Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Quantitative evaluation of macromolecular crowding environment based on translational and rotational diffusion using polarization dependent fluorescence correlation spectroscopy.

ABSTRACT: Macromolecular crowding (MMC) in cells is a hot topic in biology; therefore, well-characterized measurement standards for the evaluation of the nano-environment in MMC solutions are necessary. We propose to use polarization-dependent fluorescence correlation spectroscopy (Pol-FCS) for evaluation of macromolecular crowding in solutions. Pol-FCS can simultaneously measure the relaxation times of rotational and translational diffusion of fluorescent molecules at the same position, even in living cells with low damage. In this report, the differences in the nano-environment among solutions of small molecules, gels, and MMC solutions were evaluated by comparing their rotational and translational diffusion using Pol-FCS. Moreover, this method could distinguish the phase shift in the polyethylene glycol solution. Finally, we separately evaluated the nano-environment in the cytosol and nucleus of living cells in different cell lines and cell cycles. We expect this evaluation method to be useful in characterizing the nano-environment in MMC studies. In addition, the proposed method may be useful for other nano-environments such as liquid-liquid phase separation.

SUBMITTER: Yamamoto J

PROVIDER: S-EPMC8134472 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Rotational and translational diffusion of peptide-coated CdSe/CdS/ZnS nanorods studied by fluorescence correlation spectroscopy.

Project description:CdSe/CdS/ZnS nanorods (NRs) of three aspect ratios were coated with phytochelatin-related peptides and studied using fluorescence correlation spectroscopy (FCS). Theoretical predictions of the NRs' rotational diffusion contribution to the correlation curves were experimentally confirmed. We monitored rotational and translational diffusion of NRs and extracted hydrodynamic radii from the extracted diffusion constants. Translational and rotational diffusion constants (D(trans) and D(rot)) for NRs were in good agreement with Tirado and Garcia de la Torre's as well as with Broersma's theories when accounting for the ligand dimensions. NRs fall in the size range where rotational diffusion can be monitored with higher sensitivity than translational diffusion due to a steeper length dependence, D(rot) approximately L(-)(3) versus D(trans) approximately L(-)(1). By titrating peptide-coated NRs with bovine serum albumin, we monitored (nonspecific) binding through rotational diffusion and showed that D(rot) is an advantageous observable for monitoring binding. Monitoring rotational diffusion of bioconjugated NRs using FCS might prove to be useful for observing binding and conformational dynamics in biological systems.

| S-EPMC2535805 | biostudies-literature

Cross-correlation analysis of X-ray photon correlation spectroscopy to extract rotational diffusion coefficients.

Project description:Coefficients for translational and rotational diffusion characterize the Brownian motion of particles. Emerging X-ray photon correlation spectroscopy (XPCS) experiments probe a broad range of length scales and time scales and are well-suited for investigation of Brownian motion. While methods for estimating the translational diffusion coefficients from XPCS are well-developed, there are no algorithms for measuring the rotational diffusion coefficients based on XPCS, even though the required raw data are accessible from such experiments. In this paper, we propose angular-temporal cross-correlation analysis of XPCS data and show that this information can be used to design a numerical algorithm (Multi-Tiered Estimation for Correlation Spectroscopy [MTECS]) for predicting the rotational diffusion coefficient utilizing the cross-correlation: This approach is applicable to other wavelengths beyond this regime. We verify the accuracy of this algorithmic approach across a range of simulated data.

| S-EPMC8403868 | biostudies-literature

Hindered diffusion in polymeric solutions studied by fluorescence correlation spectroscopy.

Project description:Diffusion of molecules in the crowded and charged interior of the cell has long been of interest for understanding cellular processes. Here, we introduce a model system of hindered diffusion that includes both crowding and binding. In particular, we obtained the diffusivity of the positively charged protein, ribonuclease A (RNase), in solutions of dextrans of various charges (binding) and concentrations (crowding), as well as combinations of both, in a buffer of physiological ionic strength. Using fluorescence correlation spectroscopy, we observed that the diffusivity of RNase was unaffected by the presence of positively charged or neutral dextrans in the dilute regime but was affected by crowding at higher polymer concentrations. Conversely, protein diffusivity was significantly reduced by negatively charged dextrans, even at 0.4 ?M (0.02% w/v) dextran. The diffusivity of RNase decreased with increasing concentrations of negative dextran, and the amount of bound RNase increased until it reached a plateau of ?80% bound RNase. High salt concentrations were used to establish the electrostatic nature of the binding. Binding of RNase to the negatively charged dextrans was further confirmed by ultrafiltration.

| S-EPMC3127197 | biostudies-other

Photosystem II antenna phosphorylation-dependent protein diffusion determined by fluorescence correlation spectroscopy.

Project description:Flexibility of chloroplast thylakoid membrane proteins is essential for plant fitness and survival under fluctuating light environments. Phosphorylation of light-harvesting antenna complex II (LHCII) is known to induce dynamic protein reorganization that fine-tunes the rate of energy conversion in each photosystem. However, molecular details of how LHCII phosphorylation causes light energy redistribution throughout thylakoid membranes still remain unclear. By using fluorescence correlation spectroscopy, we here determined the LHCII phosphorylation-dependent protein diffusion in thylakoid membranes isolated from the green alga Chlamydomonas reinhardtii. As compared to the LHCII dephosphorylation-induced condition, the diffusion coefficient of LHCII increased nearly twofold under the LHCII phosphorylation-induced condition. We also verified the results by using the LHCII phosphorylation-deficient mutant. Our observation suggests that LHCII phosphorylation-dependent protein reorganization occurs along with the changes in the rate of protein diffusion, which would have an important role in mediating light energy redistribution throughout thylakoid membranes.

| S-EPMC3789154 | biostudies-literature

Macromolecular Crowding Regulates the Gene Expression Profile by Limiting Diffusion.

Project description:We seek to elucidate the role of macromolecular crowding in transcription and translation. It is well known that stochasticity in gene expression can lead to differential gene expression and heterogeneity in a cell population. Recent experimental observations by Tan et al. have improved our understanding of the functional role of macromolecular crowding. It can be inferred from their observations that macromolecular crowding can lead to robustness in gene expression, resulting in a more homogeneous cell population. We introduce a spatial stochastic model to provide insight into this process. Our results show that macromolecular crowding reduces noise (as measured by the kurtosis of the mRNA distribution) in a cell population by limiting the diffusion of transcription factors (i.e. removing the unstable intermediate states), and that crowding by large molecules reduces noise more efficiently than crowding by small molecules. Finally, our simulation results provide evidence that the local variation in chromatin density as well as the total volume exclusion of the chromatin in the nucleus can induce a homogenous cell population.

| S-EPMC5125560 | biostudies-literature

Diffusion, transport, and cell membrane organization investigated by imaging fluorescence cross-correlation spectroscopy.

Project description:Cell membrane organization is dynamic and is assumed to have different characteristic length scales. These length scales, which are influenced by lipid and protein composition as well as by the cytoskeleton, can range from below the optical resolution limit (as with rafts or microdomains) to far above the resolution limit (as with capping phenomena or the formation of lipid "platforms"). The measurement of these membrane features poses a significant problem because membrane dynamics are on the millisecond timescale and are thus beyond the time resolution of conventional imaging approaches. Fluorescence correlation spectroscopy (FCS), a widely used spectroscopic technique to measure membrane dynamics, has the required time resolution but lacks imaging capabilities. A promising solution is the recently introduced method known as imaging total internal reflection (ITIR)-FCS, which can probe diffusion phenomena in lipid membranes with good temporal and spatial resolution. In this work, we extend ITIR-FCS to perform ITIR fluorescence cross-correlation spectroscopy (ITIR-FCCS) between pixel areas of arbitrary shape and derive a generalized expression that is applicable to active transport and diffusion. ITIR-FCCS is applied to model systems exhibiting diffusion, active transport, or a combination of the two. To demonstrate its applicability to live cells, we observe the diffusion of a marker, the sphingolipid-binding domain (SBD) derived from the amyloid peptide Abeta, on live neuroblastoma cells. We investigate the organization and dynamics of SBD-bound lipid microdomains under the conditions of cholesterol removal and cytoskeleton disruption.

| S-EPMC2770616 | biostudies-literature

Statistical Analysis of Scanning Fluorescence Correlation Spectroscopy Data Differentiates Free from Hindered Diffusion.

Project description:Cells rely on versatile diffusion dynamics in their plasma membrane. Quantification of this often heterogeneous diffusion is essential to the understanding of cell regulation and function. Yet such measurements remain a major challenge in cell biology, usually due to low sampling throughput, a necessity for dedicated equipment, sophisticated fluorescent label strategies, and limited sensitivity. Here, we introduce a robust, broadly applicable statistical analysis pipeline for large scanning fluorescence correlation spectroscopy data sets, which uncovers the nanoscale heterogeneity of the plasma membrane in living cells by differentiating free from hindered diffusion modes of fluorescent lipid and protein analogues.

| S-EPMC6117752 | biostudies-literature

Polarization-dependent fluorescence correlation spectroscopy for studying structural properties of proteins in living cell.

Project description:Rotational diffusion measurement is predicted as an important method in cell biology because the rotational properties directly reflect molecular interactions and environment in the cell. To prove this concept, polarization-dependent fluorescence correlation spectroscopy (pol-FCS) measurements of purified fluorescent proteins were conducted in viscous solution. With the comparison between the translational and rotational diffusion coefficients obtained from pol-FCS measurements, the hydrodynamic radius of an enhanced green fluorescent protein (EGFP) was estimated as a control measurement. The orientation of oligomer EGFP in living cells was also estimated by pol-FCS and compared with Monte Carlo simulations. The results of this pol-FCS experiment indicate that this method allows an estimation of the molecular orientation using the characteristics of rotational diffusion. Further, it can be applied to analyze the degree of molecular orientation and multimerization or detection of tiny aggregation of aggregate-prone proteins.

| S-EPMC4973283 | biostudies-literature

Discrepancy between fluorescence correlation spectroscopy and fluorescence recovery after photobleaching diffusion measurements of G-protein-coupled receptors.

Project description:Fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) are the two most direct methods to measure the diffusion of molecules in intact living cells. Ideally, these methods should produce similar results for an identical system. We have used these methods to monitor the diffusion of two G-protein-coupled receptors and their associated proteins in the plasma membranes of cells that do not or do contain invaginated protein domains called caveolae. FRAP studies show that caveolae domains increase the immobile fraction of receptors without significantly changing their mobility. On the other hand, FCS studies show an unexpected increase the mobility of caveolae-associated proteins. Our data suggest that the geometry of caveolae domains gives rise to a confined diffusion of its attached proteins, resulting in an apparent increase in mobility.

| S-EPMC3770895 | biostudies-literature

Anomalous Diffusion Inside Soft Colloidal Suspensions Investigated by Variable Length Scale Fluorescence Correlation Spectroscopy.

Project description:The diffusion of molecules and particles inside the aqueous suspension of soft colloids (polymer microgels) is investigated using variable length scale fluorescence correlation spectroscopy (VLS-FCS). Carbopol 940 is chosen as the model matrix system, and two factors affecting diffusion are investigated: the spatial hindrance and the diffusant-matrix interaction. By studying diffusion of molecules and particles with different sizes inside the suspension, VLS-FCS reveals the restricted motion at a short length scale, that is, in the gaps between the microgels, and normal diffusion at a larger length scale. The information on the gap's length scale is also accessed. On the other hand, by tuning the pH value, the diffusant-matrix electrostatic attraction is adjusted and the results expose a short-time fast diffusion of probe molecules inside the gaps and a long-time restricted diffusion because of trapping inside the microgels. It is proved that VLS-FCS is a powerful method, investigating anomalous diffusion at different length scales and it is a promising approach to investigate diffusion in complex soft matter systems.

| S-EPMC7241028 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data