24 research outputs found
Light-regulated interaction of Dmoesin with TRP and TRPL channels is required for maintenance of photoreceptors
Recent studies in Drosophila melanogaster retina indicate that absorption of light causes the translocation of signaling molecules and actin from the photoreceptor's signaling membrane to the cytosol, but the underlying mechanisms are not fully understood. As ezrin-radixin-moesin (ERM) proteins are known to regulate actinâmembrane interactions in a signal-dependent manner, we analyzed the role of Dmoesin, the unique D. melanogaster ERM, in response to light. We report that the illumination of dark-raised flies triggers the dissociation of Dmoesin from the light-sensitive transient receptor potential (TRP) and TRP-like channels, followed by the migration of Dmoesin from the membrane to the cytoplasm. Furthermore, we show that light-activated migration of Dmoesin results from the dephosphorylation of a conserved threonine in Dmoesin. The expression of a Dmoesin mutant form that impairs this phosphorylation inhibits Dmoesin movement and leads to light-induced retinal degeneration. Thus, our data strongly suggest that the light- and phosphorylation-dependent dynamic association of Dmoesin to membrane channels is involved in maintenance of the photoreceptor cells
Calcium Handling in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes
BACKGROUND: The ability to establish human induced pluripotent stem cells (hiPSCs) by reprogramming of adult fibroblasts and to coax their differentiation into cardiomyocytes opens unique opportunities for cardiovascular regenerative and personalized medicine. In the current study, we investigated the Ca(2+)-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs). METHODOLOGY/PRINCIPAL FINDINGS: RT-PCR and immunocytochemistry experiments identified the expression of key Ca(2+)-handling proteins. Detailed laser confocal Ca(2+) imaging demonstrated spontaneous whole-cell [Ca(2+)](i) transients. These transients required Ca(2+) influx via L-type Ca(2+) channels, as demonstrated by their elimination in the absence of extracellular Ca(2+) or by administration of the L-type Ca(2+) channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca(2+) store, contributing to [Ca(2+)](i) transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca(2+)) and ryanodine (decreasing [Ca(2+)](i)). Similarly, the importance of Ca(2+) reuptake into the SR via the SR Ca(2+) ATPase (SERCA) pump was demonstrated by the inhibiting effect of its blocker (thapsigargin), which led to [Ca(2+)](i) transients elimination. Finally, the presence of an IP3-releasable Ca(2+) pool in hiPSC-CMs and its contribution to whole-cell [Ca(2+)](i) transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB) and the phospholipase C inhibitor U73122. CONCLUSIONS/SIGNIFICANCE: Our study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca(2+) store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell [Ca(2+)](i) transients in hiPSC-CMs on both sarcolemmal Ca(2+) entry via L-type Ca(2+) channels and intracellular store Ca(2+) release
Monitoring Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes with Genetically Encoded Calcium and Voltage Fluorescent Reporters
The advent of the human-induced pluripotent stem cell (hiPSC) technology has transformed biomedical research, providing new tools for human disease modeling, drug development, and regenerative medicine. To fulfill its unique potential in the cardiovascular field, efficient methods should be developed for high-resolution, large-scale, long-term, and serial functional cellular phenotyping of hiPSC-derived cardiomyocytes (hiPSC-CMs). To achieve this goal, we combined the hiPSC technology with genetically encoded voltage (ArcLight) and calcium (GCaMP5G) fluorescent indicators. Expression of ArcLight and GCaMP5G in hiPSC-CMs permitted to reliably follow changes in transmembrane potential and intracellular calcium levels, respectively. This allowed monitoring short- and long-term changes in action-potential and calcium-handling properties and the development of arrhythmias in response to several pharmaceutical agents and in hiPSC-CMs derived from patients with different inherited arrhythmogenic syndromes. Combining genetically encoded fluorescent reporters with hiPSC-CMs may bring a unique value to the study of inherited disorders, developmental biology, and drug development and testin
The characterisation of a novel nonsense mutation in RYR2 using human induced pluripotent stem cells.
The characterisation of a novel nonsense mutation in RYR2 using human induced pluripotent stem cells
The characterisation of a novel nonsense mutation in RYR2 using human induced pluripotent stem cells
Optogenetic Control of Human Induced Pluripotent Stem CellâDerived Cardiac Tissue Models
Background Optogenetics, using lightâsensitive proteins, emerged as a unique experimental paradigm to modulate cardiac excitability. We aimed to develop highâresolution optogenetic approaches to modulate electrical activity in 2â and 3âdimensional cardiac tissue models derived from human induced pluripotent stem cell (hiPSC)âderived cardiomyocytes. Methods and Results To establish lightâcontrollable cardiac tissue models, opsinâcarrying HEK293 cells, expressing the lightâsensitive cationicâchannel CoChR, were mixed with hiPSCâcardiomyocytes to generate 2âdimensional hiPSCâderived cardiac cellâsheets or 3âdimensional engineered heart tissues. Complex illumination patterns were designed with a highâresolution digital microâmirror device. Optical mapping and force measurements were used to evaluate the tissues' electromechanical properties. The ability to optogenetically pace and shape the tissue's conduction properties was demonstrated by using single or multiple illumination stimulation sites, complex illumination patterns, or diffuse illumination. This allowed to establish in vitro models for optogeneticâbased cardiac resynchronization therapy, where the electrical activation could be synchronized (hiPSCâderived cardiac cellâsheets and engineered heart tissue models) and contractile properties improved (engineered heart tissues). Next, reentrant activity (rotors) was induced in the hiPSCâderived cardiac cellâsheets and engineered heart tissue models through optogenetics programmedâ or crossâfield stimulations. Diffuse illumination protocols were then used to terminate arrhythmias, demonstrating the potential to study optogenetics cardioversion mechanisms and to identify optimal illumination parameters for arrhythmia termination. Conclusions By combining optogenetics and hiPSC technologies, lightâcontrollable human cardiac tissue models could be established, in which tissue excitability can be modulated in a functional, reversible, and localized manner. This approach may bring a unique value for physiological/pathophysiological studies, for disease modeling, and for developing optogeneticâbased cardiac pacing, resynchronization, and defibrillation approaches