This offers a potential advantage of AFM/MFM imaging over electron microscopy, which delivers a comparable resolution but cannot be used in live cells. Supporting Material Verification of functional bioavailability of ET-1 and referrals, and three numbers, are available at http://www.biophysj.org/biophysj/supplemental/S0006-3495(09)01628-2. Supporting Material Document S1. magnetic AFM. Considering its high spatial resolution and ability to observe 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 within the cell membrane and in the submembrane space. Intro Membrane surface proteins play a pivotal part in cellular function. It is well approved that most of the components of the cell membrane, e.g., ion channels, receptors, and exchangers, have a complex multimeric structure and are often localized in specialised membrane regions such as lipid rafts and caveolae. The investigation of the structure and distribution of membrane proteins and their complexes on the surface of living cells, however, presents an enormous concern and currently is definitely chiefly limited to fluorescence imaging techniques. Such techniques involve the labeling of protein macromolecules with specific antibodies, which are then targeted by fluorescent probes (1). Recent improvements in fluorescence microscopy, combined with the development of genetically coded fluorescent proteins (2) and AR-A 014418 quantum dots (3), have significantly improved our understanding of the practical and temporal dynamics of intracellular proteins. However, imaging of individual proteins remains beyond the resolution of optical tools (4). This is due to the inherent limitations of optical tools, whose resolution is restricted from the wavelength of the light. The invention of the atomic push microscope has opened up a novel approach for studying individual proteins, their topography, and protein-protein relationships in the nanoscale level. It AR-A 014418 is currently the only technique that can provide nanometer resolution under the physiological conditions required for living cells. Atomic push microscopy (AFM) imaging offers revealed fine constructions of bacteriorhodopsins in isolated bacterial membranes (5) (for a review, observe Frederix et?al. (6)), nuclear pore complexes in the nuclear envelope (7C9), space junctions (10), and receptors (11,12) overexpressed in mammalian cell lines. Also, multimeric constructions of purified isolated receptors and ion channel proteins have been shown by AFM imaging (13C18). The recent development of simultaneous topography and acknowledgement imaging (19,20) offers significantly improved the lateral resolution, permitting visualization of individual protein molecules. Despite this progress, however, the recognition of specific proteins on the surface of undamaged cells by AFM remains a challenging task and relies entirely on direct relationships between the functionalized (ligand-coated) AFM probe and a related protein target. However, the functionalized AFM tip usually has a relatively short lifetime due to the instability of attached practical ligands (for review, observe Hinterdorfer and Dufrne (21)). In recent studies using magnetically coated AFM cantilevers, relationships between streptavidin molecules attached to a self-assembled monolayer or to a modified glass surface and biotin-coated magnetic nanoparticles were shown using the magnetic mode of AFM (MFM) (22,23). In these two studies, magnetic nanoparticles were either chemically derived (22) or CCL2 isolated from magnetotactic bacteria (23). Here, we further developed this fresh methodological approach and applied it to investigate the distribution of endothelin (ET) receptors on the surface of undamaged rat aortic?clean muscle cells (SMCs). We used an innovative labeling approach to specifically tag ET-1, a potent and highly specific endogenous agonist of ET receptors, with superparamagnetic microbeads (50 nm in diameter). Magnetically labeled receptors were then recognized and imaged by AFM and MFM. The choice of cell type (ET-1 and ET receptors) for this study was dictated by a several considerations. From a functional perspective, the ET receptors (which primarily consist of two subtypes, ETA and ETB) play an important part in the cardiovascular system under physiological conditions and in various disease claims (24). Biochemical, ligand-binding, and practical studies possess shown that vascular SMCs endogenously communicate both subtypes of the ET receptors, with the ETA subtype becoming generally dominating (25). However, the distribution and corporation of receptors on the surface of undamaged vascular cells in the molecular level have only been analyzed in the vascular AR-A 014418 wall of the capybara basilar artery using electron microscopy (26). Another important thought was the highly specific and potent binding of ET-1 with the receptor and its extremely sluggish dissociation AR-A 014418 from your receptor, which make interactions between the ET receptor and ET-1 virtually irreversible (27). These features of the agonist-receptor connection should facilitate preparation of ET-1-treated cells for AFM and MFM imaging without a significant loss of.
This offers a potential advantage of AFM/MFM imaging over electron microscopy, which delivers a comparable resolution but cannot be used in live cells