Integrin alpha IIb beta 3 in a membrane environment remains the same height after Mn2+ activation when observed by cryoelectron tomography

Abstract

Integrins perform the critical function of signalling cell attachment to the extracellular matrix or to other cells. This signalling is done through a structural change propagated bidirectionally across the plasma membrane. Integrin activation has been extensively studied with ectodomain constructs, but the structural change within intact, membrane-bound molecules remains a subject of live debate. Using cryoelectron tomography, we examined the simplest predication of the different integrin activation models, i.e., the change in height of the molecules. Analysis using techniques that compensate for the missing wedge during alignment and averaging and that search for patterns in the structure of the aligned molecular subvolumes extracted from the tomogram reveals that the vast majority of molecules show no dramatic height change upon Mn(2+)-induced activation of membrane-bound integrins when compared with an inactive integrin control group. Thus, the result is inconsistent with the switchblade activation model.

Figure 1

Different predications from two integrin activation models. (A) The deadbolt model predicts that the integrin remains in the bent-over conformation after activation and that no height change is coupled to the increased ligand binding affinity of activation. (B) The integrin changes to an extended conformation after activation. The height of the molecule increases dramatically as the bent over conformation straightens and the legs separate. Headpiece denotes the large globular domain comprising the C-terminal domains of both the αIIb β-propellar domain and the β3 β-A domains (circled in the upright conformation). The genu denotes the point where the leg of the αIIb chain bends in the crystal structure. The location of the other β3 domains mentioned in the text, i.e. –hybrid, -psi and -βTD are also indicated in the upright conformationwhere they are most easily identified

Figure 2

(A). Gel pictures showing different steps of integrin purification. From left to right, lane 1 showing relative purity after the Con A column; lane 2 showing relative purity after the sepharose Q column; lane 3 shows relative purity after the mono Q column. (B). Gel photograph showing the reconstituted integrin liposomes used for the assays described in Figure S1 after sucrose gradient purification. Gels from other liposome preparations have the same appearance. Lanes as indicated in the figures are different layers on the sucrose gradient from top to bottom: Lane 1: Top layer; Lane 2: 2^nd layer from the top; Lane 3: 3^rd layer from the top; Lane 4: 4^th layer from the top; Lane 5: Layer with the visible liposome band; Lane 6: Layer 1 cm beneath the visible liposome band. The gel shows that the purified integrins are reconstituted into the liposome because they migrate with the liposome. The non-incorporated integrins form irregular aggregates, are consequently less bouyant and migrate to a position below the liposome band.

Figure 3

Results of integrin activation assay using antibody PAC-1, antibody AP5 and fibrinogen. Each measurement is the average of 3 repeats and the error bars in the figure show the range of data. (A) Assay comparing binding to PAC-1, AP5 and fibrinogen. (B) Assay comparing different loadings of PAC-1 to the assay well.

Figure 4

Active and inactive integrins incorporated into liposomes. (A) Inactive αIIbβ3 integrins in a z-section through the cryoelectron tomogram showing the top surface of the liposome. Representative molecules are circled in red. (B) z-Section through the middle of the liposome from the same tomogram as (A) illustrating side-views of inactive integrins. (C) z-Section through the middle of an activated αIIbβ3 integrin-liposome. Some representative molecules are circled, some of which appear to show paired leg domains.

Figure 5

Projections of inactive integrin molecules obtained from multivariate data analysis. Each panel is 360 Å × 360 Å. The projections are computed from 11 sections (61 Å) through the center of the subvolume. (A) 20 classes from the inactive integrins that show at least some density from the lipid bilayer. All of the classes and the class members of some classes can be seen in Supplemental Figure S2. Most of the 60 classes do not display the membrane density or show it weakly due to the effect of the missing wedge and the fact that the majority of molecules were selected from top-bottom views. Class number is indicated in the lower left hand corner of the individual panels in bold font and the number of class members in the lower right hand corner in normal font. The three horizontal lines, which are positioned identically in all panels, indicate the top of the integrin as illustrated in Figure 1, the top of the membrane and the bottom of the membrane and are separated by 110 Å and 60 Å respectively. (B) Fifteen classes of active integrins obtained from MDA that show some density from the lipid bilayer. All of the classes and the class members of some classes can be seen in Supplemental Figure S3