scholarly journals PARIS, an optogenetic method for functionally mapping gap junctions

2018 ◽  
Author(s):  
Ling Wu ◽  
Ao Dong ◽  
Liting Dong ◽  
Shi-Qiang Wang ◽  
Yulong Li
Keyword(s):  
Gap Junctions ◽  
Cell Communication ◽  
Cell Type Specificity ◽  
Spatiotemporal Resolution ◽  
Physiological Processes ◽  
Wide Range ◽  
Junctional Coupling ◽  
Chemical Coupling ◽  
And Function

ABSTRACTCell-cell communication via gap junctions regulates a wide range of physiological processes by enabling the direct intercellular electrical and chemical coupling. However, the in vivo distribution and function of gap junctions remain poorly understood, partly due to the lack of non-invasive tools with both cell-type specificity and high spatiotemporal resolution. Here we developed PARIS (pairing actuators and receivers to optically isolate gap junctions), a new fully genetically encoded tool for measuring the cell-specific gap junctional coupling (GJC). PARIS successfully enabled monitoring of GJC in several cultured cell lines under physiologically relevant conditions and in distinct genetically defined neurons in Drosophila brain, with ~10-sec temporal resolution and sub-cellular spatial resolution. These results demonstrate that PARIS is a robust, highly sensitive tool for mapping functional gap junctions and study their regulation in both health and disease.

scholarly journals PARIS, an optogenetic method for functionally mapping gap junctions

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Ling Wu ◽  
Ao Dong ◽  
Liting Dong ◽  
Shi-Qiang Wang ◽  
Yulong Li
Keyword(s):  
Gap Junctions ◽  
Cell Communication ◽  
Cell Type Specificity ◽  
Spatiotemporal Resolution ◽  
Wide Range ◽  
Junctional Coupling ◽  
Chemical Coupling ◽  
Drosophila Brain ◽  
And Function

Cell-cell communication via gap junctions regulates a wide range of physiological processes by enabling the direct intercellular electrical and chemical coupling. However, the in vivo distribution and function of gap junctions remain poorly understood, partly due to the lack of non-invasive tools with both cell-type specificity and high spatiotemporal resolution. Here, we developed PARIS (pairing actuators and receivers to optically isolate gap junctions), a new fully genetically encoded tool for measuring the cell-specific gap junctional coupling (GJC). PARIS successfully enabled monitoring of GJC in several cultured cell lines under physiologically relevant conditions and in distinct genetically defined neurons in Drosophila brain, with ~10 s temporal resolution and sub-cellular spatial resolution. These results demonstrate that PARIS is a robust, highly sensitive tool for mapping functional gap junctions and study their regulation in both health and disease.

scholarly journals Organ-on-e-chip: Three-dimensional self-rolled biosensor array for electrical interrogations of human electrogenic spheroids

2019 ◽  
Vol 5 (8) ◽  
pp. eaax0729 ◽  
Author(s):  
Anna Kalmykov ◽  
Changjin Huang ◽  
Jacqueline Bliley ◽  
Daniel Shiwarski ◽  
Joshua Tashman ◽  
...  
Keyword(s):  
Field Effect Transistors ◽  
Three Dimensional ◽  
Cell Communication ◽  
High Sensitivity ◽  
Complex Signal ◽  
Spatiotemporal Resolution ◽  
Biosensor Arrays ◽  
Biosensor Array ◽  
And Function

Cell-cell communication plays a pivotal role in coordination and function of biological systems. Three-dimensional (3D) spheroids provide venues to explore cellular communication for tissue development and drug discovery, as their 3D architecture mimics native in vivo microenvironments. Cellular electrophysiology is a prevalent signaling paradigm for studying electroactive cells. Currently, electrophysiological studies do not provide direct, multisite, simultaneous investigation of tissues in 3D. In this study, 3D self-rolled biosensor arrays (3D-SR-BAs) of either active field-effect transistors or passive microelectrodes were implemented to interface human cardiac spheroids in 3D. The arrays provided continuous and stable multiplexed recordings of field potentials with high sensitivity and spatiotemporal resolution, supported with simultaneous calcium imaging. Our approach enables electrophysiological investigation and monitoring of the complex signal transduction in 3D cellular assemblies toward an organ-on-an-electronic-chip (organ-on-e-chip) platform for tissue maturation investigations and development of drugs for disease treatment, such as arrhythmias.

Are gap junctions necessary for cell-to-cell coupling of smooth muscle?: an update

1992 ◽  
Vol 70 (4) ◽  
pp. 481-490 ◽  
Author(s):  
R. E. Garfield ◽  
G. Thilander ◽  
M. G. Blennerhassett ◽  
N. Sakai
Keyword(s):  
Smooth Muscle ◽  
Gap Junctions ◽  
Current Knowledge ◽  
Smooth Muscles ◽  
Cell Communication ◽  
Circular Muscle ◽  
Cell Coupling ◽  
And Function ◽  
Longitudinal Layer ◽  
Cell Cell

Earlier, it was questioned whether gap junctions (GJs) were necessary for cell–cell communication in smooth muscle, and GJs were not seen in some smooth muscles. We reexamined this question in the myometrium and in intestinal smooth muscle, in light of current knowledge of the presence and function of GJs. In the uterus, numerous studies show that an increase in GJ number is associated with the onset of delivery and is required for effective parturition. In all cases, this increase in GJ number and the changes in uterine contractility were correlated with increased electrical and metabolic coupling. Evidence for the much smaller, but detectable, degree of electrical coupling in the preterm uterus is explained by the small (but again detectable) number of GJs present. In the intestine, GJs are readily detected in the circular muscle layer but have not been described in the adjacent longitudinal layer. While our immunohistochemical studies failed to detect GJs in the longitudinal layer, this may not be adequate to prove their absence. Therefore, current knowledge of GJ number and function is adequate to explain cell–cell coupling in the uterus. Although it remains uncertain whether GJs are absent from the longitudinal muscle of the intestine, there is no definitive evidence that cell–cell coupling can occur by means other than GJs.Key words: gap junctions, myometrium, connexins, smooth muscle, cell communication.

scholarly journals hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function

2022 ◽  
Author(s):  
Homa Majd ◽  
Ryan M Samuel ◽  
Jonathan T Ramirez ◽  
Ali Kalantari ◽  
Kevin Barber ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Xenograft Model ◽  
Developmental Relationships ◽  
Drug Candidates ◽  
Effective Strategies ◽  
Wide Range ◽  
Functional Features ◽  
And Function

The enteric nervous system (ENS) plays a central role in gut physiology and mediating the crosstalk between the gastrointestinal (GI) tract and other organs. The human ENS has remained elusive, highlighting the need for an in vitro modeling and mapping blueprint. Here we map out the developmental and functional features of the human ENS, by establishing robust and scalable 2D ENS cultures and 3D enteric ganglioids from human pluripotent stem cells (hPSCs). These models recapitulate the remarkable neuronal and glial diversity found in primary tissue and enable comprehensive molecular analyses that uncover functional and developmental relationships within these lineages. As a salient example of the power of this system, we performed in-depth characterization of enteric nitrergic neurons (NO neurons) which are implicated in a wide range of GI motility disorders. We conducted an unbiased screen and identified drug candidates that modulate the activity of NO neurons and demonstrated their potential in promoting motility in mouse colonic tissue ex vivo. We established a high-throughput strategy to define the developmental programs involved in NO neuron specification and discovered that PDGFR inhibition boosts the induction of NO neurons in enteric ganglioids. Transplantation of these ganglioids in the colon of NO neuron-deficient mice results in extensive tissue engraftment, providing a xenograft model for the study of human ENS in vivo and the development of cell-based therapies for neurodegenerative GI disorders. These studies provide a framework for deciphering fundamental features of the human ENS and designing effective strategies to treat enteric neuropathies.  

scholarly journals Role of co-regulators in metabolic and transcriptional actions of thyroid hormone

2016 ◽  
Vol 56 (3) ◽  
pp. R73-R97 ◽  
Author(s):  
Inna Astapova
Keyword(s):  
Thyroid Hormone ◽  
Transcriptional Repression ◽  
Target Genes ◽  
Thyroid Hormone Receptor ◽  
Retinoic Acid Receptor ◽  
Specific Response ◽  
Physiological Processes ◽  
Genome Wide ◽  
Wide Range ◽  
And Function

Thyroid hormone (TH) controls a wide range of physiological processes through TH receptor (TR) isoforms. Classically, TRs are proposed to function as tri-iodothyronine (T3)-dependent transcription factors: on positively regulated target genes, unliganded TRs mediate transcriptional repression through recruitment of co-repressor complexes, while T3binding leads to dismissal of co-repressors and recruitment of co-activators to activate transcription. Co-repressors and co-activators were proposed to play opposite roles in the regulation of negative T3target genes and hypothalamic–pituitary–thyroid axis, but exact mechanisms of the negative regulation by TH have remained elusive. Important insights into the roles of co-repressors and co-activators in different physiological processes have been obtained using animal models with disrupted co-regulator function. At the same time, recent studies interrogating genome-wide TR binding have generated compelling new data regarding effects of T3, local chromatin structure, and specific response element configuration on TR recruitment and function leading to the proposal of new models of transcriptional regulation by TRs. This review discusses data obtained in various mouse models with manipulated function of nuclear receptor co-repressor (NCoR or NCOR1) and silencing mediator of retinoic acid receptor and thyroid hormone receptor (SMRT or NCOR2), and family of steroid receptor co-activators (SRCs also known as NCOAs) in the context of TH action, as well as insights into the function of co-regulators that may emerge from the genome-wide TR recruitment analysis.

scholarly journals Gap junction internalization and processing in vivo: a 3D immuno-electron microscopy study

2020 ◽  
pp. jcs.252726
Author(s):  
Rachael P. Norris ◽  
Mark Terasaki
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Connexin 43 ◽  
New Technology ◽  
Cell Communication ◽  
Membrane Vesicles ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Annular Gap

Gap junctions have well-established roles in cell-cell communication by way of forming permeable intercellular channels. Less is understood about their internalization, which forms double membrane vesicles containing cytosol and membranes from another cell, called connexosomes or annular gap junctions. Here, we systematically investigated the fate of connexosomes in intact ovarian follicles. High pressure frozen, serial sectioned tissue was immunogold labeled for Connexin 43. Within a volume corresponding to ∼35 cells, every labeled structure was categorized and its surface area was measured. Measurements support the concept that multiple connexosomes form from larger invaginated gap junctions. Subsequently, the inner and outer membranes separate, Cx43 immunogenicity is lost from the outer membrane, and the inner membrane appears to undergo fission. One pathway for processing involves lysosomes, based on localization of Cathespin B to some processed connexosomes. In summary, this study demonstrates new technology for high-resolution analyses of gap junction processing.

scholarly journals Cx50 requires an intact PDZ-binding motif and ZO-1 for the formation of functional intercellular channels

2011 ◽  
Vol 22 (23) ◽  
pp. 4503-4512 ◽  
Author(s):  
Zhifang Chai ◽  
Daniel A. Goodenough ◽  
David L. Paul
Keyword(s):  
Gap Junction ◽  
Hela Cells ◽  
Pdz Domain ◽  
Cell Communication ◽  
Normal Function ◽  
Scaffolding Protein ◽  
Binding Motif ◽  
Interacting Protein ◽  
And Function

The three connexins expressed in the ocular lens each contain PDZ domain–binding motifs directing a physical association with the scaffolding protein ZO-1, but the significance of the interaction is unknown. We found that Cx50 with PDZ-binding motif mutations did not form gap junction plaques or induce cell–cell communication in HeLa cells, whereas the addition of a seven–amino acid PDZ-binding motif restored normal function to Cx50 lacking its entire C-terminal cytoplasmic domain. C-Terminal deletion had a similar although weaker effect on Cx46 but little if any effect on targeting and function of Cx43. Furthermore, small interfering RNA knockdown of ZO-1 completely inhibited the formation of gap junctions by wild-type Cx50 in HeLa cells. Thus both a PDZ-binding motif and ZO-1 are necessary for Cx50 intercellular channel formation in HeLa cells. Knock-in mice expressing Cx50 with a PDZ-binding motif mutation phenocopied Cx50 knockouts. Furthermore, differentiating lens fibers in the knock-in displayed extensive intracellular Cx50, whereas plaques in mature fibers contained only Cx46. Thus normal Cx50 function in vivo also requires an intact PDZ domain–binding motif. This is the first demonstration of a connexin-specific requirement for a connexin-interacting protein in gap junction assembly.

scholarly journals Role of Exosomes in Islet Transplantation

2021 ◽  
Vol 12 ◽  
Author(s):  
Jordan Mattke ◽  
Srividya Vasu ◽  
Carly M. Darden ◽  
Kenjiro Kumano ◽  
Michael C. Lawrence ◽  
...  
Keyword(s):  
Islet Transplantation ◽  
Graft Function ◽  
Pancreatic Islet Transplantation ◽  
Complex Molecules ◽  
Wide Range ◽  
And Function ◽  
Antigen Presenting

Exosomes are known for their ability to transport nucleic acid, lipid, and protein molecules, which allows for communication between cells and tissues. The cargo of the exosomes can have a variety of effects on a wide range of targets to mediate biological function. Pancreatic islet transplantation is a minimally invasive cell replacement therapy to prevent or reverse diabetes mellitus and is currently performed in patients with uncontrolled type 1 diabetes or chronic pancreatitis. Exosomes have become a focus in the field of islet transplantation for the study of diagnostic markers of islet cell viability and function. A growing list of miRNAs identified from exosomes collected during the process of isolating islets can be used as diagnostic biomarkers of islet stress and damage, leading to a better understanding of critical steps of the isolation procedure that can be improved to increase islet yield and quality. Exosomes have also been implicated as a possible contributor to islet graft rejection following transplantation, as they carry donor major histocompatibility complex molecules, which are then processed by recipient antigen-presenting cells and sensed by the recipient immune cells. Exosomes may find their way into the therapeutic realm of islet transplantation, as exosomes isolated from mesenchymal stem cells have shown promising results in early studies that have seen increased viability and functionality of isolated and grafted islets in vitro as well as in vivo. With the study of exosomes still in its infancy, continued research on the role of exosomes in islet transplantation will be paramount to understanding beta cell regeneration and improving long-term graft function.

scholarly journals The Aquatic Invertebrate Hydra vulgaris Releases Molecular Messages Through Extracellular Vesicles

2021 ◽  
Vol 9 ◽  
Author(s):  
Maria Moros ◽  
Eugenio Fergola ◽  
Valentina Marchesano ◽  
Margherita Mutarelli ◽  
Giuseppina Tommasini ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Cell Communication ◽  
Population Level ◽  
Target Cells ◽  
Chemical Information ◽  
Hydra Vulgaris ◽  
Physiological Processes ◽  
Developmental Signalling ◽  
And Function ◽  
Cell Cell

Recent body of evidence demonstrates that extracellular vesicles (EVs) represent the first language of cell-cell communication emerged during evolution. In aquatic environments, transferring signals between cells by EVs offers protection against degradation, allowing delivering of chemical information in high local concentrations to the target cells. The packaging of multiple signals, including those of hydrophobic nature, ensures target cells to receive the same EV-conveyed messages, and the coordination of a variety of physiological processes across cells of a single organisms, or at the population level, i.e., mediating the population’s response to changing environmental conditions. Here, we purified EVs from the medium of the freshwater invertebrate Hydra vulgaris, and the molecular profiling by proteomic and transcriptomic analyses revealed multiple markers of the exosome EV subtype, from structural proteins to stress induced messages promoting cell survival. Moreover, positive and negative regulators of the Wnt/β-catenin signaling pathway, the major developmental pathway acting in body axial patterning, were identified. Functional analysis on amputated polyps revealed EV ability to modulate both head and foot regeneration, suggesting bioactivity of the EV cargo and opening new perspectives on the mechanisms of developmental signalling. Our results open the path to unravel EV biogenesis and function in all cnidarian species, tracing back the origin of the cell-cell, cross-species or cross-kingdom communication in aquatic ecosystems.

scholarly journals Sonogenetic stimulation of the brain at a spatiotemporal resolution suitable for vision restoration

2021 ◽  
Author(s):  
Sara Cadoni ◽  
Charlie Demene ◽  
Matthieu Provansal ◽  
Diep Nguyen ◽  
Dasha Nelidova ◽  
...  
Keyword(s):  
Visual Cortex ◽  
Neurological Disorders ◽  
Real Life ◽  
Spatiotemporal Resolution ◽  
Brain Machine Interfaces ◽  
Ultrasound Stimulation ◽  
Wide Range ◽  
The Brain ◽  
Stimulation Of

Remote, precisely controlled activation of the brain is a fundamental challenge in the development of brain machine interfaces providing feasible rehabilitation strategies for neurological disorders. Low-frequency ultrasound stimulation can be used to modulate neuronal activity deep in the brain, but this approach lacks spatial resolution and cellular selectivity and loads the brain with high levels of acoustic energy. The combination of the expression of ultrasound-sensitive proteins with ultrasound stimulation (sonogenetic stimulation) can provide cellular selectivity and higher sensitivity, but such strategies have been subject to severe limitations in terms of spatiotemporal resolution in vivo, precluding their use for real-life applications. We used the expression of large-conductance mechanosensitive ion channels (MscL) with high-frequency ultrasonic stimulation for a duration of milliseconds to activate neurons selectively at a relatively high spatiotemporal resolution in the rat retina ex vivo and the primary visual cortex of rodents in vivo. This spatiotemporal resolution was achieved at low energy levels associated with negligible tissue heating and far below those leading to complications in ultrasound neuromodulation. We showed, in an associative learning test, that sonogenetic stimulation of the visual cortex generated light perception. Our findings demonstrate that sonogenetic stimulation is compatible with millisecond pattern presentation for visual restoration at the cortical level. They represent a step towards the precise transfer of information over large distances to the cortical and subcortical regions of the brain via an approach less invasive than that associated with current brain machine interfaces and with a wide range of applications in neurological disorders.

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