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1.
Mol Biol Cell ; 32(21): ar35, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34586828

RESUMO

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.

2.
Int J Biochem Cell Biol ; 135: 105978, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33865985

RESUMO

Quantitative assessment of protein complexes, such as receptor clusters in the context of cellular signalling, has become a pressing objective in cell biology. The advancements in the field of single molecule fluorescence microscopy have led to different approaches for counting protein copy numbers in various cellular structures. This has resulted in an increasing interest in robust calibration protocols addressing photophysical properties of fluorescent labels and the effect of labelling efficiencies. Here, we want to give an update on recent methods for protein counting with a focus on novel calibration protocols. In this context, we discuss different types of calibration samples and identify some of the challenges arising in molecular counting experiments. Some recently published applications offer potential approaches to tackle these challenges.


Assuntos
Fluorescência , Microscopia de Fluorescência/métodos , Proteínas/química , Imagem Individual de Molécula/métodos , Animais , Humanos
3.
Faraday Discuss ; 228(0): 226-241, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-33586720

RESUMO

Studies of ultrafast relaxation of molecular chromophores are complicated by the fact that most chromophores of biological and technological importance are rather large molecules and are strongly affected by their environment, either solvent or a protein cage. Here we present an approach which allows us to follow transient electronic structure of complex photoexcited molecules. We use the method of time-resolved photoelectron spectroscopy in solution to follow relaxation of two prototypical aqueous chromophores, Methyl Orange and Metanil Yellow, both of which are aminoazobenzene derivatives. Using excitation by 400 nm laser pulses and ionization by wavelength-selected 46.7 nm XUV pulses from high-order harmonic generation we follow relaxation of both molecules via the dark S1 state. The photoelectron spectra yield binding energies of both ground and excited states. We combine the experimental results with surface hopping time-dependent density functional theory (TDDFT) calculations employing B3LYP+D3 and ωB97X-D functionals. The results demonstrate that the method is generally suitable for description of ultrafast dynamics in these molecules and can recover absolute binding energies observed in the experiment. The B3LYP+D3 functional appears to be better suited for these systems, especially in the case of Metanil Yellow, where it indicates the importance of an intramolecular charge transfer state. Our results pave the way towards quantitative understanding of evolving electronic structure in photo-induced relaxation processes.

4.
Thromb Haemost ; 121(11): 1435-1447, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33638140

RESUMO

Collagen has been proposed to bind to a unique epitope in dimeric glycoprotein VI (GPVI) and the number of GPVI dimers has been reported to increase upon platelet activation. However, in contrast, the crystal structure of GPVI in complex with collagen-related peptide (CRP) showed binding distinct from the site of dimerization. Further fibrinogen has been reported to bind to monomeric but not dimeric GPVI. In the present study, we have used the advanced fluorescence microscopy techniques of single-molecule microscopy, fluorescence correlation spectroscopy (FCS) and bioluminescence resonance energy transfer (BRET), and mutagenesis studies in a transfected cell line model to show that GPVI is expressed as a mixture of monomers and dimers and that dimerization through the D2 domain is not critical for activation. As many of these techniques cannot be applied to platelets to resolve this issue, due to the high density of GPVI and its anucleate nature, we used Förster resonance energy transfer (FRET) to show that endogenous GPVI is at least partially expressed as a dimer on resting and activated platelet membranes. We propose that GPVI may be expressed as a monomer on the cell surface and it forms dimers in the membrane through diffusion, giving rise to a mixture of monomers and dimers. We speculate that the formation of dimers facilitates ligand binding through avidity.

5.
Mol Cell ; 78(2): 236-249.e7, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32101700

RESUMO

The formation of silenced and condensed heterochromatin foci involves enrichment of heterochromatin protein 1 (HP1). HP1 can bridge chromatin segments and form liquid droplets, but the biophysical principles underlying heterochromatin compartmentalization in the cell nucleus are elusive. Here, we assess mechanistically relevant features of pericentric heterochromatin compaction in mouse fibroblasts. We find that (1) HP1 has only a weak capacity to form liquid droplets in living cells; (2) the size, global accessibility, and compaction of heterochromatin foci are independent of HP1; (3) heterochromatin foci lack a separated liquid HP1 pool; and (4) heterochromatin compaction can toggle between two "digital" states depending on the presence of a strong transcriptional activator. These findings indicate that heterochromatin foci resemble collapsed polymer globules that are percolated with the same nucleoplasmic liquid as the surrounding euchromatin, which has implications for our understanding of chromatin compartmentalization and its functional consequences.


Assuntos
Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Eucromatina/genética , Heterocromatina/genética , Animais , Fibroblastos , Camundongos
6.
J Phys Chem A ; 123(14): 3068-3073, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30888820

RESUMO

Rapid energy transfer from electronic to nuclear degrees of freedom underlies many biological processes and astrophysical observations. The efficiency of this energy transfer depends strongly on the complex interplay between electronic and nuclear motions. In this study, we report two-color pump-probe experiments that probe the relaxation dynamics of highly excited cationic states of naphthalene, a prototypical polycyclic aromatic hydrocarbon molecule, which are produced using wavelength-selected, ultrashort extreme ultraviolet pulses. Surprisingly, the relaxation lifetimes increase with the cationic excitation energy. We postulate that the observed effect is the result of a population trapping that leads to delayed relaxation.

7.
J Phys Chem Lett ; 9(22): 6649-6655, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30388021

RESUMO

Time-resolved valence photoelectron spectroscopy is an established tool for studies of ultrafast molecular dynamics in the gas phase. Here we demonstrate time-resolved XUV photoelectron spectroscopy from dilute aqueous solutions of organic molecules, paving the way to application of this method to photodynamics studies of organic molecules in natural environments, which so far have only been accessible to all-optical transient spectroscopies. We record static and time-resolved photoelectron spectra of a sample molecule, quinoline yellow WS, analyze its electronic structure, and follow the relaxation dynamics upon excitation with 400 nm pulses. The dynamics exhibit three time scales, of which a 250 ± 70 fs time scale is attributed to solvent rearrangement. The two longer time scales of 1.3 ± 0.4 and 90 ± 20 ps can be correlated to the recently proposed ultrafast excited-state intramolecular proton transfer in a closely related molecule, quinophthalone.

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