Nanosensing of Fcγ receptors on macrophages

Determining the distribution of specific binding sites on biological samples with high spatial accuracy (in the order of several nanometer) is an important challenge in many fields of biological science. Combination of high-resolution atomic force microscope (AFM) topography imaging with single-molecule force spectroscopy provides a unique possibility for the detection of specific molecular recognition events. The identification and localization of specific receptor binding sites on complex heterogeneous biosurfaces such as cells and membranes are of particular interest in this context. Simultaneous topography and recognition imaging was used to unravel the nanolandscape of cells of the immune system such as macrophages. The most studied phagocytic receptors include the Fc receptors that bind to the Fc portion of immunoglobulins. Here, nanomapping of FcγRs (Fc receptors for immunoglobulin G (IgG)) was performed on fixed J774.A1 mouse macrophage cell surfaces with magnetically coated AFM tips functionalized with Fc fragments of mouse IgG via long and flexible poly(ethylene glycol) linkers. Because of possible AFM tip engulfment on living macrophages, appropriate cell fixation procedure leaving the binding activity of FcγRs practically intact was elaborated. The recognition maps revealed prominent spots (microdomains) more or less homogeneously distributed on the macrophage surface with the sizes from 4 to 300 nm. Typical recognition image contained about ∼4% of large clusters (>200 nm), which were surrounded by a massive number (∼50%) of small-size (4-30 nm) and the rest by middle-size (50, 150 nm) domains. These spots were detected from the decrease of oscillation amplitude during specific binding between Fc-coated tip and FcγRs on macrophage surfaces. In addition, the effect of osmotic swelling on the topographical landscape of macrophage surfaces and on the reorganization of FcγRs was investigated.