A correlative super-resolution protocol to map the local single-channel underpinnings of fast second-messenger signals in primary cell types

2021 ◽  
Author(s):  
Miriam Hurley
Keyword(s):  
Single Channel ◽  
Super Resolution ◽  
Second Messenger ◽  
Cell Types ◽  
Primary Cell

scholarly journals A correlative super-resolution protocol to visualise structural underpinnings of fast second-messenger signalling in primary cell types

Methods ◽  
2020 ◽  
Author(s):  
Miriam E. Hurley ◽  
Thomas M.D. Sheard ◽  
Ruth Norman ◽  
Hannah M. Kirton ◽  
Shihab S. Shah ◽  
...  
Keyword(s):  
Super Resolution ◽  
Second Messenger ◽  
Cell Types ◽  
Primary Cell

scholarly journals A super-resolution protocol to correlate structural underpinnings of fast second-messenger signalling in primary cell types

2020 ◽  
Author(s):  
Miriam E. Hurley ◽  
Thomas M. D. Sheard ◽  
Ruth Norman ◽  
Hannah M. Kirton ◽  
Shihab S. Shah ◽  
...  
Keyword(s):  
Total Internal Reflection ◽  
Super Resolution ◽  
Second Messenger ◽  
Cell Types ◽  
Image Alignment ◽  
Primary Cells ◽  
Imaging Protocol ◽  
Correlative Imaging ◽  
Nanoscale Patterns ◽  
Analysis Protocol

AbstractNanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels which give rise to these Ca2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels that are locally activated during the genesis of cytoplasmic Ca2+ signals.

scholarly journals Characterization of coagulation factor synthesis in nine human primary cell types

2012 ◽  
Vol 2 (1) ◽  
Author(s):  
Monireh Dashty ◽  
Vishtaseb Akbarkhanzadeh ◽  
Clark J. Zeebregts ◽  
C. Arnold Spek ◽  
Eric J. Sijbrands ◽  
...  
Keyword(s):  
Coagulation Factor ◽  
Cell Types ◽  
Primary Cell

Properties and distribution of single voltage-gated calcium channels in adult hippocampal neurons

1990 ◽  
Vol 64 (1) ◽  
pp. 91-104 ◽  
Author(s):  
R. E. Fisher ◽  
R. Gray ◽  
D. Johnston
Keyword(s):  
Guinea Pig ◽  
Calcium Channels ◽  
Single Channel ◽  
Cell Types ◽  
Voltage Gated ◽  
Channel Current ◽  
Single Channel Current ◽  
Voltage Gated Calcium Channels ◽  
Large Conductance Channel ◽  
Small Conductance

1. The properties of single voltage-gated calcium channels were investigated in acutely exposed CA3 and CA1 pyramidal neurons and granule cells of area dentata in the adult guinea pig hippocampal formation. 2. Guinea pig hippocampal slices were prepared in a conventional manner, then treated with proteolytic enzymes and gently shaken to expose the somata of the three cell types studied. Standard patch-clamp techniques were used to record current flow through calcium channels in cell-attached membrane patches with isotonic barium as the charge carrier. 3. Single-channel current amplitudes were measured at different membrane potentials. Single-channel current-voltage plots were constructed and single-channel slope conductances were found to fall into three classes. These were (approximately) 8, 14, and 25 pS, and were observed in all three cell types. 4. The three groups of channels differed from each other in voltage dependence of activation: from a holding potential of -80, the small-conductance channel began to activate at about -40 to -30 mV, the medium-conductance channel at about -20 mV, and the large-conductance channel at approximately 0 mV. 5. Ensemble averages of single-channel currents during voltage steps revealed differences in voltage-dependent inactivation. The small-conductance channel inactivated completely within approximately 50 ms during steps from -80 to -10 mV or more positive. Steps to less positive potentials resulted in less inactivation. The medium-conductance channel displayed variable inactivation during steps from -80 to 0 mV. Inactivation of this channel during a 160-ms step ranged from virtually zero to approximately 100%. The large-conductance channel displayed no significant inactivation during steps as long as 400 ms. 6. The large-conductance channel was strikingly affected by the dihydropyridine agonist Bay K8644 (0.5-2.0 microM), resulting in a high probability of channel opening, prolonged openings, and an apparent increase in the number of channels available for activation. The medium and small-conductance channels were not noticeably affected by the drug. 7. The large-conductance channel could be induced to open at very negative membrane potentials by holding the patch for several seconds at 20 or 30 mV and stepping to -30 or -40 mV. This process was enhanced by Bay K8644, resulting in prolonged openings at potentials as negative as -100 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals A super resolution target separation and reconstruction approach for single channel sar against deceptive jamming

2021 ◽  
Author(s):  
Shi-qi Liu ◽  
Bing Li ◽  
Bo Zhao ◽  
Lei Huang ◽  
Yue-zhou Wu ◽  
...  
Keyword(s):  
Single Channel ◽  
Super Resolution ◽  
Deceptive Jamming

scholarly journals The nanoscale organization of the Wnt signaling integrator Dishevelled in the development-essential vegetal cortex domain of an egg and early embryo

2021 ◽  
Author(s):  
John H. Henson ◽  
Bakary Samasa ◽  
Charles B. Shuster ◽  
Athula H. Wikramanayake
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Super Resolution ◽  
Cell Types ◽  
Early Embryo ◽  
Ultrastructural Organization ◽  
C Terminus ◽  
Pathway Activation ◽  
Vegetal Cortex ◽  
Canonical Wnt

AbstractWnt/β-catenin (cWnt) signaling is a crucial regulator of development and Dishevelled (Dsh/Dvl) functions as an integral part of this pathway by linking Wnt binding to the frizzled:LRP5/6 receptor complex with β-catenin-stimulated gene expression. In many cell types Dsh has been localized to ill-defined cytoplasmic puncta, however in sea urchin eggs and embryos confocal fluorescence microscopy has shown that Dsh is localized to puncta present in a novel and development-essential vegetal cortex domain (VCD). In the present study, we used super-resolution light microscopy and platinum replica TEM to provide the first views of the ultrastructural organization of Dsh within the sea urchin VCD. 3D-SIM imaging of isolated egg cortices demonstrated the concentration gradient-like distribution of Dsh in the VCD, whereas higher resolution STED imaging revealed that some individual Dsh puncta consisted of more than one fluorescent source. Platinum replica immuno-TEM localization showed that Dsh puncta on the cytoplasmic face of the plasma membrane consisted of aggregates of pedestal-like structures each individually labeled with the C-terminus specific Dsh antibody. These aggregates were resistant to detergent extraction and treatment with drugs that disrupt actin filaments or inhibit myosin II contraction, and coexisted with the first division actomyosin contractile ring. These results confirm and extend previous studies and reveal, for the first time in any cell type, the nanoscale organization of plasma membrane tethered Dsh. Our current working hypothesis is that these Dsh pedestals represent a prepositioned scaffold organization that is important for canonical Wnt pathway activation at the sea urchin vegetal organization and may also be relevant to the submembranous Dsh puncta present in other eggs and embryos.

Kinetic and Pharmacological Properties of GABAAReceptors in Single Thalamic Neurons and GABAA Subunit Expression

2001 ◽  
Vol 86 (5) ◽  
pp. 2312-2322 ◽  
Author(s):  
S. H. Browne ◽  
J. Kang ◽  
G. Akk ◽  
L. W. Chiang ◽  
H. Schulman ◽  
...  
Keyword(s):  
Single Cell ◽  
Decay Time ◽  
Single Channel ◽  
Cell Types ◽  
Reticular Nucleus ◽  
Thalamic Nuclei ◽  
Rt Pcr ◽  
Structural Determinants ◽  
Thalamic Neurons ◽  
Time Constants

Synaptic inhibition in the thalamus plays critical roles in sensory processing and thalamocortical rhythm generation. To determine kinetic, pharmacological, and structural properties of thalamic γ-aminobutyric acid type A (GABAA) receptors, we used patch-clamp techniques and single-cell reverse transcriptase polymerase chain reaction (RT-PCR) in neurons from two principal rat thalamic nuclei—the reticular nucleus (nRt) and the ventrobasal (VB) complex. Single-channel recordings identified GABAAchannels with densities threefold higher in VB than nRt neurons, and with mean open time fourfold longer for nRt than VB [14.6 ± 2.5 vs. 3.8 ± 0.7 (SE) ms, respectively]. GABAA receptors in nRt and VB cells were pharmacologically distinct. Zn2+ (100 μM) reduced GABAA channel activity in VB and nRt by 84 and 24%, respectively. Clonazepam (100 nM) increased inhibitory postsynaptic current (IPSC) decay time constants in nRt (from 44.3 to 77.9 ms, P < 0.01) but not in VB. Single-cell RT-PCR revealed subunit heterogeneity between nRt and VB cells. VB neurons expressed α1–α3, α5, β1–3, γ2–3, and δ, while nRt cells expressed α3, α5, γ2–3, and δ. Both cell types expressed more subunits than needed for a single receptor type, suggesting the possibility of GABAA receptor heterogeneity within individual thalamic neurons. β subunits were not detected in nRt cells, which is consistent with very low levels reported in previous in situ hybridization studies but inconsistent with the expected dependence of functional GABAA receptors on β subunits. Different single-channel open times likely underlie distinct IPSC decay time constants in VB and nRt cells. While we can make no conclusion regarding β subunits, our findings do support α subunits, possibly α1 versus α3, as structural determinants of channel deactivation kinetics and clonazepam sensitivity. As the γ2 and δ subunits previously implicated in Zn2+sensitivity are both expressed in each cell type, the observed differential Zn2+ actions at VB versus nRt GABAA receptors may involve other subunit differences.

scholarly journals A Multiplexed Fluorescent Assay for Independent Second-Messenger Systems

2013 ◽  
Vol 18 (7) ◽  
pp. 797-806 ◽  
Author(s):  
Paul H. Tewson ◽  
Anne Marie Quinn ◽  
Thomas E. Hughes
Keyword(s):  
Real Time ◽  
Second Messengers ◽  
Basic Research ◽  
Second Messenger ◽  
Cell Types ◽  
Live Cell ◽  
Multiple Components ◽  
Second Messenger Systems ◽  
G Protein Coupled ◽  
Cell Signaling Pathways

There is a growing need in drug discovery and basic research to measure multiple second-messenger components of cell signaling pathways in real time and in relevant tissues and cell types. Many G-protein–coupled receptors activate the heterotrimeric protein, Gq, which in turn activates phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce two second messengers: diacylglycerol (DAG), which remains in the plasma membrane, and inositol triphosphate (IP3), which diffuses through the cytosol to release stores of intracellular calcium ions (Ca2+). Our goal was to create a series of multiplex sensors that would make it possible to simultaneously measure two different components of the Gq pathway in living cells. Here we describe new fluorescent sensors for DAG and PIP2 that produce robust changes in green or red fluorescence and can be combined with one another, or with existing Ca2+ sensors, in a live-cell assay. These assays can detect multiple components of Gq signaling, simultaneously in real time, on standard fluorescent plate readers or live-cell imaging systems.

scholarly journals Gating of Recombinant Small-Conductance Ca-activated K+ Channels by Calcium

1998 ◽  
Vol 111 (4) ◽  
pp. 565-581 ◽  
Author(s):  
Birgit Hirschberg ◽  
James Maylie ◽  
John P. Adelman ◽  
Neil V. Marrion
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Cell Types ◽  
K Channel ◽  
Open Probability ◽  
K Channels ◽  
Sensitive Clone ◽  
Single Channel Currents ◽  
Open States ◽  
Small Conductance

Small-conductance Ca-activated K+ channels play an important role in modulating excitability in many cell types. These channels are activated by submicromolar concentrations of intracellular Ca2+, but little is known about the gating kinetics upon activation by Ca2+. In this study, single channel currents were recorded from Xenopus oocytes expressing the apamin-sensitive clone rSK2. Channel activity was detectable in 0.2 μM Ca2+ and was maximal above 2 μM Ca2+. Analysis of stationary currents revealed two open times and three closed times, with only the longest closed time being Ca dependent, decreasing with increasing Ca2+ concentrations. In addition, elevated Ca2+ concentrations resulted in a larger percentage of long openings and short closures. Membrane voltage did not have significant effects on either open or closed times. The open probability was ∼0.6 in 1 μM free Ca2+. A lower open probability of ∼0.05 in 1 μM Ca2+ was also observed, and channels switched spontaneously between behaviors. The occurrence of these switches and the amount of time channels spent displaying high open probability behavior was Ca2+ dependent. The two behaviors shared many features including the open times and the short and intermediate closed times, but the low open probability behavior was characterized by a different, long Ca2+-dependent closed time in the range of hundreds of milliseconds to seconds. Small-conductance Ca- activated K+ channel gating was modeled by a gating scheme consisting of four closed and two open states. This model yielded a close representation of the single channel data and predicted a macroscopic activation time course similar to that observed upon fast application of Ca2+ to excised inside-out patches.

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