Simultaneous spatiotemporal computational super-resolution and multi-parametric fluorescence microscopy

Sankaran, Jagadish; Balasubramanian, Harikrushnan; Tang, Wai Hoh; Ng, Xue Wen; Röllin, Adrian; Wohland, Thorsten

doi:10.21203/rs.3.pex-1358/v1

Cited by 3 publications

(3 citation statements)

References 2 publications

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“…Details of plasmids, preparation of live cell samples, drug perturbations, and western blotting are provided in the Supporting Material. Full protocols for live cell sample preparation and data acquisition and analysis are also available on Protocol Exchange (36, 37).…”

Section: Methodsmentioning

confidence: 99%

The dependence of EGFR oligomerization on environment and structure: A camera-based N&B study

Balasubramanian

Sankaran

Goh

et al. 2022

Preprint

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Number and Brightness analysis (N&B) is a fluorescence spectroscopy technique to quantify protein oligomerization. Accurate results, however, rely on a good knowledge of non-fluorescent states of the fluorescent labels, especially of fluorescent proteins (FP), which are widely used in biology. FPs have been characterized for confocal but not camera-based N&B, which allows in principle faster measurements over larger areas. Here, we calibrate camera-based N&B implemented on a total internal reflection fluorescence (TIRF) microscope for various fluorescent proteins by determining their propensity to be fluorescent. We then apply camera-based N&B in live CHO-K1 cells to determine the oligomerization state of the epidermal growth factor receptor (EGFR), a transmembrane receptor tyrosine kinase that is a crucial regulator of cell proliferation and survival with implications in many cancers. EGFR oligomerization in resting cells and its regulation by the plasma membrane microenvironment is still under debate. Therefore, we investigate the effects of extrinsic factors, including membrane organization, cytoskeletal structure, and ligand stimulation, and intrinsic factors, including mutations in various EGFR domains, on the receptor's oligomerization. Our results demonstrate that EGFR oligomerization increases with removal of cholesterol or sphingolipids, or the disruption of GM3-EGFR interactions, indicating raft association. However, oligomerization was not significantly influenced by the cytoskeleton. Mutations in either I706/V948 residues or E685/E687/E690 residues in the kinase and juxtamembrane domains, respectively, led to a decrease in oligomerization, indicating their necessity for EGFR dimerization. Finally, EGFR phosphorylation is oligomerization-dependent involving the extracellular domain (550-580 residues). Coupled with biochemical investigations, camera-based N&B indicates that EGFR oligomerization and phosphorylation is the outcome of several molecular interactions involving the lipid content and structure of the cell membrane and multiple residues in the kinase, juxtamembrane, and extracellular domains.

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Section: Methodsmentioning

confidence: 99%

The dependence of EGFR oligomerization on environment and structure: A camera-based N&B study

Balasubramanian

Sankaran

Goh

et al. 2022

Preprint

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“…After obtaining the image stacks, data analysis was performed using a home-written GPU-accelerated Imaging FCS 1.52 plugin in ImageJ 40,43,44 . A step-by-step protocol detailing FCS data analysis is provided in Protocol Exchange 43 . The fluorescence fluctuations at all pixels were calculated with the autocorrelation functions (ACFs) and fitted with the following equation after an exponential of polynomial bleach correction.…”

Section: Estimation Of Diffusion Coefficient From Itir-fcsmentioning

confidence: 99%

Section: Estimation Of Diffusion Coefficient From Itir-fcsmentioning

confidence: 99%

Heptanol-mediated phase separation determines phase preference of molecules in live cell membranes

Gupta

Balasubramanian

et al. 2021

Preprint

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Plasma membranes contain diverse nanoscale assemblies of lipid and protein domains. Specific localization of lipids and proteins in these domains is often essential for membrane function and integrity. Due to the nanoscale size and dynamic nature of membrane domains, identification of molecules residing in domains either is not possible with modern imaging techniques or requires advanced methods with high spatiotemporal resolution. Such methods need expensive equipment making these approaches inaccessible and thus difficult to implement at large scale. Here, we present a novel membrane fluidizer-induced clustering (MFIC) approach to identify the phase-preference of molecules in intact cell membranes. Experiments in phase-separated bilayers and live cells on molecules with known phase preference demonstrate that heptanol hyperfluidizes the membrane and stabilizes phase separation in cell membranes. The domain stabilization results in a transition of nano- to micron-sized clusters of associated molecules and allows identification of molecules localized in domains using routine microscopy techniques. This assay can be carried out on both genetically and extrinsically labelled molecules in live cell membranes, does not require any invasive sample preparation and can be carried out in 10-15 minutes. This inexpensive and easy to implement assay can be conducted at large-scale and will allow easy identification of molecules partitioning into domains.

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Simultaneous spatiotemporal super-resolution and multi-parametric fluorescence microscopy

et al. 2021

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Super-resolution microscopy and single molecule fluorescence spectroscopy require mutually exclusive experimental strategies optimizing either temporal or spatial resolution. To achieve both, we implement a GPU-supported, camera-based measurement strategy that highly resolves spatial structures (~100 nm), temporal dynamics (~2 ms), and molecular brightness from the exact same data set. Simultaneous super-resolution of spatial and temporal details leads to an improved precision in estimating the diffusion coefficient of the actin binding polypeptide Lifeact and corrects structural artefacts. Multi-parametric analysis of epidermal growth factor receptor (EGFR) and Lifeact suggests that the domain partitioning of EGFR is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-EGFR interactions but is largely independent of EGFR-actin interactions. These results demonstrate that pixel-wise cross-correlation of parameters obtained from different techniques on the same data set enables robust physicochemical parameter estimation and provides biological knowledge that cannot be obtained from sequential measurements.

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Simultaneous spatiotemporal computational super-resolution and multi-parametric fluorescence microscopy

Cited by 3 publications

References 2 publications

The dependence of EGFR oligomerization on environment and structure: A camera-based N&B study

The dependence of EGFR oligomerization on environment and structure: A camera-based N&B study

Heptanol-mediated phase separation determines phase preference of molecules in live cell membranes

Simultaneous spatiotemporal super-resolution and multi-parametric fluorescence microscopy

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