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Super-Resolution Imaging of Plasma Membrane Proteins with Click Chemistry

View Article: PubMed Central - PubMed

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.

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Comparison of dSTORM images with standard fluorescent microscopy. Representative dSTORM and overlaid standard fluorescence images (upper right corner) of PM proteins at the basal membrane stained via (A) L-AHA (CuAAC), (B) L-AHA (SPAAC), (C) Ac4GalNAz (CuAAC), and (D) Ac4GalNAz (SPAAC). Comparison of L-AHA and Ac4GalNAz stained via copper-catalyzed (CuACC) and copper-free (SPAAC) show no significant differences, indicating that the presence of copper ions or THPTA do not affect the distribution of PM components. For the four staining schemes depicted, 2D projected structures lead to spatial inhomogeneties as highlighted in the lower panels, e.g., (A) one fold membrane under the nucleus, (B) two-fold membrane structure within the lamellipodia plus one filopodia, (C) membrane ruffles, and (D) projection of a vesicle located in close proximity to the plasma membrane. All images were acquired under TIRF illumination, reconstructed with a minimum localization intensity threshold of 1000 photons, and a pixel size of 10 nm. Scale bars are 5 μm (upper panels) and 1 μm (lower panels).
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Figure 2: Comparison of dSTORM images with standard fluorescent microscopy. Representative dSTORM and overlaid standard fluorescence images (upper right corner) of PM proteins at the basal membrane stained via (A) L-AHA (CuAAC), (B) L-AHA (SPAAC), (C) Ac4GalNAz (CuAAC), and (D) Ac4GalNAz (SPAAC). Comparison of L-AHA and Ac4GalNAz stained via copper-catalyzed (CuACC) and copper-free (SPAAC) show no significant differences, indicating that the presence of copper ions or THPTA do not affect the distribution of PM components. For the four staining schemes depicted, 2D projected structures lead to spatial inhomogeneties as highlighted in the lower panels, e.g., (A) one fold membrane under the nucleus, (B) two-fold membrane structure within the lamellipodia plus one filopodia, (C) membrane ruffles, and (D) projection of a vesicle located in close proximity to the plasma membrane. All images were acquired under TIRF illumination, reconstructed with a minimum localization intensity threshold of 1000 photons, and a pixel size of 10 nm. Scale bars are 5 μm (upper panels) and 1 μm (lower panels).

Mentions: The conditions presented in the given protocols constitute a robust recipe to stain and visualize large populations of PM proteins and glycans with super-resolution localization microscopy (Figure 2). Nevertheless, critical aspects, as well as limitations and future perspectives, with regard to obtain reliable quantitative data and avoid artifacts are shown in the next subsections. First, we highlight potential artifacts of dSTORM as well as the inherent problem of 2D super-resolution images due to projections of 3D structures such as membrane ruffling, filopodia, overlapping membranes, and vesicles located in close proximity to the PM. Then, we compare the fluorescence staining efficiency achieved by copper-catalyzed and copper-free click chemistry reactions for fixed metabolic labeling conditions. Finally, we show how quantitative information about the distribution of PM components can be percolated from dSTORM data using statistical spatial analysis approaches, such as pair-correlation and Ripley's K functions.


Super-Resolution Imaging of Plasma Membrane Proteins with Click Chemistry
Comparison of dSTORM images with standard fluorescent microscopy. Representative dSTORM and overlaid standard fluorescence images (upper right corner) of PM proteins at the basal membrane stained via (A) L-AHA (CuAAC), (B) L-AHA (SPAAC), (C) Ac4GalNAz (CuAAC), and (D) Ac4GalNAz (SPAAC). Comparison of L-AHA and Ac4GalNAz stained via copper-catalyzed (CuACC) and copper-free (SPAAC) show no significant differences, indicating that the presence of copper ions or THPTA do not affect the distribution of PM components. For the four staining schemes depicted, 2D projected structures lead to spatial inhomogeneties as highlighted in the lower panels, e.g., (A) one fold membrane under the nucleus, (B) two-fold membrane structure within the lamellipodia plus one filopodia, (C) membrane ruffles, and (D) projection of a vesicle located in close proximity to the plasma membrane. All images were acquired under TIRF illumination, reconstructed with a minimum localization intensity threshold of 1000 photons, and a pixel size of 10 nm. Scale bars are 5 μm (upper panels) and 1 μm (lower panels).
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Figure 2: Comparison of dSTORM images with standard fluorescent microscopy. Representative dSTORM and overlaid standard fluorescence images (upper right corner) of PM proteins at the basal membrane stained via (A) L-AHA (CuAAC), (B) L-AHA (SPAAC), (C) Ac4GalNAz (CuAAC), and (D) Ac4GalNAz (SPAAC). Comparison of L-AHA and Ac4GalNAz stained via copper-catalyzed (CuACC) and copper-free (SPAAC) show no significant differences, indicating that the presence of copper ions or THPTA do not affect the distribution of PM components. For the four staining schemes depicted, 2D projected structures lead to spatial inhomogeneties as highlighted in the lower panels, e.g., (A) one fold membrane under the nucleus, (B) two-fold membrane structure within the lamellipodia plus one filopodia, (C) membrane ruffles, and (D) projection of a vesicle located in close proximity to the plasma membrane. All images were acquired under TIRF illumination, reconstructed with a minimum localization intensity threshold of 1000 photons, and a pixel size of 10 nm. Scale bars are 5 μm (upper panels) and 1 μm (lower panels).
Mentions: The conditions presented in the given protocols constitute a robust recipe to stain and visualize large populations of PM proteins and glycans with super-resolution localization microscopy (Figure 2). Nevertheless, critical aspects, as well as limitations and future perspectives, with regard to obtain reliable quantitative data and avoid artifacts are shown in the next subsections. First, we highlight potential artifacts of dSTORM as well as the inherent problem of 2D super-resolution images due to projections of 3D structures such as membrane ruffling, filopodia, overlapping membranes, and vesicles located in close proximity to the PM. Then, we compare the fluorescence staining efficiency achieved by copper-catalyzed and copper-free click chemistry reactions for fixed metabolic labeling conditions. Finally, we show how quantitative information about the distribution of PM components can be percolated from dSTORM data using statistical spatial analysis approaches, such as pair-correlation and Ripley's K functions.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus