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Quantitative Nanoscale Visualization of Macromolecular Complexes in Live Cells using Genetic Code Expansion and High-Resolution Imaging

Periodic Reporting for period 4 - VisuLive (Quantitative Nanoscale Visualization of Macromolecular Complexes in Live Cells using Genetic Code Expansion and High-Resolution Imaging)

Reporting period: 2019-10-01 to 2021-03-31

High-resolution fluorescence imaging, including super-resolution microscopy and high-speed live cell imaging, are used to obtain quantitative information on the structural organization and kinetics of cellular processes. The contribution of these high-resolution techniques to cell biology was recently demonstrated for dynamin- and ESCRT-driven membrane fission in cells. While they advance our knowledge on membrane fission these techniques do not provide the quantitative information needed to formulate a mechanical understanding of membrane fission in a physiological context, a shortcoming that stresses the need to increase the spatiotemporal resolution and improve the live cell capabilities of these techniques. Substituting the bulky fluorescent protein tags (such as GFP) currently used in live-cell applications with much smaller fluorescent dyes that possess superior photophysical characteristics will markedly improve these advanced imaging techniques. Genetic code expansion and bioorthogonal labeling offer, for the first time, a non-invasive way to specifically attach such fluorescent dyes to proteins in live cells. In this project we aim to develop an innovative approach to label cellular proteins with fluorescent dyes via genetic code expansion for quantitative high-resolution live cell imaging of cellular protein complexes. By applying this approach to three distinguished high-resolution methodologies and by visualizing membrane fission in distinct cellular processes in live cells at milliseconds rate and at nanoscale resolution, we aim to decipher the mechanistic principles of membrane fission in cells. As numerous cellular processes rely on membrane fission for their function, such an understanding will have a broad impact on cell biology. The implications of this study reach beyond the scope of membrane fission by offering a new approach to study cellular processes at close-to-real conditions in live cells and at nanoscale resolution.
The main objective for this period was to develop the methodology for direct labeling of proteins with fluorescent dyes for quantitative high-resolution imaging of macromolecular complexes in live cells. This goal was almost fully achieved. Using tubulin as a benchmark we have demonstrated the applicability of our methodology to various cell lines and performed live cell imaging of microtubules labeled with Silicon Rhodamine (SiR). Additionally, we applied our direct labeling approach to super resolution imaging of microtubules and demonstrated that higher resolution can be reached using our approach relative to other state-of-the-art labeling approaches (Schvartz et al., MBoC, 2017). We also calibrated conditions for plasma membrane labeling for live cell and SIM imaging. And were able to perform double labeling of both the plasma membrane and the microtubules (Aloush et al.,Sci Rep, 2018). Furthermore, we demonstrated that the approach is superior over fluorescent protein tagging for particle tracking and live - super resolution microscopy (Konig, Nanoscale, 2020). Finally, to make the approach more accessible to users, we designed a short N terminal peptide tag for applying the labeling approach to a variety of proteins and generated a library of organelle markers which can be readily used by the cell biology community. To share with the community all the knowledge and expertise we have learned during the development of the approach and provided a technical guide for implementing the approach for addressing questions in cell biology (Elia, FEBS J., 2021).
Twenty-five years ago, GFP revolutionized the field of cell biology by enabling scientists to visualize, for the first time, proteins in living cells. However, when it comes to current, state-of-the-art imaging technologies, fluorescent proteins (such as GFP) have several limitations that result from their size and photophysics. Over the past decade, an elegant, alternative approach, which is based on the direct labeling of proteins with fluorescent dyes, has been introduced. In this approach, an unnatural amino acid that can covalently bind a fluorescent dye is incorporated into the coding sequence of a protein, using genetic code expansion approaches (GCE). The protein of interest is thereby site-specifically fluorescently labeled inside the cell, eliminating the need for protein- or peptide-labeling tags. In the current project we have developed this labeling approach for cell biology applications and have demonstrated its advantages for live cell imaging and super resolution microscopy. By doing so, we provide a superior labelling approach for proteins in cells with reduced size, phototoxicity and increased photostability. This approach offers an attractive, improved alternative to GFP, by enabling tracking proteins in live cell with improved spatiotemporal resolution.
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