Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A micropatterning approach for imaging dynamic Cx43 trafficking to cell-cell borders.

ABSTRACT: The precise expression and timely delivery of connexin 43 (Cx43) proteins to form gap junctions are essential for electrical coupling of cardiomyocytes. Growing evidence supports a cytoskeletal-based trafficking paradigm for Cx43 delivery directly to adherens junctions at the intercalated disc. A limitation of Cx43 localization assays in cultured cells, in which cell-cell contacts are essential, is the inability to control for cell geometry or reproducibly generate contact points. Here we present a micropatterned cell pairing system well suited for live microscopy to examine how the microtubule and actin cytoskeleton confer specificity to Cx43 trafficking to precisely defined cell-cell junctions. This system can be adapted for other cell types and used to study dynamic intracellular movements of other proteins important for cell-cell communication.

SUBMITTER: Zhang SS

PROVIDER: S-EPMC3989419 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

A micropatterning approach for imaging dynamic Cx43 trafficking to cell-cell borders.

Zhang Shan-Shan SS Hong SoonGweon S Kléber André G AG Lee Luke P LP Shaw Robin M RM

FEBS letters 20140117 8

The precise expression and timely delivery of connexin 43 (Cx43) proteins to form gap junctions are essential for electrical coupling of cardiomyocytes. Growing evidence supports a cytoskeletal-based trafficking paradigm for Cx43 delivery directly to adherens junctions at the intercalated disc. A limitation of Cx43 localization assays in cultured cells, in which cell-cell contacts are essential, is the inability to control for cell geometry or reproducibly generate contact points. Here we presen ...[more]

PMID: 24444605

Similar Datasets

Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction.

Project description:Recent results from animal studies suggest that stem cells may be able to home to sites of myocardial injury to assist in tissue regeneration. However, the histological interpretation of postmortem tissue, on which many of these studies are based, has recently been widely debated.With the use of the high sensitivity of a combined single-photon emission CT (SPECT)/CT scanner, the in vivo trafficking of allogeneic mesenchymal stem cells (MSCs) colabeled with a radiotracer and MR contrast agent to acute myocardial infarction was dynamically determined. Redistribution of the labeled MSCs after intravenous injection from initial localization in the lungs to nontarget organs such as the liver, kidney, and spleen was observed within 24 to 48 hours after injection. Focal and diffuse uptake of MSCs in the infarcted myocardium was already visible in SPECT/CT images in the first 24 hours after injection and persisted until 7 days after injection and was validated by tissue counts of radioactivity. In contrast, MRI was unable to demonstrate targeted cardiac localization of MSCs in part because of the lower sensitivity of MRI.Noninvasive radionuclide imaging is well suited to dynamically track the biodistribution and trafficking of mesenchymal stem cells to both target and nontarget organs.

| S-EPMC1456731 | biostudies-literature

Dynamic imaging of cannabinoid receptor 1 vesicular trafficking in cultured astrocytes.

Project description:Astrocytes possess GPCRs (G-protein-coupled receptors) for neuroactive substances and can respond via these receptors to signals originating from neurons as well as astrocytes. Like many transmembrane proteins, GPCRs exist in a dynamic equilibrium between receptors expressed at the plasma membrane and those present within intracellular trafficking compartments. The characteristics of GPCR trafficking within astrocytes have not been investigated. We therefore monitored the trafficking of recombinant fluorescent protein chimeras of the CB1R (cannabinoid receptor 1) that is thought to be expressed natively in astrocytes. CB1R chimeras displayed a marked punctate intracellular localization when expressed in cultured rat visual cortex astrocytes, an expression pattern reminiscent of native CB1R expression in these cells. Based upon trafficking characteristics, we found the existence of two populations of vesicular CB1R puncta: (i) relatively immobile puncta with movement characteristic of diffusion and (ii) mobile puncta with movement characteristic of active transport along cytoskeletal elements. The predominant direction of active transport is oriented radially to/from the nuclear region, which can be abolished by disruption of the microtubule cytoskeleton. CB1R puncta are localized within intracellular acidic organelles, mainly co-localizing with endocytic compartments. Constitutive trafficking of CB1R to and from the plasma membrane is an energetically costly endeavour whose function is at present unclear in astrocytes. However, given that intracellular CB1Rs can engage cell signalling pathways, it is likely that this process plays an important regulatory role.

| S-EPMC2828888 | biostudies-literature

Dynamic changes in protein interaction between AKAP95 and Cx43 during cell cycle progression of A549 cells.

Project description:Here we show that A-kinase anchoring protein 95 (AKAP95) and connexin 43 (Cx43) dynamically interact during cell cycle progression of lung cancer A549 cells. Interaction between AKAP95 and Cx43 at different cell cycle phases was examined by tandem mass spectrometry(MS/MS), confocal immunofluorescence microscopy, Western blot, and co-immunoprecipitation(Co-IP). Over the course of a complete cell cycle, interaction between AKAP95 and Cx43 occurred in two stages: binding stage from late G1 to metaphase, and separating stage from anaphase to late G1. The binding stage was further subdivided into complex binding to DNA in interphase and complex separating from DNA in metaphase. In late G1, Cx43 translocated to the nucleus via AKAP95; in anaphase, Cx43 separated from AKAP95 and aggregated between two daughter nuclei. In telophase, Cx43 aggregated at the membrane of the cleavage furrow. After mitosis, Cx43 was absent from the furrow membrane and was located in the cytoplasm. Binding between AKAP95 and Cx43 was reduced by N-(2-[P-Bromocinnamylamino]-ethyl)-5-isoquinolinesulfonmide (H89) treatment and enhanced by Forskolin. dynamic interaction between AKAP95 and Cx43 varies with cell cycle progression to regulate multiple biological processes.

| S-EPMC4754773 | biostudies-other

Microfluidics-based laser cell-micropatterning system.

Project description:The ability to place individual cells into an engineered microenvironment in a cell-culture model is critical for the study of in vivo relevant cell-cell and cell-extracellular matrix interactions. Microfluidics provides a high-throughput modality to inject various cell types into a microenvironment. Laser guided systems provide the high spatial and temporal resolution necessary for single-cell micropatterning. Combining these two techniques, the authors designed, constructed, tested and evaluated (1) a novel removable microfluidics-based cell-delivery biochip and (2) a combined system that uses the novel biochip coupled with a laser guided cell-micropatterning system to place individual cells into both two-dimensional (2D) and three-dimensional (3D) arrays. Cell-suspensions of chick forebrain neurons and glial cells were loaded into their respective inlet reservoirs and traversed the microfluidic channels until reaching the outlet ports. Individual cells were trapped and guided from the outlet of a microfluidic channel to a target site on the cell-culture substrate. At the target site, 2D and 3D pattern arrays were constructed with micron-level accuracy. Single-cell manipulation was accomplished at a rate of 150 μm s(-1) in the radial plane and 50 μm s(-1) in the axial direction of the laser beam. Results demonstrated that a single-cell can typically be patterned in 20-30 s, and that highly accurate and reproducible cellular arrays and systems can be achieved through coupling the microfluidics-based cell-delivery biochip with the laser guided system.

| S-EPMC4354940 | biostudies-literature

Laser-guided cell micropatterning system.

Project description:Employing optical force, our laser-guided cell micropatterning system, is capable of patterning different cell types onto and within standard cell research devices, including commercially available multielectrode arrays (MEAs) with glass culture rings, 35 mm Petri dishes, and microdevices microfabricated with polydimethylsiloxane on 22 mm × 22 mm cover glasses. We discuss the theory of optical forces for generating laser guidance and the calculation of optimal beam characteristics for cell guidance. We describe the hardware design and software program for the cell patterning system. Finally, we demonstrate the capabilities of the system by (1) patterning neurons to form an arbitrary pattern, (2) patterning neurons onto the electrodes of a standard MEA, and (3) patterning and aligning adult cardiomyocytes in a polystyrene Petri dish.

| S-EPMC3045411 | biostudies-other

A Simple Approach to MXene Micropatterning from Molecularly Driven Assembly.

Project description:Here, a micropatterning strategy is demonstrated to achieve stable and selective MXene adsorption through the molecularly driven assembly. MXene flakes were assembled by strong interaction with a silicon substrate, which was functionalized by microcontact printing (μCP) to create an active surface. A clear micropattern was observed by scanning electron microscopy showing uniform coverage of MXene flakes. Atomic force microscopy revealed a pattern thickness of around 50 nm, much thinner than the patterns obtained by direct μCP. The obtained micropattern presents good stability against rinsing and sonication. X-ray photoelectron spectroscopy shows that this stability can be attributed to strong covalent bonding between MXene and active molecules on a silicon substrate. The sheet resistance of the as-formed MXene layer was measured at around 154.67 (Ω/□), which is lower than those of other published techniques with a similar thickness of around 50 nm. This method can achieve a well-defined MXene pattern around the sub-100 μm scale without requiring prior MXene surface modification. Therefore, MXene can retain its intrinsic surface property, allowing further molecule adsorption as a sensing platform. Moreover, this patterning technique does not require complicated control of ink preparation and offers possible application on a substrate of any geometry with few layers of thickness.

| S-EPMC8717565 | biostudies-literature

Live cell imaging reveals continuous STAT6 nuclear trafficking.

Project description:The STAT6 transcription factor is essential for the development of protective immunity; however, the consequences of its activity can also contribute to the pathogenesis of autoimmune disease. Tyrosine phosphorylation is known to activate STAT6 in response to cytokine stimulation, but there is a gap in our understanding of the mechanisms by which it enters the nucleus. In this study, live cell imaging was used in conjunction with photobleaching techniques to demonstrate the continual nuclear import of STAT6, independent of tyrosine phosphorylation. The protein domain required for nuclear entry includes the coiled coil region of STAT6 and functions similarly before or after cytokine stimulation. The dynamic nuclear shuttling of STAT6 seems to be mediated by the classical importin-alpha-importin-beta1 system. Although STAT6 is imported to the nucleus continually, it accumulates in the nucleus following tyrosine phosphorylation as a result of its ability to bind DNA. These findings will impact diagnostic approaches and strategies to block the deleterious effects of STAT6 in autoimmunity.

| S-EPMC2891132 | biostudies-literature

Live cell imaging of duplex siRNA intracellular trafficking.

Project description:Intracellular distribution of siRNA after in vitro transfection typically depends on lipopolyplexes, which must release the siRNA into the cytosol. Here, the fate of siRNAs was monitored by FRET-based live cell imaging. Subsequent to in situ observation of uptake and release processes, this approach allowed the observation of a number of hitherto uncharacterized intracellular distribution and degradation processes, commencing with a burst of endosomal releases, followed, in some cases, by fast siRNA influx into the nucleus. The continued observation of intact siRNA against a background of free fluorophores resulting from advanced degradation was possible by a specifically developed imaging algorithm, which identified populations of intact siRNA in pixels based on FRET. This proved to be essential in the end point definition of siRNA distribution, which typically featured partially degraded siRNA pools in perinuclear structures. Our results depict the initial 4 h as a critical time window, characterized by fast initial burst release into the cytosol, which lay the foundations for subsequent intracellular distribution of siRNA. Combination with a subsequent slower, but sustained release from endosomal reservoirs may contribute to the efficiency and duration of RNAi, and explain the success of lipopolyplexes in RNAi experiments in cell culture.

| S-EPMC4482072 | biostudies-literature

Identifying epithelial borders in cholesteatoma surgery using narrow band imaging.

Project description:PurposeTo quantify changes in the perceived epithelial border with narrow band imaging (NBI) and white light imaging (WLI) during cholesteatoma surgery and to objectify possible benefits of NBI in otology.MethodsPerioperative digital endoscopic images were captured during combined approach tympanoplasty at our tertiary referral center using WLI and NBI (415 nm and 540 nm wavelengths). Sixteen otologic surgeon defined the epithelial borders within 16 identical WLI and NBI photos. Pixels of these selections were calculated to analyze the quantitative difference between WLI and NBI. A questionnaire also analyzed the qualitative differences.ResultsSixteen otologic surgeons participated in the study. Stratified per photo, only two photos yielded a significant difference: less pixels were selected with NBI than WLI. A Bland-Altman plot showed no systemic error. Stratified per otologist, four participants selected significantly more pixels with WLI than with NBI. Overall, no significant difference between selected pixels was found. Sub-analyses of surgeons with more than 5 years of experience yielded no additional findings. Despite these results, 60% believed NBI could be advantageous in defining epithelial borders, of which 83% believed NBI could reduce the risk of residual disease.ConclusionThere was no objective difference in the identification of epithelial borders with NBI compared to WLI in cholesteatoma surgery. Therefore, we do not expect the use of NBI to evidently decrease the risk of residual cholesteatoma. However, subjective assessment does suggest a possible benefit of lighting techniques in otology.Level of evidence: 3

| S-EPMC9130169 | biostudies-literature

Cell geometry guides the dynamic targeting of apoplastic GPI-linked lipid transfer protein to cell wall elements and cell borders in Arabidopsis thaliana.

Project description:During cellular morphogenesis, changes in cell shape and cell junction topology are fundamental to normal tissue and organ development. Here we show that apoplastic Glycophosphatidylinositol (GPI)-anchored Lipid Transfer Protein (LTPG) is excluded from cell junctions and flat wall regions, and passively accumulates around their borders in the epidermal cells of Arabidopsis thaliana. Beginning with intense accumulation beneath highly curved cell junction borders, this enrichment is gradually lost as cells become more bulbous during their differentiation. In fully mature epidermal cells, YFP-LTPG often shows a fibrous cellulose microfibril-like pattern within the bulging outer faces. Physical contact between a flat glass surface and bulbous cell surface induces rapid and reversible evacuation from contact sites and accumulation to the curved wall regions surrounding the contact borders. Thus, LTPG distribution is dynamic, responding to changes in cell shape and wall curvature during cell growth and differentiation. We hypothesize that this geometry-based mechanism guides wax-carrying LTPG to functional sites, where it may act to "seal" the vulnerable border surrounding cell-cell junctions and assist in cell wall fortification and cuticular wax deposition.

| S-EPMC3832655 | 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