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

Reporting period: 2018-04-01 to 2019-09-30

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. The results of this part were published in the peer reviewed journal MBoC (Schvartz et al., MBoC, 2017). Following the project plan, 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. The results of this part were recently submitted to BBA and were deposited in BioRxiv (Aloush et al., doi: Having a calibrated approach in hand, we are currently applying it for labeling and quantitative high-resolution live imaging of the ESCRT membrane remodeling complex.
Our progress so far has shown that our approach is suitable for high-resolution live cell imaging of intracellular proteins in live cells. During the next period we aim to be able to record highly dynamic cellular processes at 30 nm resolution and with minimal photobleaching and phototoxicity to cells. By doing so, we will provide an optimal tool for recording cellular events in live cells at the single molecule level and under physiological conditions. This methodology will bridge the current gap in technology by broadly expanding the use of super-resolution imaging applications to live cells.