Browse
Submit Data
Databases
API
Help

Dataset Information

7 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A method for measuring electrical signals in a primary cilium.

ABSTRACT:

Background

Most cells in the body possess a single primary cilium. These cilia are key transducers of sensory stimuli, and defects in cilia have been linked to several diseases. Evidence suggests that some transduction of sensory stimuli by the primary cilium depends on ion-conducting channels. However, the tiny size of the cilium has been a critical barrier to understanding its electrical properties. We report a novel method that allows sensitive, repeatable electrical recordings from primary cilia. Adherent cells were grown on small, spherical beads that could be easily moved within the recording chamber. In this configuration, an entire cilium could be pulled into a recording microelectrode.

Results

In 47% of attempts, suction resulted in a seal with high input resistance. Single channels could be recorded while the cilium remained attached to the cell. When the pipette was raised into the air, the cell body was pulled off at the air-bath interface. The pipette retained the cilium and could then be immersed in various solutions that bathed the cytoplasmic face of the membrane. In excised cilia, ionic currents through ciliary channels were modulated by cytoplasmic Ca(2+) and transmembrane voltage.

Conclusions

Ciliary recording is a direct way to learn the effects of second messengers and voltage changes on ciliary transduction channels.

SUBMITTER: Kleene NK

PROVIDER: S-EPMC3539729 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Melanocortin 4 receptor signals at the neuronal primary cilium to control food intake and body weight.

Project description:The melanocortin 4 receptor (MC4R) plays a critical role in the long-term regulation of energy homeostasis, and mutations in the MC4R are the most common cause of monogenic obesity. However, the precise molecular and cellular mechanisms underlying the maintenance of energy balance within MC4R-expressing neurons are unknown. We recently reported that the MC4R localizes to the primary cilium, a cellular organelle that allows for partitioning of incoming cellular signals, raising the question of whether the MC4R functions in this organelle. Here, using mouse genetic approaches, we found that cilia were required specifically on MC4R-expressing neurons for the control of energy homeostasis. Moreover, these cilia were critical for pharmacological activators of the MC4R to exert an anorexigenic effect. The MC4R is expressed in multiple brain regions. Using targeted deletion of primary cilia, we found that cilia in the paraventricular nucleus of the hypothalamus (PVN) were essential to restrict food intake. MC4R activation increased adenylyl cyclase (AC) activity. As with the removal of cilia, inhibition of AC activity in the cilia of MC4R-expressing neurons of the PVN caused hyperphagia and obesity. Thus, the MC4R signaled via PVN neuron cilia to control food intake and body weight. We propose that defects in ciliary localization of the MC4R cause obesity in human inherited obesity syndromes and ciliopathies.

| S-EPMC8087202 | biostudies-literature

Primary Cilium-Dependent Signaling Mechanisms.

Project description:Primary cilia are hair-like organelles and play crucial roles in vertebrate development, organogenesis, health, and many genetic disorders. A primary cilium is a mechano-sensory organelle that responds to mechanical stimuli in the micro-environment. A cilium is also a chemosensor that senses chemical signals surrounding a cell. The overall function of a cilium is therefore to act as a communication hub to transfer extracellular signals into intracellular responses. Although intracellular calcium has been one of the most studied signaling messengers that transmit extracellular signals into the cells, calcium signaling by various ion channels remains a topic of interest in the field. This may be due to a broad spectrum of cilia functions that are dependent on or independent of utilizing calcium as a second messenger. We therefore revisit and discuss the calcium-dependent and calcium-independent ciliary signaling pathways of Hedgehog, Wnt, PDGFR, Notch, TGF-?, mTOR, OFD1 autophagy, and other GPCR-associated signaling. All of these signaling pathways play crucial roles in various cellular processes, such as in organ and embryonic development, cardiac functioning, planar cell polarity, transactivation, differentiation, the cell cycle, apoptosis, tissue homeostasis, and the immune response.

| S-EPMC5713242 | biostudies-literature

Photon-pixel coupling: A method for parallel acquisition of electrical signals in scientific investigations.

Project description:Here we describe a novel prototype method for parallel sampling of electrical signals from 200 sensors. The amplified signal from each sensor was remotely converted into a luminous signal on a LED matrix. A digital camera supported by a duralumin skeleton, was installed at 15 cm above an LED matrix inside an opaque box. Images were sampled at discrete time intervals of 5 s. A total of 25,920 images of the LED matrix have been recorded. Thus, 5.2 million measurements have been recorded as light intensities from the LED matrix. Light intensities of individual LEDs from the images were converted into 1 pixel value/LED. Each pixel value was then converted into percentages for evaluation. We used this methodology to measure the temporal variation of the electrical current on the skin of the torso on human volunteers, to assess the presence of a correlation between the electrical activity and diabetes (Ionescu-Tirgoviste et al., 2018). This method also allowed us to compile the first high resolution map of the electrical activity generated by the human skin (Ionescu-Tirgoviste et al., 2018). •A novel method for a parallel acquisition of electrical signals which can be applied in any related field.•It provides the ability to retrieve a large number of electrical channels simultaneously.•It provides for an inexpensive and reliable way to digitize hundreds to thousands of channels at video rate frequencies.

| S-EPMC6503210 | biostudies-literature

CATCH-IT method for measuring nucleosome turnover

Project description:Direct measurement of nucleosome turnover dynamics by using co-translational incorporation of the methionine (Met) surrogate azidohomoalaine (Aha) into proteins and subsequent ligation of biotin to Aha-containing proteins through the [3+2] cycloaddition reaction between the azide group of Aha and an alkyne linked to biotin. To measure turnover rates, we treat cells briefly with Aha, couple biotin to nucleosomes containing newly incorporated histones, affinity purify with strepavidin, wash stringently to remove non-histone proteins and H2A/H2B dimers, and analyze the affinity-purified DNA using tiling microarrays. We call this strategy 'CATCH-IT' for Covalent Attachment of Tags to Capture Histones and Identify Turnover. Keywords: Chromatin affinity-purification on microarray All experiments were done using strepavidin pulldown DNA cohybridized with total input DNA to the same array. Two channels per array, Cy5 and Cy3, were used in each experiment.

2010-05-27 | E-GEOD-19788 | biostudies-arrayexpress

The primary cilium at a glance.

Project description: Not available

| S-EPMC2818190 | biostudies-literature

CATCH-IT method for measuring nucleosome turnover

2010-05-27 | GSE19788 | GEO

Low-Cost Method and Biochip for Measuring the Trans-Epithelial Electrical Resistance (TEER) of Esophageal Epithelium.

Project description:Trans-epithelial electrical resistance (TEER) is a good indicator of the barrier integrity of epithelial tissues and is often employed in biomedical research as an effective tool to assess ion transport and permeability of tight junctions. The Ussing chamber is the gold standard for measuring TEER of tissue specimens, but it has major drawbacks: it is a macroscopic method that requires a careful and labor intensive sample mounting protocol, allows a very limited viability for the mounted sample, has large parasitic components and low throughput as it cannot perform multiple simultaneous measurements, and this sophisticated and delicate apparatus has a relatively high cost. This paper demonstrates a low-cost home-made "sandwich ring" method which was used to measure the TEER of tissue specimens effectively. This method inspired the subsequent design of a biochip fabricated using standard soft lithography and laser engraving technologies, with which the TEER of pig epithelial tissues was measured. Moreover, it was possible to temporarily preserve the tissue specimens for days in the biochip and monitor the TEER continuously. Tissue responses after exposure tests to media of various pH values were also successfully recorded using the biochip. All these demonstrate that this biochip could be an effective, cheaper, and easier to use Ussing chamber substitute that may have relevant applications in clinical practice.

| S-EPMC7288155 | biostudies-literature

Synchronizing Protein Traffic to the Primary Cilium.

Project description:The primary cilium is able to maintain a specific protein composition, which is critical for its function as a signaling organelle. Here we introduce a system to synchronize biosynthetic trafficking of ciliary proteins that is based on conditional aggregation domains (CADs). This approach enables to create a wave of ciliary proteins that are transported together, which opens novel avenues for visualizing and studying ciliary import mechanisms. By using somatostatin receptor 3 (SSTR3) as model protein we studied intracellular transport and ciliary import with high temporal and spatial resolution in epithelial Madin-Darby canine kidney (MDCK) cells. This yielded the interesting discovery that SSTR3, besides being transported to the primary cilium, is also targeted to the basolateral plasma membrane. In addition, we found a similar behavior for another ciliary protein, nephrocystin-3 (NPHP3), thus suggesting a potential correlation between ciliary and basolateral trafficking. Furthermore, our CAD-based system allowed assembling a large dataset in which apical and basolateral surface SSTR3 signals could be compared to ciliary SSTR3 signals on a single cell level. This enabled to generate novel complementary evidence for the previously proposed lateral import mechanism of SSTR3 into the cilium along the plasma membrane.

| S-EPMC6419537 | biostudies-literature

Zfp423 Regulates Sonic Hedgehog Signaling via Primary Cilium Function.

Project description:Zfp423 encodes a 30-zinc finger transcription factor that intersects several canonical signaling pathways. Zfp423 mutations result in ciliopathy-related phenotypes, including agenesis of the cerebellar vermis in mice and Joubert syndrome (JBTS19) and nephronophthisis (NPHP14) in humans. Unlike most ciliopathy genes, Zfp423 encodes a nuclear protein and its developmental expression is complex, leading to alternative proposals for cellular mechanisms. Here we show that Zfp423 is expressed by cerebellar granule cell precursors, that loss of Zfp423 in these precursors leads to cell-intrinsic reduction in proliferation, loss of response to Shh, and primary cilia abnormalities that include diminished frequency of both Smoothened and IFT88 localization. Loss of Zfp423 alters expression of several genes encoding key cilium components, including increased expression of Tulp3. Tulp3 is a direct binding target of Zfp423 and reducing the overexpression of Tulp3 in Zfp423-deficient cells suppresses Smoothened translocation defects. These results define Zfp423 deficiency as a bona fide ciliopathy, acting upstream of Shh signaling, and indicate a mechanism intrinsic to granule cell precursors for the resulting cerebellar hypoplasia.

| S-EPMC5065120 | biostudies-literature

The fate of the primary cilium during myofibroblast transition.

Project description:Myofibroblasts, the culprit of organ fibrosis, can originate from mesenchymal and epithelial precursors through fibroblast-myofibroblast and epithelial-myofibroblast transition (EMyT). Because certain ciliopathies are associated with fibrogenesis, we sought to explore the fate and potential role of the primary cilium during myofibroblast formation. Here we show that myofibroblast transition from either precursor results in the loss of the primary cilium. During EMyT, initial cilium growth is followed by complete deciliation. Both EMyT and cilium loss require two-hit conditions: disassembly/absence of intercellular contacts and transforming growth factor-β1 (TGFβ) exposure. Loss of E-cadherin-dependent junctions induces cilium elongation, whereas both stimuli are needed for deciliation. Accordingly, in a scratch-wounded epithelium, TGFβ provokes cilium loss exclusively along the wound edge. Increased contractility, a key myofibroblast feature, is necessary and sufficient for deciliation, since constitutively active RhoA, Rac1, or myosin triggers, and down-regulation of myosin or myocardin-related transcription factor prevents, this process. Sustained myosin phosphorylation and consequent deciliation are mediated by a Smad3-, Rac1-, and reactive oxygen species-dependent process. Transitioned myofibroblasts exhibit impaired responsiveness to platelet-derived growth factor-AA and sonic hedgehog, two cilium-associated stimuli. Although the cilium is lost during EMyT, its initial presence contributes to the transition. Thus myofibroblasts represent a unique cilium-less entity with profoundly reprogrammed cilium-related signaling.

| S-EPMC3937090 | 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