Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Real-time investigation of dynamic protein crystallization in living cells.

ABSTRACT: X-ray crystallography requires sufficiently large crystals to obtain structural insights at atomic resolution, routinely obtained in vitro by time-consuming screening. Recently, successful data collection was reported from protein microcrystals grown within living cells using highly brilliant free-electron laser and third-generation synchrotron radiation. Here, we analyzed in vivo crystal growth of firefly luciferase and Green Fluorescent Protein-tagged reovirus ?NS by live-cell imaging, showing that dimensions of living cells did not limit crystal size. The crystallization process is highly dynamic and occurs in different cellular compartments. In vivo protein crystallization offers exciting new possibilities for proteins that do not form crystals in vitro.

SUBMITTER: Schonherr R

PROVIDER: S-EPMC4711630 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Real-time investigation of dynamic protein crystallization in living cells.

Schönherr R R Klinge M M Rudolph J M JM Fita K K Rehders D D Lübber F F Schneegans S S Majoul I V IV Duszenko M M Betzel C C Brandariz-Nuñez A A Martinez-Costas J J Duden R R Redecke L L

Structural dynamics (Melville, N.Y.) 20150522 4

X-ray crystallography requires sufficiently large crystals to obtain structural insights at atomic resolution, routinely obtained in vitro by time-consuming screening. Recently, successful data collection was reported from protein microcrystals grown within living cells using highly brilliant free-electron laser and third-generation synchrotron radiation. Here, we analyzed in vivo crystal growth of firefly luciferase and Green Fluorescent Protein-tagged reovirus μNS by live-cell imaging, showing ...[more]

PMID: 26798811

Similar Datasets

Real-time visualization of heterotrimeric G protein Gq activation in living cells.

Project description:BACKGROUND: Gq is a heterotrimeric G protein that plays an important role in numerous physiological processes. To delineate the molecular mechanisms and kinetics of signalling through this protein, its activation should be measurable in single living cells. Recently, fluorescence resonance energy transfer (FRET) sensors have been developed for this purpose. RESULTS: In this paper, we describe the development of an improved FRET-based Gq activity sensor that consists of a yellow fluorescent protein (YFP)-tagged G?2 subunit and a G?q subunit with an inserted monomeric Turquoise (mTurquoise), the best cyan fluorescent protein variant currently available. This sensor enabled us to determine, for the first time, the kon (2/s) of Gq activation. In addition, we found that the guanine nucleotide exchange factor p63RhoGEF has a profound effect on the number of Gq proteins that become active upon stimulation of endogenous histamine H1 receptors. The sensor was also used to measure ligand-independent activation of the histamine H1 receptor (H1R) upon addition of a hypotonic stimulus. CONCLUSIONS: Our observations reveal that the application of a truncated mTurquoise as donor and a YFP-tagged G?2 as acceptor in FRET-based Gq activity sensors substantially improves their dynamic range. This optimization enables the real-time single cell quantification of Gq signalling dynamics, the influence of accessory proteins and allows future drug screening applications by virtue of its sensitivity.

| S-EPMC3129320 | biostudies-other

Genetically encoded tags for real time dissection of protein assembly in living cells.

Project description:Simple methods with straightforward readouts that enable real-time interrogation of protein quaternary structure are much needed to facilitate the physicochemical characterization of proteins at the single-cell level. After screening over a series of microtubule (MT) binders, we report herein the development of two genetically encoded tags (designated as "MoTags" for the monomer/oligomer detection tag) that can be conveniently fused to a given protein to probe its oligomeric state in cellulo when combined with routine fluorescence microscopy. In their monomeric form, MoTags are evenly distributed in the cytosol; whereas oligomerization enables MoTags to label MT or track MT tips in an oligomeric state-dependent manner. We demonstrate here the broad utility of engineered MoTags to aid the determination of protein oligomeric states, dissection of protein structure and function, and monitoring of protein-target interactions under physiological conditions in living cells.

| S-EPMC6048692 | biostudies-literature

Real-time observation of light-controlled transcription in living cells

Project description:Gene expression is tightly regulated in space and time. To dissect this process with high temporal resolution, we introduce an optogenetic tool termed BLInCR (Blue Light-Induced Chromatin Recruitment) that combines rapid and reversible light-dependent recruitment of effector proteins with a real-time readout for transcription. We used BLInCR to control the activity of a reporter gene cluster in the human osteosarcoma cell line U2OS by reversibly recruiting the viral transactivator VP16. RNA production was detectable ~2 minutes after VP16 recruitment and readily decreased when VP16 dissociated from the cluster in the absence of light. Quantitative assessment of the activation process revealed biphasic activation kinetics with a pronounced early phase in cells treated with the histone deacetylase inhibitor SAHA. Comparison with kinetic models for transcription activation suggests that the gene cluster undergoes a maturation process when activated. We anticipate that BLInCR will facilitate the study of transcription dynamics in living cells.

2017-11-22 | GSE103868 | GEO

Dynamic multicolor protein labeling in living cells.

Project description:Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST, hereafter called FAST) is a 14 kDa protein tag giving a bright green-yellow fluorescent complex upon interaction with the fluorogenic dye 4-hydroxy-3-methylbenzylidene rhodanine (HMBR). Here, we report a collection of fluorogens enabling tuning of the fluorescence color of FAST from green-yellow to orange and red. Beyond allowing the multicolor imaging of FAST-tagged proteins in live cells, these fluorogens enable dynamic color switching because of FAST's reversible labeling. This unprecedented behavior allows for selective detection of FAST-tagged proteins in cells expressing both green and red fluorescent species through two-color cross-correlation, opening up exciting prospects to overcome spectral crowding and push the frontiers of multiplexed imaging.

| S-EPMC5618792 | biostudies-literature

Real-time monitoring of caspase cascade activation in living cells.

Project description:We introduce a simple, versatile and robust one-step technique that enables real-time imaging of multiple intracellular caspase activities in living cells without the need for complicated synthetic protocols. Conventional fluorogenic probes or recently reported activatable probes have been designed to target various proteases but are limited to extracellular molecules. Only a few have been applied to image intracellular proteases in living cells because most of these probes have limited cell-permeability. Our platform does not need complicated synthetic processes; instead it involves a straightforward peptide synthesis and a simple mixing step with a commercial transfection agent. The transfection agent efficiently delivered the highly quenched fluorogenic probes, comprised of distinctive pairs of dyes and quenchers, to the initiator caspase-8 and the effector caspase-3 in MDA-MB-435 cells, allowing dual-imaging of the activities of both caspases during the apoptotic process induced by TNF-related apoptosis induced ligand (TRAIL). With the combination of multiple fluorogenic probes, this simple platform can be applied to multiplexed imaging of selected intracellular proteases to study apoptotic processes in pathologies or for cell-based high throughput screening systems for drug discovery.

| S-EPMC3462246 | biostudies-literature

Real-time imaging of histone H4 hyperacetylation in living cells.

Project description:To visualize histone acetylation in living cells, we developed a genetically encoded fluorescent resonance energy transfer (FRET)-based indicator. Response of the indicator reflects changes in the acetylation state of both K5 and K8 in histone H4. Using this acetylation indicator, we were able to monitor the dynamic fluctuation of histone H4 acetylation levels during mitosis, as well as acetylation changes in response to structurally distinct histone deacetylase inhibitors.

| S-EPMC2752582 | biostudies-literature

Detecting ligand interactions in real time on living bacterial cells.

Project description:Time-resolved analysis assays of receptor-ligand interactions are fundamental in basic research and drug discovery. Adequate methods are well developed for the analysis of recombinant proteins such as antibody-antigen interactions. However, assays for time-resolved ligand-binding processes on living cells are still rare, in particular within microbiology. In this report, the real-time cell-binding assay (RT-CBA) technology LigandTracer®, originally designed for mammalian cell culture, was extended to cover Gram-positive and Gram-negative bacteria. This required the development of new immobilization methods for bacteria, since LigandTracer depends on cells being firmly attached to a Petri dish. The evaluated Escherichia coli CJ236 and BL21 as well as Staphylococcus carnosus TM300 strains were immobilized to plastic Petri dishes using antibody capture, allowing us to depict kinetic binding traces of fluorescently labeled antibodies directed against surface-displayed bacterial proteins for as long as 10-15 h. Interaction parameters, such as the affinity and kinetic constants, could be estimated with high precision (coefficient of variation 9-44%) and the bacteria stayed viable for at least 16 h. The other tested attachment protocols were inferior to the antibody capture approach. Our attachment protocol is generic and could potentially also be applied to other assays and purposes.

| S-EPMC5895670 | biostudies-literature

A versatile platform to analyze low-affinity and transient protein-protein interactions in living cells in real time.

Project description:Protein-protein interactions (PPIs) play central roles in orchestrating biological processes. While some PPIs are stable, many important ones are transient and hard to detect with conventional approaches. We developed ReBiL, a recombinase enhanced bimolecular luciferase complementation platform, to enable detection of weak PPIs in living cells. ReBiL readily identified challenging transient interactions between an E3 ubiquitin ligase and an E2 ubiquitin-conjugating enzyme. ReBiL's ability to rapidly interrogate PPIs in diverse conditions revealed that some stapled α-helical peptides, a class of PPI antagonists, induce target-independent cytosolic leakage and cytotoxicity that is antagonized by serum. These results explain the requirement for serum-free conditions to detect stapled peptide activity, and define a required parameter to evaluate for peptide antagonist approaches. ReBiL's ability to expedite PPI analysis, assess target specificity and cell permeability, and reveal off-target effects of PPI modifiers should facilitate the development of effective, cell-permeable PPI therapeutics and the elaboration of diverse biological mechanisms.

| S-EPMC4269221 | biostudies-literature

Real-time observation of flow-induced cytoskeletal stress in living cells.

Project description:The mechanical stress due to shear flow has profound effects on cell proliferation, transport, gene expression, and apoptosis. The mechanisms for flow sensing and transduction are unclear, but it is postulated that fluid flow pulls upon the apical surface, and the resulting stress is eventually transmitted through the cytoskeleton to adhesion plaques on the basal surface. Here we report a direct observation of this flow-induced stress in the cytoskeleton in living cells using a parallel plate microfluidic chip with a fluorescence resonance energy transfer (FRET)-based mechanical stress sensor in actinin. The sensing cassette was genetically inserted into the cytoskeletal host protein and transfected into Madin-Darby canine kidney cells. A shear stress of 10 dyn/cm(2) resulted in a rapid increase in the FRET ratio indicating a decrease in stress across actinin with flow. The effect was reversible, and cells were able to respond to repeated stimulation and showed adaptive changes in the cytoskeleton. Flow-induced Ca(2+) elevation did not affect the response, suggesting that flow-induced changes in actinin stress are insensitive to intracellular Ca(2+) level. The reduction in FRET ratio suggests actin filaments are under normal compression in the presence of flow shear stress due to changes in cell shape, and/or actinin is not in series with actin. Treatment with cytochalasin-D that disrupts F-actin reduced prestress and the response to flow. The FRET/flow method is capable of resolving changes of stress in multiple proteins with optical spatial resolution and time resolution >1 Hz. This promises to provide insight into the force distribution and transduction in all cells.

| S-EPMC3174563 | biostudies-literature

Real-time Imaging of Rabies Virus Entry into Living Vero cells.

Project description:Understanding the mechanism of rabies virus (RABV) infection is vital for prevention and therapy of virulent rabies. However, the infection mechanism remains largely uncharacterized due to the limited methods and viral models. Herein, we utilized a powerful single-virus tracking technique to dynamically and globally visualize the infection process of the live attenuated rabies vaccine strain-SRV9 in living Vero cells. Firstly, it was found that the actin-enriched filopodia is in favor of virus reaching to the cell body. Furthermore, by carrying out drug perturbation experiments, we confirmed that RABV internalization into Vero cells proceeds via classical dynamin-dependent clathrin-mediated endocytosis with requirement for intact actin, but caveolae-dependent endocytosis is not involved. Then, our real-time imaging results unambiguously uncover the characteristics of viral internalization and cellular transport dynamics. In addition, our results directly and quantitatively reveal that the intracellular motility of internalized RABV particles is largely microtubule-dependent. Collectively, our work is crucial for understanding the initial steps of RABV infection, and elucidating the mechanisms of post-infection. Significantly, the results provide profound insight into development of novel and effective antiviral targets.

| S-EPMC4493577 | 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