Figure 5.  Fyn and PTP-PEST modulate WASp effects on induction of actin polymerization and synapse formation. (A) Polymerization of 2.8 μM pyrene-labeled actin monomer was assayed in the presence of 20 nM Arp2/3 complex, 100 nM GST-WASp or GST-WASpΔGBD, and GST fusion proteins containing 50–500 nM either Fyn, PTP-PEST, PSTPIP, or cdc42-V12. Polymerization was monitored by the increase in prenyl-actin fluorescence. B. The pyrene actin assay was used to compare the WASp-Arp2/3–actin polymerizing activities of cdc42 at low (15 nM), medium (250 nM), or high (500 nM) concentration and Fyn at low (10 nM), medium (100 nM), or high (200 nM) concentration alone or in combination. (C) Fyn effects on synapse formation were evaluated by incubating lymphocytes from OT-II (a and b) and Fyn−/−/OT-II (c) mice with unpulsed (a) or OVA peptide–pulsed (b and c) LB27.4 cells followed by cell fixation, staining for WASp, Fyn, and actin, and visualization by immunofluorescent microscopy. Images on the far left of each panel represent a merge of the other three images within each panel. A computer-generated three-dimensional reconstruction of the synaptic region formed between wild-type T cells and APCs (d) shows the localization of Fyn in the central area of the synapse and the distribution of WASp in both the central and peripheral synaptic region. Synapses were quantified (e) by counting the numbers of T cell–APC conjugates showing clustered actin at the conjugation site. Values shown are the percent of conjugates with synapse formation and represent means (± SEM) of three independent experiments. (D) PTP-PEST effects on synapse formation were assessed using WAS−/−/OT-II lymphocytes transfected with pDSRED-WASp (a), pcDNA3-WASp and pDSRED-PSTPIP1 (b), or pcDNA3-WASp, pEGFP-PSTPIP1, and pDSRED-PTP-PEST (c). Cells were incubated with OVA peptide–pulsed LB27.4 B cells, fixed, and stained for actin and/or PKC-θ, and visualized by immunofluorescent microscopy. The image on the far right of each panel is a merged image of all other images in the panel. Synapses were quantified by counting the number of T cell–B cell conjugates showing clustered actin at the synaptic site. Values shown are the percent of conjugates with synapse formation and represent the means (± SEM) of three independent experiments.