Single-Molecule Imaging of Membrane Proteins on Vascular Endothelial Cells

Abstract

Transporting substances such as gases, nutrients, waste, and cells is the primary function of blood vessels. Vascular cells use membrane proteins to perform crucial endothelial functions, including molecular transport, immune cell infiltration, and angiogenesis. A thorough understanding of these membrane receptors from a clinical perspective is warranted to gain insights into the pathogenesis of vascular diseases and to develop effective methods for drug delivery through the vascular endothelium. This review summarizes state-of-the-art single-molecule imaging techniques, such as super-resolution microscopy, single-molecule tracking, and protein–protein interaction analysis, for observing and studying membrane proteins. Furthermore, recent single-molecule studies of membrane proteins such as cadherins, integrins, caveolins, transferrin receptors, vesicle-associated protein-1, and vascular endothelial growth factor receptor are discussed.

Analysis of Phase Heterogeneity in Lipid Membranes Using Single-Molecule Tracking in Live Cells

“…Lipid-MAP is not only limited to saturated lipids but can also be applied to various membrane components including other lipids and receptors. When recently reported intermolecular interactions (e.g., protein–protein interactions), ,,, the heterogeneity of the lipid packing of the membrane, and the curvature of plasma membrane , are considered together, we may be able to understand the chemical reactions that occur in the plasma membrane more precisely. Furthermore, using parallel observation at the single-molecule level and systematic interpretation , with cellular signals in real time, e.g., kinase phosphorylation reporters, we may be able to answer long-standing questions on how the phase separation of cell membranes regulates cellular signals.…”

From Homogeneity to Turing Pattern: Kinetically Controlled Self-Organization of Transmembrane Protein

“…To obtain D and v (which are two crucial factors for pattern formation under the RD scheme) of PLVAP simultaneously in living cells, we first visualized the lateral movement of an individual PLVAP using the single-molecule tracking (SMT) technique. , First, a monoclonal bEND3 cell line that expresses the mCherry2-PLVAP-SNAP recombinant protein was generated. Next, we specifically labeled SNAP-tagged PLVAP in living cells using “gold nanoparticles (Au NPs) in which a monovalent oligonucleotide (ssDNA) was adjoined” as a probe and “benzylguanine (BG, ligand for SNAP-tag)-conjugated complementary ssDNA” as a linker , (Figure a and Figures S5 and S6).…”