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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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Labeling efficiency of copper-catalyzed (CuAAC) and copper-free azide-alkyne cycloadditions (SPAAC). Fluorophore titration for the same metabolic labeling conditions, i.e., 4 mM L-AHA during 4 h 30 min (A,B), and 25 μM Ac4GalNAz during 48 h (C,D), show optimal staining efficiency with AF-647 alkyne and Cy5 DBCO in the range of 20 to 50 μM for 5 min CuAAC and 15 min SPAAC reactions. For each cell, detected localizations were first obtained with a sliding window analysis (diameter = 1 μm, step = 100 nm) applied to big areas defined at bottom plasma membrane under the cell nucleus as described in Figure 3B. Plotted values and error bars represent median and SE of several cells imaged and analyzed for each fluorophore concentration [(A) 7–10 cells, (B) 8–15 cells, (C) 7–8 cells, and (D) 12–16 cells].
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Figure 4: Labeling efficiency of copper-catalyzed (CuAAC) and copper-free azide-alkyne cycloadditions (SPAAC). Fluorophore titration for the same metabolic labeling conditions, i.e., 4 mM L-AHA during 4 h 30 min (A,B), and 25 μM Ac4GalNAz during 48 h (C,D), show optimal staining efficiency with AF-647 alkyne and Cy5 DBCO in the range of 20 to 50 μM for 5 min CuAAC and 15 min SPAAC reactions. For each cell, detected localizations were first obtained with a sliding window analysis (diameter = 1 μm, step = 100 nm) applied to big areas defined at bottom plasma membrane under the cell nucleus as described in Figure 3B. Plotted values and error bars represent median and SE of several cells imaged and analyzed for each fluorophore concentration [(A) 7–10 cells, (B) 8–15 cells, (C) 7–8 cells, and (D) 12–16 cells].

Mentions: The conditions given here for click chemistry staining of membrane proteins and glycoconjugates lead to maximum labeling densities ranging from 400 to 2000 localizations per μm2 (Figure 4). For the four bioconjugated systems inspected, we observed that fluorophore concentrations around 20–50 μM are required to maximize fluorescent signal. Moreover, copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) is equally efficient as CuAAC to stain Ac4GalNAz-derived glycoconjugates, and two-fold better to detect membrane proteins containing AHA. Thus, optimal conditions for click chemistry can also be achieved in absence of copper avoiding toxicity effects and simplifying the protocol.


Super-Resolution Imaging of Plasma Membrane Proteins with Click Chemistry
Labeling efficiency of copper-catalyzed (CuAAC) and copper-free azide-alkyne cycloadditions (SPAAC). Fluorophore titration for the same metabolic labeling conditions, i.e., 4 mM L-AHA during 4 h 30 min (A,B), and 25 μM Ac4GalNAz during 48 h (C,D), show optimal staining efficiency with AF-647 alkyne and Cy5 DBCO in the range of 20 to 50 μM for 5 min CuAAC and 15 min SPAAC reactions. For each cell, detected localizations were first obtained with a sliding window analysis (diameter = 1 μm, step = 100 nm) applied to big areas defined at bottom plasma membrane under the cell nucleus as described in Figure 3B. Plotted values and error bars represent median and SE of several cells imaged and analyzed for each fluorophore concentration [(A) 7–10 cells, (B) 8–15 cells, (C) 7–8 cells, and (D) 12–16 cells].
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Figure 4: Labeling efficiency of copper-catalyzed (CuAAC) and copper-free azide-alkyne cycloadditions (SPAAC). Fluorophore titration for the same metabolic labeling conditions, i.e., 4 mM L-AHA during 4 h 30 min (A,B), and 25 μM Ac4GalNAz during 48 h (C,D), show optimal staining efficiency with AF-647 alkyne and Cy5 DBCO in the range of 20 to 50 μM for 5 min CuAAC and 15 min SPAAC reactions. For each cell, detected localizations were first obtained with a sliding window analysis (diameter = 1 μm, step = 100 nm) applied to big areas defined at bottom plasma membrane under the cell nucleus as described in Figure 3B. Plotted values and error bars represent median and SE of several cells imaged and analyzed for each fluorophore concentration [(A) 7–10 cells, (B) 8–15 cells, (C) 7–8 cells, and (D) 12–16 cells].
Mentions: The conditions given here for click chemistry staining of membrane proteins and glycoconjugates lead to maximum labeling densities ranging from 400 to 2000 localizations per μm2 (Figure 4). For the four bioconjugated systems inspected, we observed that fluorophore concentrations around 20–50 μM are required to maximize fluorescent signal. Moreover, copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) is equally efficient as CuAAC to stain Ac4GalNAz-derived glycoconjugates, and two-fold better to detect membrane proteins containing AHA. Thus, optimal conditions for click chemistry can also be achieved in absence of copper avoiding toxicity effects and simplifying the protocol.

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