Ionic Strength Sensing in Living Cells.
Ontology highlight
ABSTRACT:
SUBMITTER: Liu B
PROVIDER: S-EPMC5653947 | biostudies-literature | 2017 Oct
REPOSITORIES: biostudies-literature
Publications
Ionic Strength Sensing in Living Cells.
Liu Boqun B Poolman Bert B Boersma Arnold J AJ
ACS chemical biology 20170906 10
Knowledge of the ionic strength in cells is required to understand the in vivo biochemistry of the charged biomacromolecules. Here, we present the first sensors to determine the ionic strength in living cells, by designing protein probes based on Förster resonance energy transfer (FRET). These probes allow observation of spatiotemporal changes in the ionic strength on the single-cell level. ...[more]
Similar Datasets
Project description:Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen-NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.
| S-EPMC7826950 | biostudies-literature
Project description:Luminescence thermometry usually suffer from cellular complexity of the biochemical environment (such as pH and ionic strength), and thus the accuracy and reliability of the determined intracellular temperature are directly affected. Herein, a photoluminescent nanothermometer composed of polymer encapsulated quantum dots (P-QD) has been developed. And the prepared nanothermometer exhibits some advantages: such as non-sensitivity to pH and ionic strength, as well as high detection sensitivity and ultrahigh reversibility. The intracellular temperature was accurately determined under physiological conditions with different pH and ionic strength, and direct measurement of thermogenesis in individual cells has been achieved.
| S-EPMC4597201 | biostudies-literature
Project description:Inflammation and infection can trigger local tissue Na+-accumulation. This Na+-rich environment boosts pro-inflammatory activation of monocyte/macrophage-like cells (MΦ) and their antimicrobial activity. Enhanced Na+-driven MΦ-function requires the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5), which augments NO production and contributes to increased autophagy. However, the mechanism of Na+-sensing in MΦ remained unclear. High extracellular Na+ levels (HS) trigger a substantial Na+-influx and Ca2+ loss. Here, we show that the Na+/ Ca2+-exchanger 1 (NCX1/ solute carrier family 8 member A1 (SLC8A1)) plays a critical role in HS-triggered Na+-influx, concomitant Ca2+ efflux and subsequent NFAT5 accumulation. Moreover, interfering with NCX1-activity impairs HS-boosted inflammatory signaling, infection-triggered autolysosome formation and subsequent antibacterial activity. Taken together, this demonstrates that NCX1 is able to sense Na+ and is required for amplifying inflammatory and antimicrobial MΦ responses upon HS exposure. Manipulating NCX1 offers a new strategy to regulate MΦ function.
2020-06-29 | GSE136662 | GEO
Project description:Cystathionine-beta-synthase (CBS) domains are found in >4,000 proteins in species from all kingdoms of life, yet their functions are largely unknown. Tandem CBS domains are associated with membrane transport proteins, most notably members of the ATP-binding cassette (ABC) superfamily; voltage-gated chloride channels and transporters; cation efflux systems; and various enzymes, transcription factors, and proteins of unknown function. We now show that tandem CBS domains in the osmoregulatory ABC transporter OpuA are sensors for ionic strength that control the transport activity through an electrostatic switching mechanism. The on/off state of the transporter is determined by the surface charge of the membrane and the internal ionic strength that is sensed by the CBS domains. By modifying the CBS domains, we can control the ionic strength dependence of the transporter: deleting a stretch of C-terminal anionic residues shifts the ionic strength dependence to higher values, whereas deleting the CBS domains makes the system largely independent of ionic strength. We present a model for the gating of membrane transport by ionic strength and propose a new role for CBS domains.
| S-EPMC1502282 | biostudies-literature
Project description:Integrins are major receptors for secreted extracellular matrix, playing crucial roles in physiological and pathological contexts, such as angiogenesis and cancer. Regulation of the transition between inactive and active conformation is key for integrins to fulfill their functions, and pharmacological control of those dynamics may have therapeutic applications. We create and validate a prototypic luminescent β1 integrin activation sensor (β1IAS) by introducing a split luciferase into an activation reporting site between the βI and the hybrid domains. As a recombinant protein in both solution and living cells, β1IAS accurately reports β1 integrin activation in response to (bio)chemical and physical stimuli. A short interfering RNA (siRNA) high-throughput screening on live β1IAS knockin endothelial cells unveils hitherto unknown regulators of β1 integrin activation, such as β1 integrin inhibitors E3 ligase Pja2 and vascular endothelial growth factor B (VEGF-B). This split-luciferase-based strategy provides an in situ label-free measurement of integrin activation and may be applicable to other β integrins and receptors.
| S-EPMC11861568 | biostudies-literature
Project description:Detection of specific mRNA in living cells has attracted significant attention in the past decade. Probes that can be easily delivered into cells and activated at the desired time can contribute to understanding translation, trafficking and degradation of mRNA. Here we report a new strategy termed magnetic field-activated binary deoxyribozyme (MaBiDZ) sensor that enables both efficient delivery and temporal control of mRNA sensing by magnetic field. MaBiDZ uses two species of magnetic beads conjugated with different components of a multicomponent deoxyribozyme (DZ) sensor. The DZ sensor is activated only in the presence of a specific target mRNA and when a magnetic field is applied. Here we demonstrate that MaBiDZ sensor can be internalized in live MCF-7 breast cancer cells and activated by a magnetic field to fluorescently report the presence of specific mRNA, which are cancer biomarkers.
| S-EPMC5654739 | biostudies-literature
Project description:This study investigates the impact of ionic strength on the gelation kinetics of collagen biomaterial inks and evaluates the efficiency of TGFβ-1 sequestration within these hydrogels, alongside their compatibility with bioprinting live chondrocyte and adipose-derived stem cell lines for cartilage tissue engineering. By adjusting sodium chloride and phosphate-buffered saline (PBS) concentrations, we demonstrate that reduced ionic strengths accelerate gelation, facilitating high-fidelity bioprinting while supporting high cell viability and post-printing proliferation of chondrocytes. Furthermore, at a 1% collagen concentration, the hydrogel effectively immobilized TGFβ-1, with less than 0.5% released over two weeks, indicating potent sequestration capabilities. Using adipose-derived mesenchymal stem cells, histomorphological and transcriptomic analyses reveal that the presence of TGFβ-1 significantly enhances chondrogenesis. These findings underscore the critical role of ionic strength in optimizing collagen ink properties for advanced bioprinting applications and highlight the potential of collagen hydrogels as effective carriers for sustained growth factor delivery, paving the way for successful cartilage tissue engineering strategies.
2024-10-09 | GSE271638 | GEO
Project description:Extracellular vesicles (EVs) are emerging as crucial materials for precision theragnostic applications. However, current separation methods are time-consuming, costly, and not scalable and deliver limited yields or purity. Here, we present EV precipitation by ionic strength modulation (ExoPRISM), a simple, low-cost, user-friendly, and readily adaptable approach for separating EVs in high yields without compromising their biological functions. Adding an electrolyte solution to blood plasma in small increments generates the sequential precipitation of proteins and EVs, allowing for fractional separation of EVs using low-speed centrifugation. The coprecipitated electrolytes are easily washed away, and the entire EV separation and washing process takes less than an hour. This approach successfully separates EVs from a broad range of volumes and types of biological fluids, including culture medium, urine, plasma, and serum, showing promise as a robust tool for next-generation liquid biopsies and regenerative medicine.
| S-EPMC10726304 | biostudies-literature
Project description:Understanding intracellular redox chemistry requires new tools for the site-specific visualization of intracellular oxidation. We have developed a spatially-resolved intracellular sensor of hydrogen peroxide, HyPer-Tau, for time-resolved imaging in live cells. This sensor consists of a hydrogen peroxide-sensing protein tethered to microtubules. We demonstrate the use of the HyPer-Tau sensor for three applications; dose-dependent response of human cells to exogenous hydrogen peroxide, a model immune response of mouse macrophages to stimulation by bacterial toxin, and a spatially-resolved response to localized delivery of hydrogen peroxide. These results demonstrate that HyPer-Tau can be used as an effective tool for tracking changes in spatially localized intracellular hydrogen peroxide and for future applications in redox signaling.
| S-EPMC4653655 | biostudies-literature
Project description:Cells in tissues or biofilms communicate with one another through chemical and mechanical signals to coordinate collective behaviors. Non-living cell mimics provide simplified models of natural systems; however, it has remained challenging to implement communication capabilities comparable to living cells. Here we present a porous artificial cell-mimic containing a nucleus-like DNA-hydrogel compartment that is able to express and display proteins, and communicate with neighboring cell-mimics through diffusive protein signals. We show that communication between cell-mimics allows distribution of tasks, quorum sensing, and cellular differentiation according to local environment. Cell-mimics can be manufactured in large quantities, easily stored, chemically modified, and spatially organized into diffusively connected tissue-like arrangements, offering a means for studying communication in large ensembles of artificial cells.
| S-EPMC6261949 | biostudies-literature