Abstract
Besides its function as a passive cell wall, the plasma membrane (PM) serves as a platform for different physiological processes such as signal transduction and cell adhesion, determining the ability of cells to communicate with the exterior, and form tissues. Therefore, the spatial distribution of PM components, and the molecular mechanisms underlying it, have important implications in various biological fields including cell development, neurobiology, and immunology. The existence of confined compartments in the plasma membrane that vary on many length scales from protein multimers to micrometer-size domains with different protein and lipid composition is today beyond all questions. As much as the physiology of cells is controlled by the spatial organization of PM components, the study of distribution, size, and composition remains challenging. Visualization of the molecular distribution of PM components has been impeded mainly due to two problems: the specific labeling of lipids and proteins without perturbing their native distribution and the diffraction-limit of fluorescence microscopy restricting the resolution to about half the wavelength of light. Here, we present a bioorthogonal chemical reporter strategy based on click chemistry and metabolic labeling for efficient and specific visualization of PM proteins and glycans with organic fluorophores in combination with super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM) with single-molecule sensitivity.
Highlights
The plasma membrane in eukaryotes is involved in several cell functions such as tissue formation, signal transduction, cell adhesion, and immune response
We report a chemical reporter strategy, based on metabolic labeling and click chemistry, in combination with superresolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to stain and visualize plasma membrane (PM) proteins and glycans
Overlapping membranes lead to overestimation of protein content, and vesicle-like structures located in closed proximity to the cell membrane appear as protein clusters and, can potentially result in false interpretation of PM organization
Summary
The plasma membrane in eukaryotes is involved in several cell functions such as tissue formation, signal transduction, cell adhesion, and immune response. The introduction of a bioorthogonal chemical reporter strategy, based on metabolic labeling and click chemistry, allowed the direct visualization of different membrane components by super-resolution microscopy (Letschert et al, 2014; Saka et al, 2014). This approach exploits the ability of the endogenous metabolic cellular machinery to recognize different metabolic surrogates containing small reactive chemical modifications ready to be conjugated with fluorophores. A certain degree of deviation from complete spatial randomness in PM proteins was found by Ripley’s K function analysis
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