S₁₅-Inp54p-dependent filopodial growth is PI3K-dependent. A, representative images of T cells expressing S₁₅-Inp54p that were treated with wortmannin (Wm) or treated with wortmannin, washed, and allowed to recover in growth media for 2 h (Recovery). Scale bar = 5 μm. B, quantitation of the number of filopodia in each set of conditions. The data represent the average of 90 cells measured in three separate trials. ^***, p ≤ 0.005 by Student's t test.

T cells expressing S₁₅-Inp54p exhibit increased cell spreading in a PI3K-dependent manner. A, confocal (upper panels) and differential interference contrast (DIC; lower panels) images of T cells expressing L₁₀-Inp54p, S₁₅-Inp54p, or L₁₀-GFP. One set of cells expressing S₁₅-Inp54p was treated with wortmannin (Wm) before fixation. The arrowheads in the S₁₅-Inp54p sample indicate the edges of cell spreading. Scale bars = 5 μm. B, averaged results measuring the surface area of transfected Jurkat T cells seeded onto poly-l-lysine-coated coverslips. The data are from 60 cells measured in three independent trials using differential interference contrast imaging. ^***, p ≤ 0.005 by Student's t test; NS (not significant), p > 0.05.

Ca²⁺ flux in stimulated T cells is not altered by membrane-targeted Inp54p expression. A, intracellular Ca²⁺ flux following TCR stimulation of Jurkat T cells expressing L₁₀-Inp54p, S₁₅-Inp54p, or L₁₀-GFP. T cells labeled with Indo-1 were measured by flow cytometry and stimulated with OKT3 antibody (asterisks). Ionomycin addition is indicated by the black arrowheads. B, The total intracellular Ca²⁺ flux averaged from three independent trials. C, The store-operated Ca²⁺ flux was measured the same as (A), but cells were measured in buffer containing 0.05 μm EGTA to discriminate between flux from intracellular stores and flux from store-operated channels. Ca²⁺ indicates the time point when Ca²⁺ was added. D, The Ca²⁺ flux from store-operated channels averaged from three separate trials. In B and (D), NS, p > 0.05 by Student's t test.

Cell morphologies from expression of the membrane-targeted Inp54p molecules. A, projection images of Jurkat T cells that expressed L₁₀-Inp54p, S₁₅-Inp54p, or L₁₀-GFP. The images were acquired using confocal microscopy and detected using GFP fluorescence. The white and yellow arrowheads indicate filopodia and membrane ruffling, respectively. B, quantitation of filopodia in transfected T cells. Filopodia in cells that expressed L₁₀-Inp54p, S₁₅-Inp54p, L₁₀-GFP, or S₁₅-GFP were counted using wide-field microscopy. The graph represents the average of 90 cells measured in three independent trials. The error bars represent S.E. ^***, p ≤ 0.005 by Student's t test; N.S., not significant. C, representative example of the membrane blebs observed in cells that expressed L₁₀-Inp54p. The right panel is a higher magnification of the region indicated by the square in the left panel. D, confocal images of a T cell double-labeled with S₁₅-Inp54p and cholera toxin conjugated to Texas Red (CTX-TR). The arrowheads indicate examples of double-labeled filopodia. E, confocal images of Jurkat cells double-labeled with S₁₅-Inp54p and DiIC₁₆. The arrowheads indicate filopodia labeled with DiIC₁₆ but not S₁₅-Inp54p. Scale bars = 5 μm.

L₁₀-Inp54p inhibits actin capping in stimulated T cells. A, differential interference contrast (DIC) and confocal images of stimulated Jurkat T cells expressing L₁₀-Inp54p, S₁₅-Inp54p, L₁₀-GFP, or L₁₀-GFP treated with neomycin (Neo). Following stimulation, the samples were stained with Texas Red-conjugated phalloidin to detect F-actin. The fluorescence intensities are represented by pseudocolor, and the color assignment for intensity values is shown at the bottom. The asterisks indicate the positions of the antibody-coated beads. The arrows indicate actin-enriched membrane caps at the cell-bead interface. B, histograms of the relative enrichment of F-actin at the bead-cell interface measured in ∼100 bead-cell conjugates. C, quantitation of the frequency of F-actin capping of each sample measured using confocal microscopy. A cell was scored as positive for capping when the mean fluorescence intensity of the bead-cell interface divided by the mean fluorescence intensity of the rest of the cell was 1.5 or greater. Percent cells positive for capping for each sample is graphed. The results were averaged from 90 separate bead-cell conjugates measured in three separate trials. ^**, p ≤ 0.01 by Student's t test; ^***, p ≤ 0.005; NS (not significant), p > 0.05.

Membrane-targeted Inp54p molecules cause distinct changes in raft and nonraft PIP₂. A, targeting of the PIP₂-specific phosphatase Inp54p to raft and nonraft membrane fractions. The constructs contained either the first 10 amino acids of Lck (L₁₀) or the first 15 amino acids of c-Src (S₁₅) for membrane association, followed by GFP and finally the soluble domain of Inp54p (L₁₀-Inp54p and S₁₅-Inp54p, respectively). L₁₀ targets proteins to the detergent-resistant raft fraction, and S₁₅ restricts proteins to the detergent-soluble nonraft fraction (21). Myr and Palm indicate sites of myristoylation and palmitoylation, respectively, of the indicated residues in the membrane-anchoring signals. A third construct consisting of membrane-anchored GFP (L₁₀-GFP) was used as a control. B, measurement of changes in membrane PIP₂ pools by L₁₀-Inp54p and S₁₅-Inp54p (upper panel). 293T cells expressing either L₁₀-Inp54p or S₁₅-Inp54p were lysed with Triton X-100, and the raft and nonraft membrane fractions were separated by sucrose gradient equilibrium centrifugation. Following separation, the gradient fractions containing the respective membrane fractions were pooled and extracted, and PIP₂ was measured in each by immunoblotting. PIP₃, PIP₂, and phosphatidylinositol 4-phosphate (PI4P) represent purified lipids that were measured in parallel as controls for antibody specificity. Immunoblotting a range of the PIP₂ standards together with a set of samples from cells that expressed S₁₅-Inp54p showed that PIP₂ extracted from cells was within the dynamic range of the measure (lower panel). In the accompanying plot, values for the S₁₅-Inp54p raft and nonraft fractions are represented by open circles. A.U. denotes arbitrary units. C, quantitation of the immunoblot in B. D, measurement of membrane PIP₂ pools for cells expressing L₁₀-GFP and S₁₅-GFP as a control for any sequestering of PIP₂ by the S₁₅ sequence. Quantitation of the dots is shown in the graph below. E, average of measurements from three separate trials. These results are represented as the fraction of total PIP₂ that was raft-associated. F, total lipids measured for PIP₂.In E and F, the error bars represent S.E. ^*, p ≤ 0.05 by Student's t test; NS (not significant), p > 0.38.