1,721,060 research outputs found
A Protocol for the Automated Assessment of Cutaneous Pathology in a Mouse Model of Hemichannel Dysfunction
No full textIn this chapter, we provide detailed instructions to perform quantitative reflectance imaging in a mouse model of a rare epidermal disorder caused by hyperactive connexin 26 hemichannels. Reflectance imaging is a versatile and powerful tool in dermatology, offering noninvasive, high-resolution insights into skin pathology, which is essential for both clinical practice and research. This approach offers several advantages and applications. Unlike traditional biopsy, reflectance imaging is noninvasive, allowing for real-time, in vivo examination of the skin. This is particularly valuable for monitoring chronic conditions or assessing the efficacy of treatments over time, enabling the detailed examination of skin morphology. This is crucial for identifying features of skin diseases such as cancers, inflammatory conditions, and infections. In therapeutic applications, reflectance imaging can be used to monitor the response of skin lesions to treatments. It can help in identifying the most representative area of a lesion for biopsy, thereby increasing the diagnostic accuracy. Reflectance imaging can also be used to diagnose and monitor inflammatory skin diseases, like psoriasis and eczema, by visualizing changes in skin structure and cellular infiltration. As the technology becomes more accessible, it has potential in telemedicine, allowing for remote diagnosis and monitoring of skin conditions. In academic settings, reflectance imaging can be a powerful research tool, enabling the study of skin pathology and the effects of novel treatments, including the development of monoclonal antibodies for therapeutic applications
Measurement of Ca2+ Uptake Through Connexin Hemichannels
No full textIncreasing evidence points to deregulated flux of ionized calcium (Ca2+) mediated by hyperactive mutant connexin (Cx) hemichannels (HCs) as a common gain-of-function etiopathogenetic mechanism for several diseases, ranging from skin disorders to nervous system defects. Furthermore, the opening of nonmutated Cx HCs is associated with an impressive list of widespread diseases including, but not limited to, ischemia/stroke, Alzheimer’s disease, and epilepsy. HC inhibitors are attracting a growing attention due to their therapeutic potential for numerous pathologies. This chapter describes a quantitative method to measure Ca2+ uptake though HCs expressed in cultured cells. The assay we developed can be used to probe HC activity as wells as to test HC inhibitors. Furthermore, with minor changes it can be easily adapted to high-throughput high-content platforms and/or primary cells and microtissues
Connexin Hemichannels: Methods and Protocols.
No full textThis volume explores the latest developments in the study of connexin hemichannels (HCs). The chapters in this book cover topics such as purification, reconstitution, and functional analysis of connexin HCs; spatial and temporal localization of connexins in cells using confocal microscopy; evaluation of connexin HC activity in vivo; generation of connexin-expressing stable cell pools; and automated assessment of cutaneous pathology in a mouse model of HC dysfunction. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls
Molecular Dynamics Simulation of Permeation Through Connexin Channels
No full text: Molecular dynamics (MD) simulations are a collection of computational tools that can be used to trace intermolecular interactions at the sub-nanometer level. They offer possibilities that are often unavailable to experimental methods, making MD an ideal complementary technique for the understanding a plethora of biological processes. Thanks to significant efforts by many groups of developers around the world, setting up and running MD simulations has become progressively simpler. However, simulating ionic permeation through membrane channels still presents significant caveats.MD simulations of connexin (Cx) hemichannels (HCs) are particularly problematic because HCs create wide pores in the plasma membrane, and the lateral sizes of the extracellular and intracellular regions are quite different. In this chapter, we provide a detailed instruction to perform MD simulations aimed at computationally modeling the permeation of inorganic ions and larger molecules through Cx HCs
Inner ear connexins, intercellular signalling and deafness.
No full textThe genes GJB2 and GJB6 respectively encoding transmembrane proteins connexin 26 (C×26) and connexin 30 (C×30) are found within 50 kb of each other in the DFNB1 complex deafness locus on chromosome 13ql2. Up to 50% of all patients with autosomal recessive non-syndromic prelingual deafness in different populations present with mutations or deletions in this locus. C×26 and C×30 are abundantly expressed in the inner ear and, in recent years, have been shown to form hemichannels that release ATP from the endolymphatic surface of supporting and epithelial cells of the organ of Corti (OoC), as well as gap junction (GJ) channels that allow the concomitant intercellular diffusion of Ca2+ mobilizing second messengers. Released ATP in turn activates G-protein coupled P2Y2 and P2Y4 receptors, PLC-dependent generation of IP3, release of Ca2+ from intracellular stores, ensuing in the regenerative propagation of intercellular Ca2+ signals (ICS) across these coupled cells. The range of ICS propagation in the OoC is sensitive to the concentration of extracellular divalent cations and to ectonucleotidase activity. Strictly related oscillations of the intracellular free Ca2+ concentration ([Ca2+]i) in cochlear supporting cells are also evoked by nanomolar concentrations of ATP on the endolymphatic surface of the OoC. ICS and [Ca2+]i oscillations are of great interest in relation to the responses evoked by damaging stimuli delivered to hair cells, and may play a crucial role in development of the OoC and the acquisition of hearing
Commercially derived versatile optical architecture for two-photon STED, wavelength mixing and label-free microscopy
Full text linkMultimodal microscopy combines multiple non-linear techniques that take advantage of different optical processes to generate contrast and increase the amount of information that can be obtained from biological samples. However, the most advanced optical architectures are typically custom-made and often require on-site adjustment of optical components performed by trained personnel for optimal performance. Here, we describe a hybrid system we built based on a commercial upright microscope. We show that our multimodal imaging platform can be used to seamlessly perform two-photon STED, wavelength mixing and label-free microscopy in both ex vivo and in vivo turbid samples. The system is stable and endowed with remote alignment hardware that ensures long-term operability also for non-expert users, using the alignment protocol described in this article and in the related material. This optical architecture is an important step forward towards a wider practical applicability of non-linear optics to bioimaging
Dynamics of intracellular Calcium in hair cells isolated from the frog
No full textK and Ca ions play an essential role in hair cell transduction mechanisms. K carries the transduction current at the apical ciliated pole of the cell, where localised cytosolic free Ca concentration ([Ca ]i) changes modulate the open probability of the mechanosensitive channels and thus regulate the amplitude of the receptor potential. This, in turn, activates Ca and K basolateral conductances, which ultimately control transmitter release at the cell's cytoneural junction. Some vertebrate hair cells display cytosolic microdomains where the [Ca]i increases markedly in response to depolarisation. These Ca hotspots are thought to depend on focal Ca entry through clusters of voltage-activated Ca channels colocalised with Ca -activated K channels at presynaptic active zones. Ca microdomains have been studied in hair cells of the frog sacculus and in auditory hair cells of the turtle, but no studies of Ca dynamics have been performed on hair cells of the semicircular canal. To further characterise the spatial and temporal features of the [Ca]i changes associated to the gating of voltage-activated Ca channels, we have applied fast CCD imaging of intracellular Ca and patch clamp recording to hair cells from the crista arnpullaris of Rana esculenta. Changes in [Ca]i were optically monitored using the membrane-impermeant form of the Ca-selective dye Oregon Green 488 BAPTA-1. Cells were stimulated by depolarizing voltage commands during whole cell voltage clamp recordings and concurrently imaged at 247 Hz frame rate. Cells stimulated by depolarisation revealed Ca entry at selected sites (hotspots) located mostly in the lower (synaptic) half of the celi body. [Ca]i at individual hotspots rose with a time constant of about 70 ms and decayed with a bi-exponential time-course (time constants: 160 and 2500 ms) following a 160 ms depolarisation to -20 mV. With repeated stimulation [Ca]i underwent independent amplitude changes at distinct hotspots, suggesting that the underlying Ca channel clusters can be regulated differentially by intracellular signalling pathways. Block by nifedipine indicated that the L-type Ca channels are distributed at different densities in distinct hotspots. The study of diffusion away from the hotspot indicates that no major diffusional barriera other than the nuclear region exists in the cytoplasm, and the Ca clearance machinery is also likely clustered at selected locations. Therefore, during a prolonged depolarisation (lasting up to 1 s), Ca was able to reach the cell apical ciliated pole. The effective Ca diffusion constant, measured from the progression of Ca wavefronts in the cytosol, was about 57 micron2/s. Our results indicate that, in these hair cells, buffered diffusion of Ca proceeds evenly from the source point to the cell interior and is dominated by the diffusion constant of the endogenous mobile buffers
Generation of Connexin-Expressing Stable Cell Pools
No full textStable cell pools have the advantage of providing a definite, consistent, and reproducible transmission of a transgene of interest, compared to transient expression from a plasmid transfection. Stably expressing a transgene of interest in cells under induction is a powerful way to (switch on and) study a gene function in both in vitro and in vivo assays. Taking advantage of the ability of lentivirus (LV) to promote transgene delivery, and genomic integration and expression in both dividing and nondividing cells, a doxycycline-inducible transfer vector expressing a bicistronic transgene was developed to study the function of connexins in HeLa DH cells. Here, delving on connexin 32 (Cx32), we report how to use the backbone of this vector as a tool to generate stable pools to study the function of a gene of interest (GOI), especially with assays involving Ca2+ imaging, employing the GCaMP6s indicator. We describe a step-by-step protocol to produce the LV particle by transient transfection and the direct use of the harvested LV stock to generate stable cell pools. We further present step-by-step immunolabeling protocols to characterize the transgene protein expression by confocal microscopy using an antibody that targets an extracellular domain epitope of Cx32 in living cells, and in fixed permeabilized cells using high affinity anti-Cx32 antibodies. Using common molecular biology laboratory techniques, this protocol can be adapted to generate stable pools expressing any transgene of interest, for both in vitro and in vivo functional assays, including molecular, immune, and optical assays
Ca2+ Extrusion in Frog Semicircular Canal Hair Cells Probed with Flash Photolysis of Novel Ca-caged Compounds and Fast Calcium Imaging
No full textCa ions play an essential role in hair cell functioning, since localized intracellular Ca changes modulate the transduction process and regulate the amplitude of the receptor potential. This, in turn, activates Ca and K basolateral conductances, which ultimately control transmitter release at the cell's cytoneural junction. Several studies have been recently reported on Ca entry via the voltage-gated Ca channels of hair cells, and on Ca extrusion at the level of the stereocilia mediated by highly packed Ca pumps. On the other hand, the mechanism of Ca clearance at the cell basolateral pole is less known, therefore the Ca extrusion via putative Na:Ca or Na:Ca,K exchanger was investigated in hair cells ìsolated from frog semicircular canals. Cells concurrently imaged during whole celi recordings using a fluorescent dye selective for Ca showed that the voltage-gated Ca entry occurred at selected sites (hotspots) located at the cell synaptic pole. Since the rate of Ca extrusion by any exchanger working in the forward mode is speeded up by cell hyperpolarization, voltage dependence of Ca clearance dynamics was studied following a standard 160 ms depolarization pulse to -20 mV to elicit Ca entry. In any hotspot and in any of the three cell types found in crista ampullaris (club-like, pear-like and cylinder-like), hyperpolarization did not accelerate at all Ca clearance kinetics. Reverse exchange was probed in patch-clamped cells, dialyzed with a Ca-, K-free solution containing 115 Na (concentrations in mM), upon switching from Ca-free, 115 Li external solution to 4 Ca + 115 Li or to 4 Ca + 20 K + 95 Li solution. The K currents were blocked with external 15 TEA and 5 Cs; the holding potential was -70 mV to block the Ca current. After correcting for the leak change upon switching from the external Ca-free solution to 4 Ca, no attual current changes were evoked by the solutions tested. In lizard rod photoreceptors in the same conditions, peak exchange currents exceeding 100 pA were recorded in the Ca + K external solution. The forward exchange mode was probed by rapidly increasing the intracellular Ca concentration by means of flash photolysis of two novel caged Ca complexes of 4-(2-nitrophenyl)-3,6-dioxaoctanedioic acid and 4-(4,5-dimethoxy-2-nitrophenyl)-3,6-dioxaoctanedioic acid in the presence of internal K and external Na. No currents were evoked by the Ca jump, whereas in rod photoreceptors, forward exchange currents up to 50 pA were recorded in the same conditions. It was concluded that neither Na:Ca nor Na:Ca,K electrogenic exchanger exists in this hair celi type
CA2+ dynamics in auditory and vestibular hair cells: Monte carlo simulations and experimental results
No full textWe developed a simulation code in the Matlab environment for the study, using the Monte Carlo method, of cellular phenomena involving diffusion, buffering, extrusion and release of Ca2+. In particular we simulated the entry of Ca2+ at individual presynaptic active zones (hotspots) of auditory and vestibular hair cells, where Ca2+ plays a fundamental role in the transduction of mechanical stimuli, due to sound or acceleration, into electrical signals to be sent to the brain. The realistic reconstruction, in three dimensions, of the cellular boundaries and the derivation of the virtual fluorescence ratio ?F/F0 (equivalent to the one computed from fluorescence microscopy experiments) allowed us (i) to directly compare simulations to experimental data, (ii) to supply an estimate of the equivalent concentration of Ca2+ reactants (buffers) and (iii) to show how the mass action law hypothesis brakes down because of the local non equilibrium of the system
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