measured for C₁₆-DiO and C₁₆-DiI in untreated control cells and cells that were treated with filipin, Lat B, or blebbistatin (Blebb). The cells contained the indicated D:A ratio. The acceptor intensity was similar to that in A. Indicated in A and B are the average FRET efficiency for each sample (horizontal bars) and the S.E. (error bars). *, p < 0.05 by two-tailed Student's t test; NS, 0.05 < p. Each data set passed the Bartlett's test for equal variance (ns, 0.05 < p).

To determine whether co-clustering of C₁₆-anchored DiO and DiI was sensitive to disruption of the actomyosin cytoskeleton, we measured cells treated with either latrunculin B (Lat B) to disrupt actin filaments, or blebbistatin to inhibit nonmuscle myosin II (NM II). Each of these conditions was as efficient as filipin in reducing FRET between C₁₆-DiO and C₁₆-DiI, and each resulted in the FRET efficiency becoming independent of the D:A ratio (Fig. 2B). Importantly, the decrease in probe co-clustering by filipin and blebbistatin was not due to a loss of F-actin, as treated and untreated cells showed similar amounts of staining by phalloidin (Fig. 3). Altogether, these data show that ordered lipid domains detected by FRET between C₁₆-anchored probes are sensitive to conditions that either disrupt F-actin or inhibit NM II activity.

FIGURE 3.

Neomycin, neomycin co-treatment with filipin, and blebbistatin do not affect the F-actin content in Jurkat cells. A, cells were stained with Texas Red-labeled phalloidin (Phalloidin-Texas Red) following the respective treatments, and the labeling was measured by flow cytometry (as described under “Experimental Procedures”). Cells treated with Lat B served as a positive control to show detection of depletion of F-actin. An unstained sample (Unstained) was measured to control for nonspecific fluorescence. The y axis (% of max) represents normalized values 1602 of cell number. B, averaged values of staining intensity measured by flow cytometry. The data represent histogram peak intensity values were averaged from three separate trials. Error bars represent S.D. For the neomycin-, neomycin + filipin-, blebbistatin-, and filipin-treated samples, 0.05 ≤ p relative to the untreated cells by Dunnett's multiple comparison test. p < 0.05 for all samples relative to the Lat B-treated sample. Rel. Fluor Inten., relative fluorescent intensity.

Cytoskeleton Elevates Interactions between L_o- and L_d-preferring Probes

In contrast to our results with C₁₆-DiO and C₁₆-DiI, FRET between C₁₆-DiO and C₁₂-DiI was not affected by filipin, Lat B, or blebbistatin (supplemental Fig. S2). This suggests that co-clustering of C₁₆-DiO and C₁₂-DiI is minimal and therefore not further reduced by the respective treatments. Alternatively, lipid heterogeneities co-labeled with C₁₆-DiO and C₁₂-DiI may exist but were not distinguished by the FRET analysis in Fig. 2. To discriminate these separate interpretations, we employed an alternative approach to assess probe co-clustering using FRET efficiency values. This consisted of measuring the effect of acceptor concentration (F) on FRET efficiency, which can distinguish clustering events not resolved by measuring FRET over a narrow range of acceptor concentrations (32). Co-clustering of each donor-acceptor pair was quantitated as described (13), which consisted of fitting FRET efficiency (E) values to the isothermal binding equation,

where K in Equation 2 is analogous to a disassociation constant for the donor and acceptor (13), decreasing in value as their co-clustering increases.

Fitted curves generated from FRET between C₁₆-DiO and either C₁₆-DiI or C₁₂-DiI are shown in Fig. 4A, and K determined for each experiment is plotted in Fig. 4B. Variance in the E values that is inherent in this approach was addressed by using large data sets for the curve fitting, which minimized the 95% confidence intervals in the fitted curve (blue dashed lines). Consistent with our findings in Fig. 2 showing an elevated co-clustering of C₁₆-anchored probes, K from FRET between C₁₆-DiO and C₁₆-DiI was 5-fold less than K for C₁₆-DiO and C₁₂-DiI. To test the specificity of the curve fitting, FRET efficiency values measured for C₁₆-DiO and C₁₂-DiI were fit to Equation 2 using K determined for C₁₆-DiO to C₁₆-DiI FRET. This resulted in a fitted curve that deviated from the experimental values and produced large and nonrandom residuals (supplemental Fig. S3), thus showing that resolved K values were specific to the respective data sets. In summary, the separate FRET donor-acceptor pairs produced distinct sets of FRET efficiency values over a range of a acceptor values, and this resulted in unique values for K.

FIGURE 4.

Co-clustering of L_o and L_d phase probes is sensitive to cytoskeletal treatments. A, curve fitting of FRET efficiency values to Equation 2. FRET was measured over a range of acceptor concentrations using cells with a D:A ratio of 1:1. The red line represents the curve fit of experimental data to Equation 1, and the blue dashed lines outline the 95% confidence level for the curve fitting. The plot at the top of each curve is the residuals between the experimental and calculated values for the respective curve fitting. (B, K values determined for the indicated FRET pairs by fitting FRET efficiency values to Equation 1. *, p < 0.05 by a two-tailed Student's t test for distributions of unequal variance, measured for the indicated populations of FRET efficiency values and also by a Mann-Whitney nonparametric test. ns, p > 0.05 by the same tests. The fitted curves for each FRET pair and in each set of conditions are shown in supplemental Fig. S4. For C₁₆-DiO to C₁₆-DiI FRET, K ± the estimated S.D. of each fit value (error bars) was 167 ± 62, 942 ± 407, 1602 ± 801, and 1926 ± 742 for untreated, filipin-, Lat B-, and blebbistatin-treated cells, respectively. For C₁₆-DiO and C₁₂-DiI, K was 570 ± 217, 724 ± 302, 1445 ± 702, and 2374 ± 1500 for untreated, and filipin-, Lat B-, and blebbistatin-treated cells.

Also plotted in Fig. 4B are K values determined for cells treated with filipin, Lat B, or blebbistatin, each resolved from FRET between C₁₆-DiO and either C₁₆-DiI or C₁₂-DiI. Summarizing our results, filipin increased K for the C₁₆-DiO and C₁₆-DiI FRET pair alone, whereas K trended toward even larger values for both sets of FRET pairs when cells were treated with either Lat B or blebbistatin. Furthermore, the fitted curves for the Lat B- and blebbistatin-treated samples were linear over much of the range of acceptor values, making K approximate to the largest acceptor intensity value (Fig. 4B and supplemental Fig. S4). This property is indicative of a random or nonclustered distribution of the donor and acceptor (13), showing efficient demixing of both L_d and L_o phase probes by Lat B and blebbistatin. Thus, the cytoskeleton affected co-clustering of probes independent of their affinity for ordered lipid environments, indicating that cytoskeletal modulation of lipid interactions was not restricted to the raft L_o phase.

Clustering of Lipophilic Probes Requires PIP₂

To identify membrane signals that activate lipid clustering, we measured FRET in cells where the lipid co-factor phosphatidylinositol 4,5-bisphosphate (PIP₂) was sequestered using neomycin (33, 34). FRET efficiency and K values determined in this experiment are plotted in Fig. 5. These data show that treatment with neomycin decreased co-clustering of C₁₆-DiO and C₁₆-DiI, evidenced by a decrease in FRET efficiency (Fig. 5A) and an increase in K (Fig. 5B and supplemental Fig. S5). Furthermore, this effect was specific because FRET efficiency (Fig. S2) and K (Fig. 5B) in cells double-labeled with C₁₆-DiO and C₁₂-DiI were not changed significantly by the neomycin. In a separate experiment, we observed that sequestering PIP₂ by overexpressing the pleckstrin homology domain of phospholipase C-δ caused a measurable and significant decrease in the generalized polarization of Laurdan (supplemental Fig. S6), which is a signature of decondensation of membrane lipids associated with disruption of rafts (35, 54).

FIGURE 5.

Neomycin with filipin disrupts co-clustering of L_o and L_d phase probes. Shown is the FRET efficiency (A) and K values (B) determined for untreated control cells, cells treated with neomycin (Neo) alone, and cells treated with filipin following pretreatment with neomycin (Neo+filipin). Measurements and statistics are as described for Figs. 2 and 3. The fitted curves for each FRET pair and in each set of conditions are shown in supplemental Fig. S5. K ± the first S.D. from FRET between C₁₆-DiO and C₁₆-DiI was 81 ± 131, 689 ± 321, and 2780 ± 2300 for untreated, neomycin, and neomycin+filipin-treated cells, respectively. For C₁₆-DiO and C₁₂-DiI, K for the same conditions was 493 ± 280, 709 ± 357, and 2122 ± 1440.

Because PIP₂ signals that structure the cytoskeleton are cholesterol-dependent (34), we also measured the effect of co-treating cells with neomycin and filipin on probe co-clustering. As we show in Fig. 5, these conditions disrupted co-clustering of C₁₆-DiO with both C₁₆-DiI and C₁₂-DiI, indicated by K values that trended toward the maximum acceptor intensity for each donor-acceptor pair (Fig. 5B and supplemental Fig. S5). Similarly, FRET efficiency for C₁₆-DiO and C₁₆-DiI became minimal and independent of the D:A ratio (Fig. 5A). Importantly, neither neomycin alone, nor neomycin plus filipin, affected phalloidin staining (Fig. 3), again showing that the changes in probe co-clustering were not due to a decrease in the amount of F-actin. Altogether, these data are evidence that cytoskeletal effects on lipid ordering require cholesterol-dependent PIP₂ signals.

SFK Phosphoregulation by Signals That Modulate Lipid Interactions

SFKs undergo regulation through alternate phosphorylation and dephosphorylation of separate regulatory tyrosines. For Lck, this consists of phosphorylation of its regulatory C-terminal Tyr⁵⁰⁵ by Csk to down-regulate activity (36) and dephosphorylation of phospho-Tyr⁵⁰⁵ (pTyr⁵⁰⁵) by CD45 for activation (37).

Previous findings suggest membrane rafts are necessary to maintain negative regulation of Lck. For example, detergent fractionation studies show Csk co-associates with Lck in a detergent-resistant membrane fraction, which is posited to be representative of the composition of rafts (5, 38). Conversely, CD45 is excluded from detergent-resistant membranes (4). Similarly, treating T cells with either filipin or Lat B using conditions that disrupt rafts results in dephosphorylation of pTyr⁵⁰⁵ of Lck (19). To determine whether either neomycin or blebbistatin also affected Lck regulation, we measured phosphorylation of Tyr⁵⁰⁵ (pTyr⁵⁰⁵) in cells treated with neomycin alone, neomycin plus filipin, or blebbistatin alone. Whole cell lysates prepared from treated and untreated control Jurkat cells were immunoblotted with separate antibodies that recognized either pTyr⁵⁰⁵ or the Lck N terminus to report total Lck. Detection of Lck and pTyr⁵⁰⁵ was specific because signal associated with Lck was absent in immunoblots of Lck-deficient JCaM1.6 cells (Fig. 6A). Representative data from separate immunoblotting measurements are shown in Fig. 6B, showing that neomycin, neomycin plus filipin, and blebbistatin were each as effective as Lat B in reducing the pTyr⁵⁰⁵ content of Lck.

FIGURE 6.

Modulation of Lck tyrosine phosphorylation by the cytoskeleton. A, whole cell lysates from Jurkat and JCaM1.6 cells immunoblotted with antibody to the N terminus of Lck, Lck pTyr⁵⁰⁵, or Src pTyr⁴¹⁶. Molecular weight, in thousands, is indicated on the right. B–D, measurement of Lck Tyr⁵⁰⁵ and Tyr³⁹⁴ phosphorylation, and total Lck, in cell lysates from either Jurkat cells (B and C) or J45.01 cells (D). Each sample received the indicated treatment immediately before cell lysis. The pTyr signal divided by that from the Lck immunoblot is reported at the bottom of each lane.

Another site of phosphoregulation in SFKs is a conserved tyrosine present in the activation loop of the kinase domain, which is phosphorylated to up-regulate SFK activity (39). In Lck, this residue is Tyr³⁹⁴, and we therefore measured the effect of the separate conditions on the phospho-Tyr³⁹⁴ (pTyr³⁹⁴) content of Jurkat cells. We detected the pTyr³⁹⁴ by immunoblotting with an antibody made to the phosphorylated form of the equivalent site in Src, Tyr⁴¹⁶. Immunoblotting lysate from JCaM1.6 cells showed signal from the anti-pTyr⁴¹⁶ antibody was specific to Lck expression (Fig. 6A), thus representing pTyr³⁹⁴. In Fig. 6, C and D, are representative immunoblots with the anti-pTyr³⁹⁴ antibody, again measuring lysates of cells that were treated with the indicated conditions. These data show that treatment with either neomycin alone, or neomycin plus filipin, increased the pTyr³⁹⁴ content of Lck by 2-fold and 30%, respectively. Conversely, blebbistatin produced only a nominal increase in pTyr³⁹⁴, and Lat B had no effect (Fig. 6D).

pTyr³⁹⁴ is a substrate for CD45 (40), and CD45 may therefore quench increases in pTyr³⁹⁴ that result from treatment with either Lat B or blebbistatin. We therefore also measured pTyr³⁹⁴ levels in Lat B- and blebbistatin-treated J45.01 cells, which are a CD45-deficient clone of Jurkat cells (41). This showed that blebbistatin caused a robust increase in the pTyr³⁹⁴ (Fig. 6D), whereas Lat B caused a modest decrease in pTyr³⁹⁴. In summary, Fig. 6 shows that conditions that disrupt lipid interactions that we detected by FRET results in activation of Lck, indicating that interactions between Lck and its regulators are modulated by lipid domains that are maintained by the cytoskeleton. The results with Lat B-treated cells, however, indicate that other, lipid-independent mechanisms are also important for regulating Lck during homeostasis.

DISCUSSION

We report here findings showing that nanoscopic lipid domains composed of ordered lipids are maintained by the actomyosin cytoskeleton, evidenced by declustering of C₁₆-anchored DiO and DiI by either depolymerizing actin filaments with Lat B, or by inhibiting NM II activity with blebbistatin. Probe co-clustering was sensitive to sequestration of cholesterol with filipin, evidence that lipid domains detected by C₁₆-DiO to C₁₆-DiI FRET fall within the broad generalization of rafts, namely, lipid domains that form through interactions between cholesterol, other membrane lipids, and membrane proteins (42).

The phosphoinositide PIP₂ and its PI3K produce phosphatidylinositol 3,4,5-trisphosphate are critical co-factors in actin polymerization and attachment of actin filaments to cell membranes. Furthermore, we observed that co-clustering of the C₁₆-anchored dyes was reduced in cells treated with neomycin to sequester PIP₂. Similarly, sequestering PIP₂ by overexpressing pleckstrin homology domain of phospholipase C-δ produced a decondensation of plasma membrane lipids as reported by the generalized polarization of Laurdan. The augmented effect in disrupting probe clustering from co-treating cells with neomycin and filipin is consistent with findings that show cytoskeletal architecture is regulated by both PIP₂ and cholesterol (34) and suggests PIP₂ signals that modulate the cytoskeleton are compartmentalized to rafts. Similarly, we previously showed that selectively elevating or depleting raft pools of PIP₂ by expressing separate forms of the polyphosphate 5′-phosphatase Inp54p resulted in robust and distinct changes in cell morphology and adhesion (22), which are products of cytoskeletal architecture regulated by PIP₂.

The plasma membrane is suggested to exist at a critical point that is near the transition between separate fluid phases with different degrees of lipid ordering, such that small changes in composition, temperature, or other physical properties of the membrane favor formation of ordered lipid domains (42, 43). Furthermore, protein binding to the PIP₂ headgroup imparts an ordering effect in its lipid hydrocarbon chains (44), which may, in turn, impact lipid interactions proximal to PIP₂. Accordingly, we posit that interactions between the cytoskeleton and plasma membrane via PIP₂ favor formation of ordered lipid domains of various sizes and compositions, producing the elevated FRET between the lipophilic probes that was reported here (Fig. 7). Proteins that bind both phosphoinositides and the cytoskeleton and are therefore predicted to be important in the cytoskeletal modulation of lipid ordering include ezrin-radixin-moesin, Rho GTPases, annexins, and filamin A (16, 45, 46). Finally, the lipid ordering by the cytoskeleton to stabilize rafts contrasts with the notion that cytoskeleton facilitates raft formation through caging or direct binding of proteins to the actin network that underlies the plasma membrane (47).

FIGURE 7.

Membrane lipid ordering by the cytoskeleton. Association of the cytoskeleton with the plasma membrane activates lipid interactions in the underlying membranes to produce ordered lipid domains that include L_o phase rafts and cholesterol-independent L_d* domains. Arrows indicate sites of PIP₂-dependent interactions between the cytoskeleton and plasma membrane that are necessary for cytoskeletal effects on lipid ordering. PIP₂-dependent lipid ordering is predicted to be mediated by cytoskeletal proteins that bind simultaneously to F-actin and PIP₂, such as ezrin-radixin-moesin proteins, filamin A, and some species of annexins. These are indicated in the schematic as the “PIP₂-binding protein,” the different colors indicating separate species of these factors that may reside in different membrane compartments. NM II-dependent increases in lipid ordering may occur through tension that is imparted in the cytoskeleton and transduced to the membrane surface.

Disruption of probe co-clustering by inhibiting NM II without effecting the F-actin content suggests that mechanical properties of the cytoskeleton such as tension generated by myosin activity is important for lipid ordering effects by the cytoskeleton. It is interesting to note that actin filaments can exert considerable compressive force upon lipid bilayers, evidenced by robust changes in the shape of liposomes and lipid droplets that contain attached F-actin (48, 49). Furthermore, compression applied to membrane surfaces can order underlying fluid phase lipids (50). However, further studies are needed to show whether compression applied by a combination of crosslinked actin filaments and NM II activity contribute to lipid ordering and formation rafts in cell membranes.

Previous interpretations have stressed the similarities between L_o phase lipids in model membranes and that deduced for lipids in rafts. Namely, rafts, similar to L_o phase lipids, form by interactions between fluid lipids and cholesterol to form ordered lipid domains (9, 51). However, recent studies of membrane blebs show that the notion that rafts are represented by L_o phase lipids alone is likely an oversimplification. For example, blebs with different compositions and degrees of lipid ordering can be produced by relatively modest changes in preparation (52), and some species of blebs produce ordered domains that do not exclude L_d markers (11). These properties suggest that the plasma membrane is composed of a continuum of domains with separate degrees of lipid ordering, each established by a combination of their respective protein and lipid composition. Similarly, we showed here a cytoskeleton-dependent co-clustering of the L_o/L_d FRET pair C₁₆-anchored DiO and C₁₂-anchored DiI that was not affected by filipin. This suggests co-labeling of cholesterol-independent lipid domains that are also influenced by the cytoskeleton (Fig. 7). Alternatively, the cytoskeleton may produce a global effect that increases lipid ordering regardless of lipid phase.

Conditions that decreased co-clustering of the raft markers also decreased pTyr⁵⁰⁵ levels in Lck. Furthermore, we showed for the first time that, in some instances, conditions that disrupted rafts also caused activation of Lck as reported by an elevation of pTyr³⁹⁴. We interpret these findings as evidence that cyoskeletal rafts are important for maintaining signal quiescence in Lck. Detergent fractionation studies show evidence that Csk co-associates with Lck in rafts and that rafts sequester Lck from CD45. Both of these properties would underlie inhibition of raft pools of Lck, first by phosphorlyation of Tyr⁵⁰⁵ by Csk and then sequestration of the resulting pTyr⁵⁰⁵ from CD45. Accordingly, disruption of rafts is predicted to elevate interactions between Lck and CD45 while decreasing its interactions with Csk. This would account for the observed decrease in Lck pTyr⁵⁰⁵ content in cells treated with Lat B, neomycin, and blebbistatin. The corresponding increase in pTyr³⁹⁴ that was observed in most conditions could occur by activation of Lck from release of its autoinhibition by pTyr⁵⁰⁵ dephosphorylation. Other proteins likely participate in the down-regulation of raft Lck because blebbistatin increased pTyr³⁹⁴ in CD45-deficient J45.01 cells. One candidate is PEP (PEST domain-enriched tyrosine phosphatase), which associates with Csk to inhibit SFK signaling in T cells (53). Interactions between Lck and PEP via Csk may therefore be disrupted by blebbistatin. Altogether, further study is necessary to identify these candidates and the role of rafts in regulating their interactions with Lck.

In summary, we measured FRET between lipophilic carbocyanine dyes to assess lipid heterogeneities occurring in the plasma membrane of T cells. Our data show a cholesterol-dependent lipid ordering that is sensitive to disruption of F-actin and inhibition of either NM II or PIP₂. Measuring tyrosine phosphorylation of Lck, we showed that conditions that disrupt lipid domains formed by the cytoskeleton also cause deregulation of Lck. Altogether, these data show cholesterol-dependent rafts that occur as a result of interactions between the cytoskeleton and plasma membrane and that contribute to Lck regulation.