Calcium Sensor Research Articles

Placental extracellular vesicles from severe preeclamptic women alter calcium homeostasis in cardiomyocytes: An ex vivo study.

Women with severe preeclampsia (sPE) exhibit a heightened risk of postpartum cardiovascular disease compared to those with normotensive pregnancies (NTP). While placental extracellular vesicles (EV) play a crucial role in feto-maternal communication, their impact on cardiomyocytes, particularly in the context of sPE, remains unclear. This study investigated the effect of sPE-associated placental EV (sPE-Plex EV) on cardiomyocyte calcium dynamics. We hypothesized that sPE-Plex EV mediates cardiomyocyte dysfunction by disrupting calcium signaling. EV were isolated from plasma and placental explant culture (Plex) using precipitation methods, and confirmed as Plex EV by PLAP activity and electron microscopy. Moreover, confocal microscopy confirmed the uptake of plasma EV in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and Plex EV by human AC-16 cardiomyocytes (hAC-16CM) cells. hiPSC-CM cells treated with sPE-EV and hAC-16CM cells treated with sPE-Plex EV exhibited significantly lower levels of Stromal interaction molecule 1 (STIM1) and Phospholamban (PLN) proteins compared to those treated with normotensive controls EV, as confirmed by western blot analysis. Treatment with sPE-Plex EV also resulted in the downregulation of STIM1 and PLN proteins in murine cardiomyocyte (mCM) cells compared to treatment with NTP-Plex EV. Our findings suggest that both plasma EV and Plex EV from sPE may alter calcium signaling in cardiac cells by downregulating calcium sensor proteins (STIM1 and PLN). Therefore, plasma EV and Plex EV from sPE pregnancies have adverse effects by altering calcium dynamics in hiPSC-CM, hAC-16CM, and mCM compared to normotensive control and potential impairment of cardiomyocyte function ex vivo.

The calcium sensor AtCML8 contributes to Arabidopsis plant cell growth by modulating the brassinosteroid signaling pathway.

Calcium signaling plays an essential role in integrating plant responses to diverse stimuli and regulating growth and development. While some signaling components and their roles are well-established, such as the ubiquitous calmodulin (CaM) sensor, plants possess a broader repertoire of calcium sensors. Notably, CaM-like proteins (CMLs) represent a poorly characterized class for which interacting partners and biological functions remain largely elusive. Our work investigates the role of Arabidopsis thaliana CML8 that exhibits a unique expression profile in seedlings. A reverse genetic approach revealed a function of CML8 in regulating root growth and hypocotyl elongation. RNA-seq analyses highlighted CML8 association with the regulation of numerous genes involved in growth and brassinosteroid (BR) signaling. Using co-immunoprecipitation experiments, we demonstrated that CML8 interacts with the BR receptor, BRI1, in planta in a ligand-dependent manner. This finding suggests the existence of a novel regulatory step in the BR pathway, involving calcium signaling.

Stress survival and longevity of Caenorhabditis elegans lacking NCS-1.

Although dysfunctional Ca2+ signaling can trigger biochemical reactions that lead to cell death, the role of calcium-binding proteins (CBPs) in this process is still a topic of debate. Neuronal calcium sensor 1 (NCS-1) is a CBP that is highly conserved and has been shown to increase cell survival against various types of injuries. As such, we hypothesized that NCS-1 could also be a stress-responsive protein with potential effects on survival and longevity. To explore this possibility, we conducted experiments to examine how Caenorhabditis elegans ncs-1 mutant nematodes fared under three different stress conditions: hyperosmotic, thermal, and chemical oxidant challenges. Our results showed that while the lack of NCS-1 had no effect on survival responses to hyperosmotic and thermal stresses, ncs-1 worms demonstrated remarkable resistance to the oxidant paraquat in a dose-dependent manner. Based on these findings, we conclude that C. elegans may employ adaptive mechanisms in the absence of NCS-1 to survive specific oxidative stress stimuli.

Diacylated 1,10‐diaza‐18‐crown‐6 as an Alternative Template for Fluorescent Sensors of Calcium Ions

ABSTRACTThe performance of many known fluorescent sensors for Ca2+ is pH‐dependent. We set out to address this by preparing a novel calcium sensor based on diacylated 1,10‐diaza‐18‐crown‐6 and containing no basic centres. The sensor is sensitive to sub‐millimolar levels of Ca2+ and partially tolerant of an aqueous environment. However, it suffers from a lack of selectivity over some other divalent cations, and retained pH sensitivity in the acidic range. The relations between these properties and the sensor's structure are discussed.

A large field-of-view, single-cell-resolution two- and three-photon microscope for deep and wide imaging

In vivo imaging of large-scale neuronal activity plays a pivotal role in unraveling the function of the brain's circuitry. Multiphoton microscopy, a powerful tool for deep-tissue imaging, has received sustained interest in advancing its speed, field of view and imaging depth. However, to avoid thermal damage in scattering biological tissue, field of view decreases exponentially as imaging depth increases. We present a suite of innovations to optimize three-photon microscopy for large field-of-view imaging at depths unreachable by two-photon microscopy. These techniques enable us to image neuronal activities of transgenic animals expressing protein calcium sensors in a ~ 3.5-mm diameter field-of-view with single-cell resolution in the deepest cortical layer of mouse brains. We further demonstrate simultaneous large field-of-view two-photon and three-photon imaging, subcortical imaging in the mouse brain, and whole-brain imaging in adult zebrafish. The demonstrated techniques can be integrated into typical multiphoton microscopes to enlarge field of view for system-level neural circuit research.

Correlation between evoked neurotransmitter release and adaptive functions in SYT1-associated neurodevelopmental disorder

Pathogenic missense variants in the essential synaptic vesicle protein synaptotagmin-1 (SYT1) cause a neurodevelopmental disorder characterised by motor delay and intellectual disability, hyperkinetic movement disorder, episodic agitation, and visual impairments. SYT1 is the presynaptic calcium sensor that triggers synchronous neurotransmitter release. We have previously shown that pathogenic variants around the calcium-sensing region of the critical C2B domain decrease synaptic vesicle exocytosis in neurons. Here, we have used cultured hippocampal neurons transfected with SYT1-pHluorin to examine how variants within the C2A and C2B domain of SYT1 impact evoked exocytosis. We show that recently identified variants within the facilitatory C2A domain of the protein (L159R, T196K, E209K, E219Q), as well as additional variants in the C2B domain (M303V, S309P, Y365C, G369D), share an underlying pathogenic mechanism, causing a graded and variant-dependent dominant-negative impairment in exocytosis. We establish that the extent of evoked exocytosis observed invitro in the presence of SYT1 variants correlates with neurodevelopmental impacts of this disorder. Specifically, the severity of motor and communication impairments exhibited by individuals harbouring these variants correlates with multiple measures of exocytic efficiency. Together, this suggests that there is a genotype-function-phenotype relationship in SYT1-associated neurodevelopmental disorder, centring impaired evoked neurotransmitter release as a common pathogenic driver. Moreover, this points toward a direct link between control of neurotransmitter release and development of adaptive functions, providing a tractable target for therapeutic amelioration. Australian National Health and Medical Research Council, UK Medical Research Council, Great Ormond Street Hospital Children's Charity, University of Melbourne.

Simultaneous mesoscopic measurement and manipulation of mouse cortical activity.

Dynamics of activity across the cerebral cortex at the mesoscopic scale - coordinated fluctuations of local populations of neurons - are essential to perception and cognition and relevant to computations like sensorimotor integration and goal-directed task engagement. However, understanding direct causal links between population dynamics and behavior requires the ability to manipulate mesoscale activity and observe the effect of manipulation across multiple brain regions simultaneously. Here, we develop a novel system enabling simultaneous recording and manipulation of activity across the dorsal cortex of awake mice, compatible with large-scale electrophysiology from any region across the brain. Transgenic mice expressing the GCaMP calcium sensor are injected systemically with an adeno-associated virus driving expression of the ChrimsonR excitatory opsin. This strategy drives expression of the blue-excited calcium indicator, GCaMP, in excitatory neurons and red-excited Chrimson opsin in inhibitory neurons. We demonstrate widefield single-photon calcium imaging and simultaneous galvo-targeted laser stimulation over the entire dorsal cortical surface. The light channels of the imaging and the opsin do not interfere. We characterize the spatial and temporal resolution of the method, which is suitable for targeting specific cortical regions and specific time windows in behavioral tasks. The preparation is stable over many months and thus well-suited for long-term behavioral experiments. This technique allows for studying the effect of cortical perturbations on cortex-wide activity, on subcortical spiking activity, and on behavior, and for designing systems to control cortical activity in closed-loop.

Characterization of the calmodulin-like protein family in Chara braunii and their conserved interaction with the calmodulin-binding transcription activator family.

Calcium sensor proteins play important roles by detecting changes in intracellular calcium and relaying that information onto downstream targets through protein-protein interaction. Very little is known about calcium sensors from plant species that predate land colonization and the evolution of embryophytes. Here, we examined the genome of the multicellular algae, Chara braunii, for orthologs to the evolutionarily-conserved calcium sensor calmodulin (CaM), and for CaM-like proteins (CMLs). We identified one CaM and eight CML isoforms which range in size from 16.4 to 21.3 kDa and are predicted to have between two to four calcium-binding (EF-hand) domains. Using recombinant protein, we tested whether CbCaM and CbCMLs1-7 possess biochemical properties of typical calcium sensors. CbCaM and the CbCMLs all displayed high-affinity calcium binding with estimated global KD,app values in the physiological µM range. In response to calcium binding, CbCaM and the CbCMLs exhibited varying degrees of increase in exposed hydrophobicity, suggesting different calcium-induced conformational changes occur among isoforms. We found many examples of putative CaM targets encoded in the C. braunii genome and explored the ability of CbCaM and CbCMLs to interact in planta with a representative putative target, a C. braunii CaM-binding transcription factor (CbCAMTA1). CbCaM, CbCML2, and CbCML4 associated with the C-terminal region of CbCAMTA1. Collectively, our data support the hypothesis that complex calcium signaling and sensing networks involving CaM and CMLs evolved early in the green lineage. Similarly, it seems likely that calcium-mediated regulation of transcription occurs in C. braunii via CAMTAs and is an ancient trait predating embryophytic emergence.

A Fully Printable and Integrated System with Long-Term Stability for Versatile and Multimodal Perspiration Tracking.

Real-time and continuous monitoring of physiological status via noninvasive sweat sensing shows promise for personalized healthcare and fitness management. However, the largely varied perspiration rates in different body statuses introduce challenges for effective sweat collection and accurate sensing. Herein, a fully printable strategy was developed to realize fully integrated patches for wireless sensing of sweat biomarker levels and perspiration rates with desirable stability and versatility. The printable calcium sensors with modified ion-selective membranes displayed an ultrawide linear range of 0.1-100 mM and a long-term stability with minimized drift down to 0.083 mV/h for around 40 h. Moreover, the microfluidic channels in versatile configurations were capable of a minimum sweat rate monitoring of 0.5 μL/min and a large sweat storage volume of up to 200 μL. The as-proposed fully printable sensing platforms provide high compatibility for sensor integration to achieve versatile perspiration tracking and comprehensive health monitoring.

Deep-prior ODEs augment fluorescence imaging with chemical sensors

To study biological signalling, great effort goes into designing sensors whose fluorescence follows the concentration of chemical messengers as closely as possible. However, the binding kinetics of the sensors are often overlooked when interpreting cell signals from the resulting fluorescence measurements. We propose a method to reconstruct the spatiotemporal concentration of the underlying chemical messengers in consideration of the binding process. Our method fits fluorescence data under the constraint of the corresponding chemical reactions and with the help of a deep-neural-network prior. We test it on several GCaMP calcium sensors. The recovered concentrations concur in a common temporal waveform regardless of the sensor kinetics, whereas assuming equilibrium introduces artifacts. We also show that our method can reveal distinct spatiotemporal events in the calcium distribution of single neurons. Our work augments current chemical sensors and highlights the importance of incorporating physical constraints in computational imaging.

Integration of single-photon miniature fluorescence microscopy and electrophysiological recording methods for <i>in vivo</i> studying hippocampal neuronal activity

The miniature single-photon fluorescent microscope (miniscope) enables the visualization of calcium activity in vivo in freely moving laboratory animals, providing the capability to track cellular activity during the investigation of memory formation, learning, sleep, and social interactions. However, the use of calcium sensors for in vivo imaging is limited by their relatively slow (millisecond-scale) kinetics, which complicates the recording of high-frequency spike activity. The integration of methods from single-photon miniature fluorescent microscopy with electrophysiological recording, which possesses microsecond resolution, represents a potential solution to this issue. Such a combination of techniques allows for the simultaneous recording of optical and electrophysiological activity in a single animal in vivo. In this study, a flexible polyimide microelectrode was developed and integrated with the gradient lens of the miniscope. The in vivo tests conducted in this research confirmed that the microelectrode combined with the gradient lens facilitates simultaneous single-photon calcium imaging and local field potential recording in the hippocampus of an adult mouse.

Interactions of Li+ ions with NCS1: A potential mechanism of Li+ neuroprotective action against psychotic disorders

Li+ based drugs have been used for the treatment of psychiatric disorders due to their mood stabilizing role for decades. Recently, several studies reported the protective effect of Li+ against severe neuropathologies such as Parkinson's, Alzheimer's, and Huntington's disease. Surprisingly, despite a broad range of Li+ effects on neurological conditions, little is known about its molecular mechanism. In this study, we propose that neuronal calcium sensor 1 (NCS1), can be an effective molecular target for Li+ action. Here we show that the EF-hands in ApoNCS1 have submillimolar affinity for Li+ with Kd = 223 ± 19 μM. Li+ binding to ApoNCS1 quenches Trp emission intensity, suggesting distinct Trp sidechains environment in Li+NCS1 compared to ApoNCS1 and Ca2+NCS1. Li+ association also stabilizes the protein α-helical structure, in a similar way to Ca2+. Li+ association does not promote NCS1 dimerization. Association of Li+ increases NCS1 affinity for the D2R receptor binding peptide, in a similar way to Ca2+, however, the affinity of NCS1 for chlorpromazine is reduced with respect to Ca2+NCS1, possibly due to a decrease in solvent exposed hydrophobic area on the NCS1 surface in the presence of Li+. MD simulation data suggests that Li+ ions are coordinated by four oxygens from Asp and Glu sidechains and one carbonyl oxygen, in a similar way as reported previously for Li+ binding to DREAM. Overall, the data shows that Li+ binds to EF-hands of NCS1 and Li+NCS1 interactions may be involved in the potential neuroprotective role of Li+ against psychotic disorders.

Tomato Synaptotagmin F accelerates fruit ripening, shortens fruit shelf-life and increases susceptibility to Penicillium expansum

Synaptotagmins (SYTs), initially identified as calcium sensors for regulating synaptic vesicle exocytosis and endocytosis in mammalian neurons, play crucial role in biotic and abiotic stresses in plants. However, the function of SYTs in fruit ripening is unclear. In this study, a tomato Synaptotagmins gene, SlSYTF, was found to accelerate tomato fruit ripening. SlSYTF encodes an endoplasmic reticulum localized protein whose transcription is continuously enhanced during fruit ripening. Overexpression SlSYTF in tomato resulted in accelerated ripening progress, increased carotenoid content as well as decreased the firmness of tomato fruit, whereas the mutant slsytf-c exhibited the opposite phenotype. Importantly, SlSYTF could increase ethylene production by activating the expression of ethylene synthesis genes and prompt cell wall degradation by increasing pectinase and cellulase activities. Nevertheless, the accelerated cell wall degradation and thinned cuticle due to SlSYTF results in reduced shelf life and pathogen resistance. Collectively, we revealed a new SYTs gene that plays a dual role in tomato fruit ripening and pathogen response. This finding may shed new light on the relationship between maturation and immunity.

STIM1 functions as a proton sensor to coordinate cytosolic pH with store-operated calcium entry

The meticulous regulation of intracellular pH (pHi) is crucial for maintaining cellular function and homeostasis, impacting physiological processes such as heart rhythm, cell migration, proliferation, and differentiation. Dysregulation of pHi is implicated in various pathologies such as arrhythmias, cancer, and neurodegenerative diseases. Here, we explore the role of STIM1, an ER calcium (Ca2+) sensor mediating Store Operated Ca2+ Entry (SOCE), in sensing pHi changes. Our study reveals that STIM1 functions as a sensor for pHi changes, independent of its Ca2+-binding state. Through comprehensive experimental approaches including confocal microscopy, FRET-based sensors, and mutagenesis, we demonstrate that changes in pHi induce conformational alterations in STIM1, thereby modifying its subcellular localization and activity. We identify two conserved histidine within STIM1 essential for sensing pHi shifts. Moreover, intracellular alkalization induced by agents such as Angiotensin II or NH4Cl enhances STIM1-mediated SOCE, promoting cardiac hypertrophy. These findings reveal a novel facet of STIM1 as a multi-modal stress sensor that coordinates cellular responses to both Ca2+ and pH fluctuations. This dual functionality underscores its potential as a therapeutic target for diseases associated with pH and Ca2+ dysregulation.

Neuromodulator and neuropeptide sensors and probes for precise circuit interrogation in vivo.

To determine how neuronal circuits encode and drive behavior, it is often necessary to measure and manipulate different aspects of neurochemical signaling in awake animals. Optogenetics and calcium sensors have paved the way for these types of studies, allowing for the perturbation and readout of spiking activity within genetically defined cell types. However, these methods lack the ability to further disentangle the roles of individual neuromodulator and neuropeptides on circuits and behavior. We review recent advances in chemical biology tools that enable precise spatiotemporal monitoring and control over individual neuroeffectors and their receptors in vivo. We also highlight discoveries enabled by such tools, revealing how these molecules signal across different timescales to drive learning, orchestrate behavioral changes, and modulate circuit activity.

Miniscope Recording Calcium Signals at Hippocampus of Mice Navigating an Odor Plume.

Mice navigate an odor plume with a complex spatiotemporal structure in the dark to find the source of odorants. This article describes a protocol to monitor behavior and record Ca2+ transients in dorsal CA1 stratum pyramidale neurons in the hippocampus (dCA1) in mice navigating an odor plume in a 50 cm x 50 cm x 25 cm odor arena. An epifluorescence miniscope focused through a gradient-index (GRIN) lens imaged Ca2+ transients in dCA1 neurons expressing the calcium sensor GCaMP6f in Thy1-GCaMP6f mice. The paper describes the behavioral protocol to train the mice to perform this odor plume navigation task in an automated odor arena. The methods include a step-by-step procedure for the surgery for GRIN lens implantation and baseplate placement for imaging GCaMP6f in CA1. The article provides information on real-time tracking of the mouse position to automate the start of the trials and delivery of a water reward. In addition, the protocol includes information on using an interface board to synchronize metadata describing the automation of the odor navigation task and frame times for the miniscope and a digital camera tracking mouse position. Moreover, the methods delineate the pipeline used to process GCaMP6f fluorescence movies by motion correction using the NorMCorre algorithm followed by identification of regions of interest with EXTRACT. Finally, the paper describes an artificial neural network approach to decode spatial paths from CA1 neural ensemble activity to predict mouse navigation of the odor plume.

Synaptotagmin-1 in phase: Condensate biology reveals new insights into the synaptic calcium sensor.

Two recent papers by Mehta et al. and Zhu et al. in this issue (https://doi.org/10.1083/jcb.202311191) discover that synaptotagmin-1, the primary calcium sensor at the synapse, forms biomolecular condensates, identifying a new layer of regulation in calcium-triggered synaptic vesicle exocytosis.

Vesicle docking and fusion pore modulation by the neuronal calcium sensor Synaptotagmin-1.

Synaptotagmin-1 (Syt1) is a major calcium sensor for rapid neurotransmitter release in neurons and hormone release in many neuroendocrine cells. It possesses two tandem cytosolic C2 domains that bind calcium, negatively charged phospholipids, and the neuronal SNARE complex. Calcium binding to Syt1 triggers exocytosis, but how this occurs is not well understood. Syt1 has additional roles in docking dense core vesicles (DCV) and synaptic vesicles (SV) to the plasma membrane (PM) and in regulating fusion pore dynamics. Thus, Syt1 perturbations could affect release through vesicle docking, fusion triggering, fusion pore regulation, or a combination of these. Here, using a human neuroendocrine cell line, we show that neutralization of highly conserved polybasic patches in either C2 domain of Syt1 impairs both DCV docking and efficient release of serotonin from DCVs. Interestingly, the same mutations resulted in larger fusion pores and faster release of serotonin during individual fusion events. Thus, Syt1's roles in vesicle docking, fusion triggering, and fusion pore control may be functionally related.

Role of Calmodulin in Cardiac Disease: Insights on Genotype and Phenotype.

Calmodulin, a protein critically important for the regulation of all major cardiac ion channels, is the quintessential cellular calcium sensor and plays a key role in preserving cardiac electrical stability. Its unique importance is highlighted by the presence of 3 genes in 3 different chromosomes encoding for the same protein and by their extreme conservation. Indeed, all 3 calmodulin (CALM) genes are among the most constrained genes in the human genome, that is, the observed variants are much less than expected by chance. Not surprisingly, CALM variants are poorly tolerated and accompany significant clinical phenotypes, of which the most important are those associated with increased risk for life-threatening arrhythmias. Here, we review the current knowledge about calmodulin, its specific physiological, structural, and functional characteristics, and its importance for cardiovascular disease. Given our role in the development of this knowledge, we also share some of our views about currently unanswered questions, including the rational approaches to the clinical management of the affected patients. Specifically, we present some of the most critical information emerging from the International Calmodulinopathy Registry, which we established 10 years ago. It appears growingly evident as further progress requires the collection of deep phenotypic information through international contributions to the registry as the best way to expand our knowledge about Calmodulinopathies with the goal of acquiring the information necessary to guide clinical management.

Viral vector eluting lenses for single-step targeted expression of genetically-encoded activity sensors for in vivo microendoscopic calcium imaging.

Optical methods for studying the brain offer powerful approaches for understanding how neural activity underlies complex behavior. These methods typically rely on genetically encoded sensors and actuators to monitor and control neural activity. For microendoscopic calcium imaging, injection of a virus followed by implantation of a lens probe is required to express a calcium sensor and enable optical access to the target brain region. This two-step process poses several challenges, chief among them being the risks associated with mistargeting and/or misalignment between virus expression zone, lens probe and target brain region. Here, we engineer an adeno-associated virus (AAV)-eluting polymer coating for gradient refractive index (GRIN) lenses enabling expression of a genetically encoded calcium indicator (GCaMP) directly within the brain region of interest upon implantation of the lens. This approach requires only one surgical step and guarantees alignment between GCaMP expression and lens in the brain. Additionally, the slow virus release from these coatings increases the working time for surgical implantation, expanding the brain regions and species amenable to this approach. These enhanced capabilities should accelerate neuroscience research utilizing optical methods and advance our understanding of the neural circuit mechanisms underlying brain function and behavior in health and disease.