Dataset Information

Photobleaching step analysis for robust determination of protein complex stoichiometries.

ABSTRACT: The counting of discrete photobleaching steps in fluorescence microscopy is ideally suited to study protein complex stoichiometry in situ. The counting range of photobleaching step analysis has been significantly improved with more-sophisticated algorithms for step detection, albeit at an increasing computational cost and with the necessity for high-quality data. Here, we address concerns regarding robustness, automation, and experimental validation, optimizing both data acquisition and analysis. To make full use of the potential of photobleaching step analysis, we evaluate various labeling strategies with respect to their molecular brightness, photostability, and photoblinking. The developed analysis algorithm focuses on automation and computational efficiency. Moreover, we validate the developed methods with experimental data acquired on DNA origami labeled with defined fluorophore numbers, demonstrating counting of up to 35 fluorophores. Finally, we show the power of the combination of optimized trace acquisition and automated data analysis by counting labeled nucleoporin 107 in nuclear pore complexes of intact U2OS cells. The successful in situ application promotes this framework as a new resource enabling cell biologists to robustly determine the stoichiometries of molecular assemblies at the single-molecule level in an automated manner.

SUBMITTER: Hummert J

PROVIDER: S-EPMC8693960 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Photobleaching step analysis for robust determination of protein complex stoichiometries.

Hummert Johan J Yserentant Klaus K Fink Theresa T Euchner Jonas J Ho Yin Xin YX Tashev Stanimir Asenov SA Herten Dirk-Peter DP

Molecular biology of the cell 20210929 21

The counting of discrete photobleaching steps in fluorescence microscopy is ideally suited to study protein complex stoichiometry in situ. The counting range of photobleaching step analysis has been significantly improved with more-sophisticated algorithms for step detection, albeit at an increasing computational cost and with the necessity for high-quality data. Here, we address concerns regarding robustness, automation, and experimental validation, optimizing both data acquisition and analysis ...[more]

PMID: 34586828

Similar Datasets

Project description:BackgroundFluorescence loss in photobleaching (FLIP) is a widely used imaging technique, which provides information about protein dynamics in various cellular regions. In FLIP, a small cellular region is repeatedly illuminated by an intense laser pulse, while images are taken with reduced laser power with a time lag between the bleaches. Despite its popularity, tools are lacking for quantitative analysis of FLIP experiments. Typically, the user defines regions of interest (ROIs) for further analysis which is subjective and does not allow for comparing different cells and experimental settings.ResultsWe present two complementary methods to detect and quantify protein transport and aggregation in living cells from FLIP image series. In the first approach, a stretched exponential (StrExp) function is fitted to fluorescence loss (FL) inside and outside the bleached region. We show by reaction-diffusion simulations, that the StrExp function can describe both, binding/barrier-limited and diffusion-limited FL kinetics. By pixel-wise regression of that function to FL kinetics of enhanced green fluorescent protein (eGFP), we determined in a user-unbiased manner from which cellular regions eGFP can be replenished in the bleached area. Spatial variation in the parameters calculated from the StrExp function allow for detecting diffusion barriers for eGFP in the nucleus and cytoplasm of living cells. Polyglutamine (polyQ) disease proteins like mutant huntingtin (mtHtt) can form large aggregates called inclusion bodies (IB's). The second method combines single particle tracking with multi-compartment modelling of FL kinetics in moving IB's to determine exchange rates of eGFP-tagged mtHtt protein (eGFP-mtHtt) between aggregates and the cytoplasm. This method is self-calibrating since it relates the FL inside and outside the bleached regions. It makes it therefore possible to compare release kinetics of eGFP-mtHtt between different cells and experiments.ConclusionsWe present two complementary methods for quantitative analysis of FLIP experiments in living cells. They provide spatial maps of exchange dynamics and absolute binding parameters of fluorescent molecules to moving intracellular entities, respectively. Our methods should be of great value for quantitative studies of intracellular transport.

Dataset Information

Photobleaching step analysis for robust determination of protein complex stoichiometries.

Publications

Photobleaching step analysis for robust determination of protein complex stoichiometries.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets