Single protein molecule mapping with magnetic atomic force microscopy

Abstract

Understanding the structural organization and distribution of proteins in biological cells is of fundamental importance in biomedical research. The use of conventional fluorescent microscopy for this purpose is limited due to its relatively low spatial resolution compared to the size of a single protein molecule. Atomic force microscopy (AFM), on the other hand, allows one to achieve single-protein resolution by scanning the cell surface using a specialized ligand-coated AFM tip. However, because this method relies on short-range interactions, it is limited to the detection of binding sites that are directly accessible to the AFM tip. We developed a method based on magnetic (long-range) interactions and applied it to investigate the structural organization and distribution of endothelin receptors on the surface of smooth muscle cells. Endothelin receptors were labeled with 50-nm superparamagnetic microbeads and then imaged with magnetic AFM. Considering its high spatial resolution and ability to "see" magnetically labeled proteins at a distance of up to 150 nm, this approach may become an important tool for investigating the dynamics of individual proteins both on the cell membrane and in the submembrane space.

Figure 1

Labeling method and imaging of biotinylated intact rat aortic SMCs. (A) Schematics of the cell biotinylation and AFM/MFM imaging procedure, showing the key steps of cell membrane biotinylation with biotin XX (step 1), labeling with anti-biotin-coated superparamagnetic microbeads (step 2), and imaging with the AFM/MFM probe (step 3). (B and C) Confocal images of freshly isolated (B) and primary cultured (C) rat aortic SMCs were biotinylated as shown in A (step 1), but labeled with anti-biotin-conjugated FITC instead of magnetic microbeads (step 2). Confocal images were used to verify the experimental procedure before magnetic labeling. The left and right panels in B and C compare fluorescent and superimposed fluorescent and transmitted light images, respectively. Scale bars are 50 μm.

Figure 2

Dependence of the magnetic signal on the lift height of the MFM probe. (A) AFM image of biotinylated cultured aortic SMCs in tapping mode (left) and a 3D reconstruction (right) of a 4 × 2 μm area of the surface of a biotinylated cell. (B) MFM images of the same area at different lift heights as indicated above each panel. (C) Line analysis of the AFM and MFM responses across the selected area shown by straight lines in A and B, and by an arrowhead in the 3D reconstruction image in A.

Figure 3

Magnetic nature of MFM responses. (A) Schematic of the experiment with the MFM probe polarized vertically up (left picture) and vertically down (right picture). (B) AFM images of two areas of a primary cultured biotinylated SMC measured with the MFM tip polarized “up” (left column) and “down” (right column). (C) Corresponding MFM images for the two selected cell areas. Note that for magnetic repolarization, the MFM probe had to be removed from the instrument; therefore, the images on the left and right do not exactly represent the same area of the cell. (D) Comparison of the topographic (AFM) and magnetic (MFM) profiles (respectively shown by straight lines in B and C) for a single superparamagnetic microbead for different polarizations of the MFM tip. Black and blue lines depict variations in the AFM height and the MFM voltage, respectively.

Figure 4

Method for visualizing ET receptors in biotinylated cultured SMCs, and AFM/MFM imaging verified by confocal microscopy. (A) Schematics of the experimental procedure. The key steps include biotinylation of ET-1 (step 1), labeling of the surface ET receptors with bET-1 (step 2), labeling of bET-1 bound to the receptor with anti-biotin-coated superparamagnetic microbeads (step 3), and imaging of the labeled receptors with AFM/MFM (step 4). (B) Confocal imaging of cultured cells treated with 100 nM bET-1-and then labeled with anti-biotin FITC-conjugated antibodies instead of anti-biotin-coated superparamagnetic microbeads. The bottom panel shows superimposed fluorescent and transmitted light images. Scale bars are 50 μm.

Figure 5

Specificity of bET-1 labeling. (A–C) Confocal images of primary cultured aortic SMCs: nontreated (control) (A), pretreated with 200 nM of nonmodified ET-1 and then treated with bET-1 (100 nM) (B), and treated with bET-1 (100 nM) (C).

Figure 6

AFM/MFM imaging of ET receptors on the surface of aortic SMCs. (A) AFM image (50 × 30 μm) of the surface area of a cultured SMC. (B) AFM images of three selected areas at high spatial resolution (2.5 × 2.5 μm) shown by squares 1–3 in A (upper panels) and their corresponding 3D reconstruction (bottom panels). (C) Corresponding MFM images of the three areas shown in B. Note a correlation between amplitude heights measured with AFM and magnetic responses measured with MFM, confirming the specificity of the receptor labeling. (D) Line section analysis of the AFM topographic (black lines) and MFM magnetic (blue lines) responses for two individual ET receptors (i) and two receptor complexes (ii and iii) marked by straight lines in the third selected area (right columns in B and C).