Talin recruitment to αIIbβ3 can be visualized in living cells by BiFC. (A) Principle of BiFC. The top panel depicts the two fusion proteins, αIIb-VC and VN-talin, used for BiFC. Also depicted are wild-type β3 and β3 (Δ724), which were cotransfected into CHO cells with αIIb-VC to enable the expression of αIIb-VCβ3 and αIIb-VCβ3 (Δ724), respectively. TM, transmembrane domain. The bottom panel illustrates the concept of BiFC when VN-talin successfully interacts with αIIb-VCβ3 as the result of inside-out signaling. See Results for further discussion. (B) Flow cytometry with antibody D57 was used to determine αIIbβ3 surface expression in cells stably transfected with αIIbβ3, αIIb-VCβ3, or αIIb-VCβ3 (Δ724). Parental CHO cells were used as a negative control. Expression of the chimeric integrins was equivalent to that of wild-type αIIbβ3. (C) Western blotting illustrates the molecular sizes of the αIIb and β3 subunits obtained from cells shown in B. (D) VN-talin complements Venus fluorescence better than talin-VC. The N-terminal half of Venus was fused to either the talin N terminus (VN-talin) or the talin C terminus (talin-VN) and transiently transfected into αIIb-VCβ3 cells. BiFC was then measured by flow cytometry. The same experiment was performed in parallel with αIIb-VCβ3 (Δ724) cells, which lack a major talin interaction site in β3. Data are means ± SEM (error bars) of three experiments. (E) αIIb-VCβ3–CHO cells and αIIb-VCβ3 (Δ724)–CHO cells were cotransfected with VN-talin and Rap1a (V12). 48 h later, cells were plated on fibrinogen for 60 min, fixed, and analyzed by deconvolution microscopy. Note that in the αIIb-VCβ3 cells, BiFC fluorescence (green) was colocalized with vinculin (red) to lamellipodial edges (arrow), focal complexes (arrowheads), and focal adhesions (double arrowhead). Only minor colocalization was noted in the αIIb-VCβ3 (Δ724) cells. Bars, 10 μm.

Structural requirements in talin and β3 for talin recruitment and inside-out αIIbβ3 activation in living cells. (A) Effects of talin mutation on BiFC and PAC-1 binding. VN-talin (either wild-type, L325R, or W359A talin) was cotransfected with Rap1a (V12) into CHO cells expressing αIIb-VCβ3 or αIIb-VCβ3 (Δ724). As a control, cells were transfected with Rap1a (V12) but not VN-talin (white bars). 48 h later, BiFC fluorescence, PAC-1 binding, and talin expression in transfected cells were quantified by flow cytometry as described in Materials and methods. Responses are expressed as fold increase compared with cells not transfected with VN-talin. (B) Effects of β3 mutation on BiFC and PAC-1 binding. VN-talin was cotransfected with Rap1a (V12) into CHO cells expressing αIIb-VCβ3, αIIb-VCβ3 (Y747A), or αIIb-VCβ3 (Δ724). As a control, cells were transfected with empty vector (mock). 48 h later, BiFC fluorescence, PAC-1 binding, and talin expression in transfected cells were quantified by flow cytometry. Responses are expressed as fold increase compared with mock controls. (A and B) Data are means ± SEM (error bars) of four experiments.

RIAM and lamellipodin promote talin recruitment and αIIbβ3 activation. (A) αIIb-VCβ3 CHO cells were engineered to conditionally express VN-talin under the control of a tetracycline-regulated promoter as described in Materials and methods. Cells were then incubated for 24 h in the absence (Dox (−)) or presence (Dox (+)) of 0.75 ng/ml doxycycline, a concentration that induced approximately half-maximal expression of VN-talin. Concomitantly, cells were transiently transfected as indicated with empty vector (mock) or vectors for Rap1a (V12), RIAM, or lamellipodin (Lpd). Then, BiFC and PAC-1 binding were analyzed and expressed as fold increase compared with Dox (−) mock controls. Data represent means ± SEM (error bars) of four experiments. Double asterisks indicate means that were significantly different from the Dox (+) mock control at P ≤ 0.01. (B) Protein expression was analyzed in cell lysates by Western blotting. Immunoblotting for β-actin was used to assess gel loading. A representative Western blot is shown.

Knockdown of RIAM but not lamellipodin blocks talin recruitment and αIIbβ3 activation induced by Rap1a (V12). αIIbβ3-CHO cells were transduced with lentivirus encoding RIAM-specific shRNA, lamellipodin-specific shRNA (Lpd), or an irrelevant control (CTRL) shRNA. 48 h later, some cells were transfected with Rap1a (V12) as indicated, and, 24 h after that, 0.75 ng/ml doxycycline was added to induce VN-talin expression. (A) Protein expression was analyzed in cell lysates by Western blotting. (B) BiFC and PAC-1 binding were measured and expressed as fold increase compared with noninduced cells (no VN-talin). Data are means ± SEM (error bars) of three experiments.

Effect of RIAM or lamellipodin knockdown on PAR1-mediated αIIbβ3 activation in CHO cells. (A) αIIbβ3-CHO cells were engineered to conditionally express PAR1 and talin under the control of a tetracycline-regulated promoter. Cells were then transduced with lentivirus encoding RIAM shRNA, lamellipodin (Lpd) shRNA, or control shRNA (CTRL). After PAR1 and talin induction for 24 h with doxycycline, induced (Dox (+)) and noninduced (Dox (−)) cells were incubated for 10 min at room temperature with PAC-1 in the absence or presence of a subsaturating (0.5 μM) or saturating (50 μM) concentration of PAR1 agonist peptide, SFLLRN. Then, specific PAC-1 binding to the lentiviral-transduced cells was determined. PAC-1 binding is expressed as fold increase compared with unstimulated Dox (−) cells transduced with control shRNA. Data are means ± SEM (error bars) of three experiments. (B) Protein expression was analyzed by Western blotting. (C) Flow cytometry histograms depicting surface expression of recombinant PAR1 before (open histograms) and after (closed histograms) doxycycline induction detected with an antibody specific for the Flag epitope.

Localization and function of RIAM in platelets and megakaryocytes. (A) RIAM localization in platelets. Washed human platelets were plated on fibrinogen for up to 45 min. Cells in a–c were stained with antibodies to RIAM (red) and vinculin (green). Cells in d–f were stained for RIAM (green), vinculin (blue), and F-actin (red). Platelets were imaged by deconvolution microscopy. Each panel represents a platelet representative of the range of platelets observed. As spreading proceeded, RIAM was prominent within vinculin-rich filopodia (arrows), lamellipodial edges (arrowhead), and focal adhesion-like structures (double arrowhead). Bar, 10 μm. (B–D) RIAM is required for agonist-induced αIIbβ3 activation in primary megakaryocytes. Murine megakaryocytes derived from embryonic stem cells were infected with lentivirus encoding control (CTRL) or RIAM shRNA. 7 d later, megakaryocytes were evaluated for RIAM expression in cell lysates by Western blotting (B), αIIbβ3 surface expression by flow cytometry using antibody D57 (closed histograms) and an IgG control (open histograms; C), and specific fibrinogen binding by flow cytometry (D). Megakaryocytes were either unstimulated (no agonist) or stimulated for 30 min at room temperature with 1 mM PAR4 agonist peptide (AYPGFK) or a combination of AYPGFK, 50 μM ADP, and 50 μM epinephrine. Fibrinogen binding is expressed as fold increase compared with unstimulated megakaryocytes transduced with control shRNA. Data are means ± SEM (error bars) of three experiments.