(a) Schematic representation of the full-length Fyb/SLAP2, the EST clone (IMAGE clone ID 221953; RZPD IMAGp998F02441), the murine clone 5/7, and the three GST fusion proteins encoding for different regions of Fyb/SLAP2 (Fyb/SLAP I, III, and IX). Potential EVH1 domain–binding sites, SH3-like domain, and the 138-bp insertion are also shown. (b) RT-PCR of the COOH terminus of Fyb/SLAP as obtained from HL60 cells shows two bands running at ∼700 and 840 bp (lanes 1–3, RT-PCRs of three independent experiments; lane 4, 100 bp marker). (c) Immunodetection of Fyb/SLAP in extracts of human platelets and Jurkat T cells. The affinity-purified polyclonal antibody #51 specifically reacts with a single band on Jurkat T cells lysate and with two bands on platelet extract, whereas the polyclonal antibody #81 reacts with a single band on platelet extract. (d) Amino acid sequence and structural motifs of Fyb/SLAP2. Single letter amino acids sequence of Fyb/SLAP2. Boxes represent potential EVH1 domain–binding sites, stretch of bold-faced amino acids represents the insertion not present in Fyb/SLAP1. Potential SH3-binding regions (underlined), potential tyrosine phosphorylation sites (bold and boxed), and the SH3-like domain (dotted) are shown. The sequence of Fyb/SLAP2 is available from EMBL/GenBank/DDBJ under accession no. AF198052.

(a) Alignment of protein sequences of the NH₂ terminus of ActA (from L. monocytogenes), iActA (from L. ivanovii), and Fyb/SLAP. Identical amino acids are indicated with bold letters, homologous amino acids are underlined. Amino acids are numbered according to Domann et al. 1992 for ActA and to Kreft et al. 1995 for iActA. (b) Alignment of the G-actin binding sites of thymosin β4, villin, dematin, the potential G-actin binding site of Mena, and Fyb/SLAP. Amino acids involved in the binding to G-actin are indicated with bold letters. Homologous amino acids are underlined.

(a) Blot overlay assay of in vitro translated ³⁵S-labeled VASP on GST fusion proteins of Fyb/SLAP and the murine clone 5/7. (lane 1) GST-ActA proline-rich repeats (positive control), (lane 2) Fyb/SLAP I GST, (lane 3) Fyb/SLAP III GST, (lane 4) Fyb/SLAP IX GST, and (lane 5) murine clone 5/7 GST. Note that bands representing degradation products are visible in lanes 1, 4, and 5. (b) Peptide spot analysis of the full-length Fyb/SLAP1 and of the COOH terminus of Fyb/SLAP2. Peptides were synthesized on a cellulose membrane and incubated with ³⁵S-labeled VASP. Positive spots were detected with a PhosphorImager. (c) Single letter amino acid sequences of the peptides corresponding to the positive spots shown in b. (d) Coimmunoprecipitation of Fyb/SLAP and VASP from a lysate of Jurkat T cells. Extracts of activated Jurkat T cells were incubated with mAbs against VASP. Immune complexes were collected using anti–mouse IgG agarose. Bound proteins were separated by SDS-PAGE, blotted, and probed with the affinity-purified antibody #51 to Fyb/SLAP. (lane 1) Lysate of activated Jurkat T cells (positive control), (lane 2) proteins immunoprecipitated using the mAb (358C1) raised against a bacterial protein, and (lane 3) proteins immunoprecipitated using anti-VASP specific mAbs. Molecular weight markers are indicated on the left in a and d.

Fluorescence micrographs showing the localization of Fyb/SLAP and cytoskeletal proteins in human platelets. Platelets were allowed to attach and spread on glass coverslips, fixed, and double stained for Fyb/SLAP (a–d), actin (a′), VASP (b′), zyxin (c′), or vinculin (d′). (b′′) Merged picture of Fyb/SLAP (b) and VASP (b′). Bars, 5 μm.

Distribution of Fyb/SLAP in activated T cells. Jurkat T cells were incubated with anti-CD3–coated beads (a′, asterisk) and allowed to settle on glass coverslips. T cells were then double stained for F-actin (a–g), Fyb/SLAP (a′), Evl (b′), WASP (c′), Arp3 (d′), Vav (e′), vinculin (f′), or zyxin (g′). Fyb/SLAP is enriched at the interface between the activated T cell and the anti-CD3–coated bead (a′, arrow). F-actin also colocalizes with Evl, WASP, Arp3, and Vav (arrows in b′, c′, d′, and e′ respectively). In contrast, vinculin and zyxin do not accumulate at the interface between T cells and beads (e′ and f′). Note that beads are visible in the panels e′–g′ due to the binding of the secondary goat anti–mouse antibody to the mAbs against CD3 used to coat the beads. Bar, 5 μm.

(a–b′) Displacement of Ena/VASP family proteins by the proline-rich repeats of ActA. HeLa cells were transfected with GFP-ActA repeats (a and a′) or with the mutant GFP-ActA repeats F > A (b and b′), fixed, and labeled with antibodies to VASP (a and b, VASP labeling; a′ and b′, GFP signal). In untransfected cells, VASP localizes to focal adhesions (a, arrows). In cells expressing the GFP-ActA repeats (a, asterisks), VASP is displaced from the focal adhesions. In contrast, cells transfected with the mutated ActA repeats (for instance, asterisks in b) show a normal VASP localization (b, arrows). (c–h′) Inhibition of F-actin accumulation at the interface between Jurkat T cells and anti-CD3–coated beads. Jurkat T cells were transfected with GFP-ActA repeats (c and c′) or with the mutant GFP-ActA repeats F > A (d and d′). Note the lack of actin accumulation at the T cell–bead interface and the reduced zone of contact between T cell and bead (c, arrow). Such actin accumulation is not affected in Jurkat T cells that were transfected with the mutated ActA repeats (d, arrow; c and d, fluorescent phalloidin; c′ and d′, GFP signal). (e–h′) The localization of Evl but not that of Fyb/SLAP, WASP, and the Arp2/3 complex at the T cell–bead interface is affected by the proline-rich repeats of ActA. Jurkat T cells were transfected with GFP-ActA repeats, incubated with anti-CD3–coated beads, fixed, and labeled with antibodies to Evl (e), Fyb/SLAP (f), WASP (g), and Arp3 (h) (e′–h′, GFP signal). In cells expressing the GFP-ActA repeats, Evl is displaced from the T cell–bead contact sites (e, arrow). In contrast, the localization of Fyb/SLAP, WASP, and the Arp2/3 complex is unaffected. Bars: 10 μm (a–b′); and 5 μm (c–h′).

Scanning EM analysis of the interaction between Jurkat T cell and anti-CD3–coated beads. Untransfected Jurkat T cells (a), T cells transfected with GFP-ActA repeats (b), and T cells transfected with GFP-ActA repeats F > A (c) were incubated with anti-CD3–coated beads, fixed, and processed for scanning EM. Jurkat T cells, which expressed GFP-ActA repeats, formed a very loose contact with the bead. In contrast, control T cells or cells which expressed the mutated GFP-ActA repeats extended sheet-like protrusions that almost completely surrounded the beads. Bar, 5 μm.

(a–b′) Delocalization of the Arp2/3 complex by the COOH terminus of Scar1 inhibits the actin reorganization at the T cell–bead interface. Jurkat T cells were transfected with myc-tagged ScarWA (a and a′) or ScarW (b and b′), incubated with anti-CD3–coated beads, fixed, and stained with fluorescent phalloidin and anti-myc antibodies. In Jurkat T cells, which expressed ScarWA, the actin accumulation at the T cell–bead interface is impaired (a, arrow), whereas T cells transfected with ScarW show a normal actin rearrangement (b, arrow; a and b, fluorescent phalloidin; a′ and b′, myc tag labeling). (c–f′) In T cells expressing ScarWA, Evl, Fyb/SLAP, and WASP accumulated at the T cell–bead interface. Jurkat T cells were transfected with myc-tagged ScarWA, incubated with anti-CD3–coated beads, fixed, and stained with antibodies to Evl (c), Fyb/SLAP (d), WASP (e), Arp3 (f), and myc tag (c′–f′). Note that the Arp2/3 complex did not localize at the T cell–bead interface. Bar, 5 μm.

(a) Fyb/SLAP coimmunoprecipitates with WASP. Lysates of activated Jurkat T cells were incubated with the WASP-specific antiserum Fus3. Immune complexes were collected using protein A–agarose. Bound proteins were resolved by SDS-PAGE, blotted, and probed with polyclonal antibodies #51 to Fyb/SLAP. (lane 1) Cell extract of activated Jurkat T cells; (lane 2) immunoprecipitation with a nonimmune serum; and (lane 3) immunoprecipitation using a WASP-specific antiserum. (b) Fyb/SLAP, SLP-76, Nck, and WASP form a multiprotein complex in Jurkat T cells. Lysates from nonstimulated (NST) or stimulated (ST) Jurkat T cells were immunoprecipitated using the WASP mAb #67B4, resolved by SDS-PAGE, blotted, and probed with antibodies to Fyb/SLAP, SLP-76, and Nck. As negative control, lysates were immunoprecipitated using an antibody to a bacterial protein. To exclude nonspecific trapping, WASP immunoprecipitated from lysates of activated T cells were probed using a zyxin mAb. (c) The stimulation of Jurkat T cells results in the association of additional tyrosine-phosphorylated proteins with the complex. WASP immunoprecipitates from lysates of nonstimulated (NST) or stimulated (ST) Jurkat T cells were resolved by SDS-PAGE and probed with an antibody to phosphotyrosine. Note the appearance of three additional bands in stimulated T cells. hc, heavy chain; lc, light chain. Molecular weight markers are indicated on the left in (a and b) and on the right in (c).

(a) Model describing intermolecular interactions and signal transduction pathways leading to the cytoskeletal remodeling and T cell activation upon TCR ligation (adapted from Koretzky 1997). Ena/VASP family proteins and WASP are linked through a molecular chain formed by Fyb/SLAP, SLP-76, and Nck (red highlighted proteins) to the T cell–bead interface (see text for further details). The green line in the Fyb/SLAP molecule indicates the EVH1 domain–binding site. (b) Schematic representation of the proteins involved in the actin-based motility of Listeria monocytogenes showing the recruitment of Ena/VASP family proteins and Arp2/3 complex to the bacterial surface protein ActA.