The Integrin Reconstruction Act: reconsolidating, towards the synthetic recreation of integrin-based focal adhesions

I just delayed my lunch for complete reading of this paper!

Very very admiring / starving …

J Cell Biol. 2010 Jan 5. [Epub ahead of print]

Recreation of the terminal events in physiological integrin activation.

Ye F, Hu G, Taylor D, Ratnikov B, Bobkov AA, McLean MA, Sligar SG, Taylor KA, Ginsberg MH.

Department of Medicine, University of California, San Diego, La Jolla, CA 92093.

Increased affinity of integrins for the extracellular matrix (activation) regulates cell adhesion and migration, extracellular matrix assembly, and mechanotransduction. Major uncertainties concern the sufficiency of talin for activation, whether conformational change without clustering leads to activation, and whether mechanical force is required for molecular extension. Here, we reconstructed physiological integrin activation in vitro and used cellular, biochemical, biophysical, and ultrastructural analyses to show that talin binding is sufficient to activate integrin alphaIIbbeta3. Furthermore, we synthesized nanodiscs, each bearing a single lipid-embedded integrin, and used them to show that talin activates unclustered integrins leading to molecular extension in the absence of force or other membrane proteins. Thus, we provide the first proof that talin binding is sufficient to activate and extend membrane-embedded integrin alphaIIbbeta3, thereby resolving numerous controversies and enabling molecular analysis of reconstructed integrin signaling.

PMID: 20048261

We are human because we are making tools to achieve goals!

Purification of inactive integrin IIbβ3

Integrin IIbβ3 was purified from outdated human platelets based on a modified protocol from Ye et al. (2008).

Purification of recombinant talin head

The recombinant human talin head expression plasmid construct was obtained from Dr. Steve Lam (Patil et al., 1999).

Liposome reconstitution and purification

Integrin–talin head liposomes were made with a protocol modified from Ye et al. (2008). In brief, 450 nmol 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 450 nmol 1,2-dimyristoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DMPG), and 200 nmol cholesterol were solubilized in chloroform or a chloroform/methanol mixture, mixed thoroughly, and dried onto a glass tube under steady flow of nitrogen. The homogeneous lipid mixture was then solubilized with 20 mM Tris, 150 mM NaCl, pH 7.4, 0.1% Triton X-100, and 1 mM β-mercaptoethanol. 0.2 mg of IIbβ3 with or without 0.5 mg of talin head construct in the same buffer was added to the lipid solution. The final volume of the entire mixture was 1 ml. To induce liposome formation, Triton X-100 in the solution was removed by several additions (in 3-h intervals) of 85 mg SM-2 Biobeads prewashed with methanol and water until the solution became visibly cloudy. The reconstituted liposomes were purified on a sucrose gradient to separate the free proteins from the reconstituted protein-liposome. The visible liposome band was extracted and dialyzed against three changes of 20 mM Tris and 150 mM NaCl, pH 7.4. Successful incorporations of talin head and integrins were verified by SDS-PAGE and Cryo-EM tomography.

Recombinant talin F3 expression and liposome cosedimentation assay

Talin F3 and F3(K322D) were expressed and purified as described previously (Wegener et al., 2007). In brief, GST-F3 or GST-F3(K322D) was expressed with pGEX-6P vector and purified with glutathione-conjugated agarose beads. F3 or F3(K322D) was then cleaved off from the GST using PreScission protease (GE Healthcare), and further purified with a Superdex 200 size exclusion column. The molecular weights of purified F3 or F3 mutant measured by matrix-assisted laser desorption mass spectrometry varied by less than 0.1% from calculated values. For the liposome cosedimentation assay, liposomes were prepared as described above except that no integrins were incorporated. 1 ml of 5 µg/ml purified F3 or F3 (K322D) was incubated with 100 µl of liposomes at room temperature for 1 h. The liposomes were centrifuged at 14,000 rpm for 30 min. The liposome pellets were washed and solubilized with 1x SDS-PAGE loading buffer and analyzed by SDS-PAGE. Coomassie-stained protein bands were scanned and quantified using an infrared fluorescence spectrometer (LI-COR Biosciences).

Liposome integrin activity assays

PAC1 binding was measured by fluorescence-activated cell sorting (FACS). For the analysis, 9 µl of purified liposomes were mixed with 6 µl of 25 µg/ml FITC-conjugated PAC1. After incubation for 30 min, the sample was diluted into 150 µl 20 mM Hepes and 150 mM NaCl, pH 7.4 (HBS buffer) and analyzed using FACSCalibur (BD). For each sample, separate FACS analysis was performed with addition of 1 mM MnCl2 as a control for full activation and MnCl2 plus 20 µM eptifibatide as a negative control. Because larger liposomes would have more integrin incorporated and thus exhibit higher PAC1 binding, we plotted PAC1 binding against liposome size using forward scattering (FSC) as a size measurement. This plot helped us compare the PAC1 binding of liposomes with the same size. The PAC1-FSC plots were further analyzed by dividing the liposomes into 11 subsets along the FSC axis. The mean FSC and mean fluorescent intensity (MFI) of PAC1 binding was calculated for each subset. The activation indexes were calculated as 100 x (F – F0) / (Fmax – F0), where F = PAC1 binding, F0 = binding in the presence of 20 µM eptifibatide, and Fmax = binding in the presence of 1 mM MnCl2. The PAC1 MFI or the activation index was then plotted against the FSC.

Assaying the effect of THD in CHO cells stably expressing IIbβ3

Using a lentiviral cloning vector pRRLSIN.cPPT.PGK-IRES-GFP.WPRE. (Addgene, plasmid ID 12252), viruses containing IIb or β3 gene were generated separately as described previously (Wiznerowicz and Trono, 2003). CHO cells stably expressing integrin IIbβ3 were established by co-infection of CHO K1 cells with the IIb and β3 viruses. The Y747A and Y759A mutations were introduced into the β3 gene with the QuikChange Mutagenesis kit. To assess the effects of THD on the activation of integrins, 3 µg THD expression construct and 0.1 µg D-tomato (a transfection marker) was cotransfected. After 24 h, the cells were trypsinized, stained with PAC1 for 30 min, washed, stained with APC-conjugated anti–mouse IgM, and then analyzed by FACS. Separate FACS was also performed in the presence of anti-LIBS6 as full activation control and in presence 20 µM eptifibatide as negative control. To assess the effect of kindlin-2 on those cells, 3 µg THD expression construct, 0.5 µg of kindlin-2 construct, and 0.1 µg D-tomato construct were cotransfected into the cells, and the cells were stained as described above and analyzed by FACS.

Assay for calpain cleavage

To cleave β3 tail from the purified IIbβ3, 25 µl of 1 mg/ml recombinant calpain-II (EMD) was added to 500 µl of 2 mg/ml purified IIbβ3 in a buffer of TBS plus 0.1% Triton X-100, 1 mM CaCl2, and 1 mM MgCl2. The mixture was incubated at room temperature overnight. The next morning, the calpain was neutralized by adding protease inhibitor E-64 (Sigma-Aldrich) at a final concentration of 10 µM plus calpain inhibitor calpeptin at a final concentration of 5 µM. An ELISA assay was developed to measure the effectiveness of the cleavage. In brief, ELISA plates were coated with 5 µg/ml AP3 antibody overnight at 4°C, blocked with BSA for 1 h at 37°C, and incubated with 6 µg/ml of cleaved or uncleaved IIbβ3 for 1 h at room temperature. After removing the protein solution and washing the ELISA plates, either Ab8053 against the whole protein, Ab8276 against the IIb, or Ab8275 against the β3 tail were added to the captured IIbβ3. After 1 h of incubation, the unbound antibodies were removed, the wells were washed again, and horseradish peroxidase (HRP)–conjugated goat anti–rabbit Ig antibodies were added for another hour of room temperature incubation. The amount of antibody binding was measured using enhanced chemiluminescence (ECL) reagent as peroxidase substrate (BD) and read on a Victor2 plate reader (PerkinElmer).

Integrin activity assay

For the integrin activity assay, ELISA plates were coated with 5 µg/ml AP3 antibody overnight at 4°C, blocked with BSA for 1 h at 37°C. After washing the plate, 6 µg/ml integrin with different amounts of talin head were added on to the plate. The plate was incubated for 2.5 h at room temperature. The wells were then washed and detected with either PAC1 antibody to measure the activity or anti-β3 Ab8053 to measure the capture. 1 mM MnCl2 and 1 mM MnCl2 plus 20 µM eptifibatide were used as positive and negative controls. After 1 h of incubation, the wells were again washed and HRP-conjugated anti–mouse IgM (for PAC1 wells) or HRP-conjugated anti–rabbit Igs (for Ab8053 wells) were added for 1 more hour of incubation. After the final wash, ECL reagent was added to the wells and the plate was read on a Victor2 plate reader (PerkinElmer). The procedure for the alternative activity assay configuration was similar to that described above, except that the plates were coated with 25 µg/ml of PAC1 antibody and that anti-β3 Ab8053 followed by HRP-conjugated anti–rabbit antibody was used to detect the amount of bound integrin.

Integrin nanodiscs assembly and purification

Integrin nanodiscs were assembled based on a protocol adapted from previous papers (Denisov et al., 2004 PMID: 15025475; Nath et al., 2007 PMID: 17263563). In brief, DMPC and DMPG were solubilized in chloroform or a chloroform/methanol mixture, mixed thoroughly, and dried onto a glass tube under steady flow of nitrogen. The homogeneous lipid mixture was then solubilized in 100 mM cholate in 10 mM Tris and 100 mM NaCl, pH 7.4, giving a lipid concentration of 50 mM. 72 µl of the lipid solution was then mixed with 200 µl of 200 µM membrane scaffold protein (MSP) in dH2O and 200 µl of 10 µM purified inactive integrin (described earlier). The final ratio of lipids/MSP/protein is 90:1:0.05 in a total volume of 472 µl. The integrin nanodiscs were assembled by removing the detergents with SM-2 Biobeads overnight at room temperature. The assembled integrin nanodiscs were then purified with a hi-load 16/60 Superdex 200 size exclusion column with 20 mM Tris, 150 mM NaCl, and 0.5 mM CaCl2, pH 7.4, as the column buffer. The integrin nanodiscs and empty nanodiscs were readily separated (Fig. S2) and the successful assembly was verified by SDS-PAGE analysis. Two MSP constructs, MSP1D1 and MSP1E3D1 (Denisov et al., 2004) expressed and purified from bacteria, were used to make integrin nanodiscs and similar patterns of integrin activation results were obtained with both constructs.

Analytical ultracentrifugation

Analytical ultracentrifugation was performed with a ProteomeLab (model XL-I) centrifuge according to the instructions from Beckman Coulter and established theories (Lebowitz et al., 2002).

Integrin nanodisc activation assay

Integrin nanodisc activation assays were performed in a similar manner to that used to study the detergent-solubilized integrin as described above. In brief, ELISA plates were coated with 5 µg/ml AP3 antibody overnight at 4°C, blocked with BSA for 1 h at 37°C. After washing the plate, integrin nanodiscs were added to the plate. The plate was incubated for 3 h at room temperature. The integrin nanodiscs were then activated by various concentrations of THD as indicated, and activation was detected by binding of PAC1 antibody. PAC1 binding in the presence of anti-LIBS6 antibody was used as control for full activation and PAC1 binding in the presence of 20 µM eptifibatide was used as negative control. After 3 h of incubation, the wells were again washed and HRP-conjugated anti–mouse IgM (for PAC1 wells) was added for 1 more hour of incubation. After the final wash, ECL reagent was added to the wells and the luminescence of each well was read on a Victor2 plate reader. The activation indices were calculated as 100 x (L – L0) / (Lmax – L0), where L = luminescence intensity, L0 = luminescence in presence of 1 µM eptifibatide, and Lmax = luminescence in the presence of anti-LIBS6 antibody. Increase in activation was calculated as AIwith-THD – AIintegrin-alone, where AI stands for activation indices.

Integrin nanodiscs binding to fibrin and negatively stained EM images of integrin nanodiscs

2D fibrin was prepared by lipid monolayer technique as described previously (Taylor and Taylor, 1999; Taylor et al., 2007). In brief, wells in a Teflon block were filled with a fibrinogen solution. Thrombin was added to each well and a positively charged lipid solution was immediately applied to the solution surface to form the lipid monolayer. The samples were incubated at room temperature for 1 h while fibrin polymerized, and were then stored at 4°C. The fibrin was imaged to verify its 2D integrity and appropriate concentration. To test their binding to fibrin, the integrin nanodiscs were first incubated (with a THD concentration of either 0 or 5 µM) for 2 h then injected into the fibrin sample well where the final buffer concentration was 20 mM Tris, 50 mM NaCl, and 0.2 mM CalCl2, pH 7.6, and incubated for 24 h at 4°C. The resulting lipid monolayer sample was lifted on a reticulated carbon film and stained with 2% uranyl acetate. Images of the fibrin with integrin nanodiscs were taken on a transmission electron microscope (model 1200EX; JEOL) at a magnification of 65,000. The images were recorded on Kodak film and were digitized with a film scanner (Coolscan 9000; Nikon) to give a final pixel size of 3.9Å. The numbers of bound integrin nanodiscs and of unbound integrin nanodiscs were counted manually. The length of the fibrin was measured in ImageJ by tracing the fibrin fiber with segmented line.

Cryoelectron tomography

Specimens were prepared on glow-discharged, 200-mesh copper grids with reticulated carbon support film with a 2-µm hole size (Quantifoil, Inc.). The grids were plunge-frozen in liquid ethane and examined at a temperature of –180°C. The data were collected at a magnification of 24,000 on an electron microscope (model CM300-FEG; FEI) (integrin liposomes) and at a magnification of 31,000 on an electron microscope (Polara; FEI) (integrin + THD liposomes), each equipped with a 4 x 4-k CCD camera. The tomography tilt series was collected at an underfocus of 6–8 µm. The tilt range covered –65° to 65° with Saxton scheme angle increments with an initial angle of 2.0° and 1.5°, respectively (Saxton et al., 1984). The raw tilt images were processed and aligned with a marker-free alignment algorithm and the 3D reconstruction was calculated by weighted back projection (Winkler and Taylor, 2006).

Particle selection and 2D averaging

All images processing were performed with the EMAN1.9 package (Ludtke et al., 1999). Integrin nanodisc molecular images were identified in images. Images of well-resolved integrin nanodiscs not bound to fibrin were used for further image processing. In total, we obtained 1,283 images for integrin nanodiscs alone and 1,553 images for integrin nanodiscs in the presence of THD. The extracted molecular images were normalized, contrast transfer function (CTF) corrected by phase flipping, and low-pass filtered to 10Å resolution. The images were then subjected to an iterative classification, alignment, and averaging algorithm. No initial model or references were used and the data robustly converged to a final set of class averages. Minimum number of particles per class was set to be 20. Typical command used for 2D average and classification is: “refine2d.py–iter=41–ninitcls=10–finalsep=2–nbasis=64–minptcl=20″. The integrin nanodiscs activated by THD separated into class averages of either compact or extended conformations. However, due to the smaller number and the lower percentage of extended conformation in the sample of integrin nanodiscs alone, the algorithm failed to generate any classes attributable to the extended conformation despite repeated efforts with various parameter settings. The total numbers of extended or compact integrin nanodiscs were obtained by summing the number of images in the classes showing extended or compact conformation. The class averages showing only nanodisc density were treated as contaminating empty nanodiscs and their numbers were not included the activity calculation.

Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200908045/DC1.