Single-molecule Labeling and Tracking of FcγRIIA on Human Platelets Reveals Differential Mobility Dynamics, which Depends on Platelet Cytoskeletal Integrity

Scientific Research Question: Is the FcγRIIA mobile in the platelet membrane and how is this mobility regulated?

Methodology: For single molecule FcγRIIA labelling, fluorescent monovalent quantum dots (mQD) were prepared by conjugating QD (3-5 nm in hydrodynamic radius) to monoclonal anti-FcγRIIA Fab in 1:1 molar ratio. Visualization and tracking of single mQD tagged FcγRIIA on human platelets was performed by high-speed fluorescence imaging (11.9Hz - 82.7ms/image) using a cooled back-illuminated electron multiplying CCD camera. Quantification of FcγRIIA mobility dynamics (e.g. single receptor lateral trajectories, track length and speed) on platelet filopodia, lamellipodia and platelet body were performed. To assess the role of FcγRIIA mobility for sensing of IgG opsonized particles by platelets, we used micropatterned arrays coated with aggregated IgG, or IgG coated bacteria-mimetic beads, or IgG opsonized live bacteria and analysed platelet morphodynamics. The relevance of platelet cytoskeletal integrity was assessed by blocking cytoskeletal functions with cytochalasin D and latrunculin B.

Findings: We report for the first time visualization and tracking of single FcγRIIA molecules on human platelets using mQD. The single FcγRIIA molecules moved longer distances on filopodia (median 0.93 µm n = 238) and lamellipodia (median 0.4 µm n =  303) compared to the FcγRIIA molecules on platelet body (median 0. 25 µm n = 248; < P = 0.0001) (Figure 1A). FcγRIIA molecules also moved faster on filopodia (median 1.08 µm/s n = 989) and lamellipodia (0.89 µm/s n = 928) than on the platelet body (median 0.83 µm/s n = 895; < P = 0.0001) Figure 1B). Disruption of F-actin assembly using cytoclalasin D and latrunculin B markedly decreased the track length and speed of single FcγRIIA movement in the platelet membrane. Cytoskeletal integrity was indispensable for FcγRIIA mediated ligand sensing, platelet adhesion, spreading and activation on aggregated IgG, IgG coated bacteria-mimetic beads, and IgG opsonized live bacteria.

Conclusions: Continuous actin-based “treadmilling” mechanism controls the dynamics of FcγRIIA lateral mobility on the platelet surface. This facilitates rapid ligand engagement by FcγRIIA and may have wide range of implications for the response of platelets to opsonized bacteria and IgG coated autologous cells, e.g. in immune thrombocytopenia or the anti-phospholipid syndrome.

Fig. 1 Track length and speed of FcγRIIA single molecules on human platelets.