Structural analysis of receptors and actin polarity in platelet protrusions

Abstract

During activation the platelet cytoskeleton is reorganized, inducing adhesion to the extracellular matrix and cell spreading. These processes are critical for wound healing and clot formation. Initially, this task relies on the formation of strong cellular-extracellular matrix interactions, exposed in subendothelial lesions. Despite the medical relevance of these processes, there is a lack of high-resolution structural information on the platelet cytoskeleton controlling cell spreading and adhesion. Here, we present in situ structural analysis of membrane receptors and the underlying cytoskeleton in platelet protrusions by applying cryoelectron tomography to intact platelets. We utilized three-dimensional averaging procedures to study receptors at the plasma membrane. Analysis of substrate interaction-free receptors yielded one main structural class resolved to 26 Å, resembling the α_IIbβ₃ integrin folded conformation. Furthermore, structural analysis of the actin network in pseudopodia indicates a nonuniform polarity of filaments. This organization would allow generation of the contractile forces required for integrin-mediated cell adhesion.

Keywords: actin; cryoelectron tomography; platelets; receptors.

Fig. 1.

Cryo-ET of platelet pseudopodia. (A and B) Cryo-EM of human platelets spread on fibrinogen-functionalized SiO₂-coated gold grids. Pseudopodia are detected in single projections (arrows). (C) x-y slices, 10 nm in thickness, through cryotomograms of pseudopodia. The membrane is decorated with protein densities (arrowheads) while the cytoskeleton is seen in the cytoplasm. Insets show magnified membrane receptors (white arrowheads). Fiducial gold clusters are seen as 10-nm black densities.

Fig. 2.

The 3D architecture of platelet pseudopodia. Surface rendering of three pseudopodia. Three of the volumes were rendered and all elements composing pseudopodia are shown. Actin (light brown), microtubules (purple), membrane (light turquoise), and receptors (green) were segmented. (Scale bar, 100 nm.)

Fig. 3.

A nonuniform actin polarity in pseudopodia. (A) Four 2D class averages of the in situ actin segments (SI Appendix, Fig. S2). A 14-Å resolved actin structure (shown in gray) allows identification of actin polarity, barbed (+) and the pointed (−) ends are indicated. (Scale bar, 10 nm.) (B) Back-mapping of the actin structure into the tomogram revealed a mixed actin polarity in each tomogram. Actin filaments pointing to the pseudopodia tip (blue) were found to compose 74% ± 10% of the total filament numbers, while actin filaments pointing toward the cell body (red) represent 26% ± 10%. (C) The pseudopodia shown in Fig. 2 were used to overlay the polarity of the total actin filament content within protrusions. Actin filaments and segments pointing to the plasma membrane are colored in blue while the actin pointing toward the cell is colored in red. The total segmented actin filaments are depicted in light brown. Additionally, microtubules (purple), membrane (light turquoise) and receptors (green) were segmented as well. (Scale bar, 100 nm.)

Fig. 4.

Structural analysis of platelets receptors. (A) Subtomogram averaged map of platelet receptors in different orientations, the membrane was colored in blue and the protein receptor in green. The bent integrin structure (PDB ID code 3FCS, in ribbon) was fitted into the density map obtained by in situ structural determination. (B) Surface rendering of three pseudopodia where subtomograms corresponding to the platelet receptor (green) were placed back in the original tomograms using the refined coordinates and orientations. Actin (light brown), microtubules (purple) and membrane (light turquoise). (Scale bar, 100 nm.)