Cell Calcium

Neuronal populations connected by gap junctions can be revealed via dye coupling of small molecules like neurobiotin and lucifer yellow. However, the extent of dye diffusion between neurons varies with connexin subtype, loading method, and neuromodulation. Due to the increasing availability of GCaMP transgenic animals, we explore the possibility of revealing gap junctional coupling using Ca2+ imaging in the Xenopus laevis tadpole motor system. Reliable axo-axonal electrical coupling was previously found in excitatory descending interneurons (dINs) using paired recordings but not with neurobiotin dye coupling. Here, we made whole-cell patch-clamp recordings with Ca2+-supplemented intracellular solution to load Ca2+ into GCaMP6s-expressing neurons, followed by Ca2+ imaging to detect potential Ca2+ diffusion across coupled neurons. Successful membrane breakthroughs led to transient fluorescence increases in the patched neuron. However, increasing the Ca2+ concentration promoted membrane resealing and rapid loss of whole-cell recordings. Regardless of recording duration, loading-triggered fluorescence only lasted up to three minutes, suggesting rapid Ca2+ clearance. Pharmacologically blocking sarcoplasmic /endoplasmic reticulum Ca2+-ATPases and plasma membrane Na+/Ca2+ exchangers did not prolong fluorescence, although sustained fluorescence was achieved with positive current injections. Counter to our expectations, fluorescence increases in Ca2+-loaded dINs did not spread to neighboring dINs. Robust intracellular Ca2+ regulation mechanisms, membrane resealing, and long dIN axons likely hindered intercellular Ca2+ diffusion. Therefore, this approach is not appropriate for revealing electrical coupling within this system.

The question of whether islet neogenesis occurs in adult humans has been a subject of long-standing debate. To explore the characteristics of islet endocrine cells associated with pancreatic ducts, we employed imaging mass cytometry to examine pancreatic tissues from individuals across different age groups, including those with prediabetes or type 2 diabetes (T2D). Our analysis revealed the presence of all five pancreatic islet endocrine cell types, along with two types of non-hormone-expressing endocrine cells, located within or immediately adjacent to the ducts. These cells were most abundant in infancy, with a gradual decline observed through adulthood. Notably, ductal {beta} cells predominated in infancy, whereas ductal cells became more prevalent in adulthood, and significantly increased in the group aged over 60 years. Obesity further increased the ductal {beta} cells in the subjects aged over 60 years. Under prediabetic and T2D conditions, an increase in all duct-related endocrine cells was observed. These findings indicate that ductal cells may serve as a reservoir for new pancreatic endocrine cells, offering potential insights into the promotion of endogenous {beta} cell regeneration in diabetic patients. Highlights{bigcirc} Characterization of various islet endocrine cell types related to ducts in human pancreas. {bigcirc}The insulin-positive cells are the dominant cells among all duct-related islet endocrine cell types during the infancy period, however, the glucagon-positive cells become the dominant cells in adulthood. {bigcirc}T2D, Obesity, and aging are involved in the increase in the number of duct-related endocrine cells.

Cardiac function is dependent upon the ability of cardiomyocytes to adapt their contractions to meet the demands of the body. Increased preload lengthens the sarcomere, altering the efficiency of contraction. The surface topography of cardiomyocytes is distinct from other cell types. T-tubules are key membrane structures which protrude into the cell body, aligned with the edges of the sarcomere. These are key signalling domains which ensure efficient and adaptable excitation-contraction coupling. It has been shown that T-tubules are dynamic structures which deform during the contraction cycle however, how the T-tubule structure adapts to increased preload has not been realised. Here we demonstrate a methodology for the measurement of the surface topography and sub-cellular signalling of isolated adult cardiomyocytes under diastolic stretch. We track individual T-tubule openings, showing that increased load causes them to shift, increase diameter and become stiffer. Future applications of this system include experimental modelling of preload-reducing therapies, for the treatment of acute and chronic heart failure.

Effective insulin secretion and blood glucose homeostasis depend on the multistep maturation of insulin secretory granules (ISGs), a process that includes lumen acidification, enzymatic insulin processing, and biophysical remodeling of the granule. An under studied aspect of ISG maturation is the role of inter-organelle contacts in organelle remodeling. While a correlation between ISG-mitochondria contacts and ISG maturation has been observed, many questions remain on how this interaction may impact maturation (1-5). We sought to address this gap in knowledge by using multi-scale imaging approaches (fluorescent microscopy, soft X-ray tomography, and cryo-electron tomography) to examine how the biophysical properties and spatial organization of ISGs change around the mitochondrial network. Our data suggests that ISGs in proximity to mitochondria exhibit lower pH, higher biomolecular density, and smaller vesicle diameter. Time-resolved imaging using a SNAP tag labelling system also shows that as ISGs age, their proximity to the mitochondria network is increased between 3-6 hours after biosynthesis, suggesting that ISG-mitochondria association is dynamically spatiotemporally regulated in pancreatic {beta}-cells. These data suggest that mitochondrial proximity contributes to the maturation and remodeling of ISGs in pancreatic beta cells.

In LGI1-linked animal models of inherited autosomal dominant lateral temporal lobe epilepsy, increased neuronal excitability is accompanied by modifications in the AMPA/NMDA receptor ratio and a large decrease in Kv1 type potassium channels. However, the mechanism which links the absence of LGI1 to reduced expression of key neuronal ion channels is unknown. We observed multiple conserved canonical ganglioside-binding domains (GBDs) within human LGI1, mainly located in the EPTP domain. We show that GT1b is co-captured from native rat brain extracts by human LGI1 antibodies and, using SPR analysis, that recombinant full length LGI1 interacted with liposomes containing GT1b and GM1, but not GM3, lyso-lactosylceramide, phosphatidylserine or phosphatidylcholine. The ganglioside binding capacity of GBD peptide sequences exposed at the surface of LGI1 were confirmed using SPR and Langmuir film balance. Our data suggest that LGI1 interacts with gangliosides and may be involved in organizing lipid membrane platforms accommodating functional protein complexes. The loss of LGI1 could destabilize these platforms and contribute to reduced expression of key ion channels in Lgi1-/- mice.

Pancreatic beta cells have the unique function of synthesizing and secreting high amounts of the inhibitory neurotransmitter {gamma}-aminobutyric acid (GABA). The mechanism of GABA secretion, whether vesicular or channel-mediated, is debated. Our study reveals surprising temporal complexity in the pattern of islet GABA secretion. We used insulin secretion modulators to demonstrate that GABA release is not directly correlated with insulin secretion. VGAT reporter mice also showed that beta cells do not express the requisite vesicular GABA transporter (VGAT) for vesicular GABA release. Instead, GABA is secreted from the cytosol in pulses by the LRRC8A/D isoform of the volume regulatory anion channel (VRAC). We further demonstrate the dynamic coordination of GABA release with calcium influx in beta cells and dependence on beta cell depolarization. These results suggest a model where GABA is released during the peaks of beta cell calcium oscillations to provide feedback which strengthens and reinforces the oscillation waveform.

G protein-coupled receptors (GPCRs) perceive spatially and temporally diverse stimuli and activate G protein heterotrimers comprising , {beta}, and {gamma} subunits, which broadcast signals through a broad range of effectors at various subcellular compartments. Therefore, understanding endogenous G protein activity dynamics at the subcellular level, thereby recapitulating in vivo signaling paradigms, will facilitate the identification of pathological signaling pathways. However, the lack of sensors for endogenous G proteins has been an obstacle. Here, we demonstrate the engineering of sensors to probe endogenous GiGTP and GqGTP. Compared to examining overexpressed and fluorescently tagged G, our sensors capture the magnitude and kinetics of endogenous GGTP dynamics, including their generation, equilibrium signaling, and hydrolysis, with native fidelity. Using the translocation-based GiGTP sensor, we show that heterotrimer dissociation upon Gi-GPCR activation is G{gamma}-subtype dependent. Confirming our previous findings, the GqGTP sensor showed that Gq expression is low and tightly regulated in most cells. Using optogenetic tools, we demonstrate that our sensors detect GGTP generation and hydrolysis during asymmetric GPCR-G protein activation, a capability that will be particularly useful in morphologically diverse cells such as neurons. Therefore, our engineered novel GGTP sensors can be highly beneficial in decoding subcellularly resolved endogenous G protein signaling dynamics.

Pancreatic islets contain -, {beta}-, {gamma}- and {delta}-cells as sensors and actuators regulating glucose homeostasis. Despite the known importance of -cells, they are seemingly required for glucose tolerance only under metabolic stress. In an inducible model of -cell ablation in mice (GluDTR), glucose tolerance was considerably decreased by physiological addition of amino-acids mimicking meals. Analysis of islet {beta}-cell secretion and electrical activities using microelectrode arrays (MEA) detected only minor differences in GluDTR mice for glucose but revealed a major reduction upon addition of amino acids. Analysis of functional islet {beta}-cell networks by high density MEA revealed leading regions in different locations, a high degree of synchrony and the activation of large cell clusters. The characteristics of leading regions were preserved in GluDTR islets, but synchrony, cluster size and signal propagation speed were largely reduced. Thus, even without metabolic stress, -cells are required for nutrient homeostasis by regulating the dynamics of {beta}-cell networks. TeaserIslet -cells are required for meal tolerance by adjusting synchrony, cluster size and signal propagation of {beta}-cell networks.

Insulin-producing {beta}-cells demonstrate remarkable heterogeneity in their individual responsiveness to glucose, and that cellular heterogeneity contributes to coordinating islet activity and glucose homeostasis. Our current understanding of how variation in cell-intrinsic factors control cellular excitability and insulin secretion is informed by foundational experiments conducted on dispersed single {beta}-cells. Such studies are limited in their ability to link multiple electrical or metabolic properties within a single cell and preclude the ability to relate, post hoc, each parameters contribution to glucose responsiveness. Computational modelling represents a unique and underutilized tool to integrate and investigate the role of natural {beta}-cell heterogeneity in physiologic glucose responses. Herein, we utilize a high-volume single-cell electrophysiology "patch-seq" dataset to define the physiologically relevant sources of variability in human {beta}-cell electrophysiology and model their influence on single-cell glucose responses. Three thousand in silico human {beta}-cells were fitted to physiologically relevant variations in glucokinase activity, K+ current, Na+ current, Ca2+ current, and exocytotic function. Four dominant electrical phenotypes arose at low (2 mM) and high (20 mM) glucose: silent, bursting, spiking, and depolarized. Approximately 50% of uncoupled {beta}-cells remained electrically silent at high glucose. Furthermore, Na+ channel half-inactivation voltage was a major predictor of the silent and spiking phenotypes at each glucose concentration, and of cells that transition from silent to spiking when glucose increased. Indeed, experimentally observed variation in Na+ channel voltage dependence was second only to variation in ATP-sensitive potassium channel conductance in determining {beta}-cell excitability. Our data-driven computational modelling highlights the functional importance of electrical heterogeneity in human {beta}-cell glucose responses, and provides a useful tool for generating testable hypotheses.

Trypanosoma cruzi is the etiological agent of Chagas disease (CD), a neglected tropical disease that affects millions of people worldwide. During its life cycle, T. cruzi undergoes several key differentiation processes that are essential for its survival. The precise mechanisms that regulate these processes remain elusive, and any interference in this cycle would represent a breakthrough in the development of effective therapy against CD. Here, after disrupting a single IP6K allele of T. cruzi, we observed that key differentiation processes (metacyclogenesis, amastigogenesis and trypomastigogenesis) were profoundly impaired. Epimastigote forms of IP6K-deficient T. cruzi exhibited morphological alterations and reduced metacyclogenesis. IP6K-deficient metacyclic forms had reduced infective potential in human cardiomyocytes. IP6K-deficient amastigote forms showed impaired ability to transform into trypomastigotes, with most of the population egressing from human cardiomyocytes without completing trypomastigogenesis. Together, our results suggest that IP6K is critical to sustain the T. cruzi life cycle. Since disruption of both IP6K alleles was lethal and the primary structure of IP6K shares only [~]15% similarity with its human homolog, this kinase emerges as a promising target for drug development against CD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=153 HEIGHT=200 SRC="FIGDIR/small/700787v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1c6e87corg.highwire.dtl.DTLVardef@1c95a1eorg.highwire.dtl.DTLVardef@3b8221org.highwire.dtl.DTLVardef@dc79fd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Insulin secretion from pancreatic {beta}-cells is a tightly controlled process where hormone synthesis, granule formation and release are regulated in order to maintain whole body glucose homeostasis. Failure to produce or release insulin results in hyperglycemia that may develop into diabetes. Insulin-containing granules exist in different pools that have different propensity for release, yet what determined the fate of a granule after initial formation is not clear. In this study we aimed to identify key steps in the early life of an insulin granule that directs it towards release. Using two different methods for time-dependent labeling, we found that insulin granules shortly after budding from the trans-Golgi network associate with mitochondria. This organelle interaction involves the voltage-dependent anion channel (VDAC) and the vesicular nucleotide transporter (VNUT). Reduced VNUT expression prevented the recruitment of VDAC to insulin granules and redirected granules towards autophagy-dependent lysosomal degradation, resulting in reduced insulin content and impaired insulin secretion. These results show the requirement of granule-mitochondria crosstalk for normal progression through the early stages of the secretory pathway.

Voltage-gated Na+ (Nav) channels, including Nav1.5, are responsible for the initiation of cardiac and neuronal action potentials. Regulation of Nav1.5 inactivation is linked to multiple accessory proteins that bind its C-terminal domain (CTD) including calmodulin (CaM) and intracellular fibroblast growth factors (iFGF). Previous results demonstrate that Ca2+-bound CaM preferentially binds to iFGF12A. The role of intracellular Ca2+ ([Ca2+]i) in regulating Nav1.5 gating, either directly or via auxiliary proteins like CaM, is controversial. We hypothesize that CaM binding to the Nav1.5 CTD and iFGF12A synergistically alters channel inactivation in a previously unobserved calcium-dependent manner. We performed Fluorescence Resonance Energy Transfer (FRET) imaging in live cells to observe the interaction between the Nav1.5 alpha subunit, CaM and iFGF12A. At resting [Ca2+]i, a 2-fold difference between acceptor and donor FRET efficiency was observed, implying that a single CaM acceptor is present on the Nav1.5 CTD even in the presence of FGF12A. After increasing [Ca2+]i, the donor and acceptor FRET efficiencies equalize, suggesting a 2:1:1 ratio between CaM, FGF12A, and the Nav1.5 CTD. We then compared the voltage-dependent gating kinetics of Nav1.5 with FGF12A in the presence/absence of calcium. With low [Ca2+]i, the steady-state inactivation of Nav1.5 with FGF12A was significantly shifted toward hyperpolarized potential compared to resting [Ca2+]i. Thus, the FGF12A:CaM complex confers a Ca2+-dependent mechanism enabling FGF12A modulates the Nav1.5 steady-state inactivation. Additionally, the ability of multiple subunits to bring CaM to the Nav1.5 CTD implies biological redundancy to prevent major alteration to Nav1.5 inactivation in the absence of CaM.

Local field potentials (LFPs) measured in the extracellular matrix of the brain are postulated to arise from the integration of synaptic ionic currents and spread by volume conduction. However, there is a lack of consensus on whether these spatiotemporal voltage gradients are just an epiphenomenon of spiking or if the LFPs play a functional role in information processing. To examine a potential functional role of LFPs in information processing, we developed a microfluidic device that allows neurons from the hippocampal formation to self-wire through microfluidic channels, effectively isolating the activity of single axons between subregions of the network. We recorded spontaneous theta-band activity (4-10 Hz) in these axons whose power spectra were independent of simultaneous spiking activity. A sparse set of axons from the CA3 into the CA1 had the highest theta amplitudes. Source neurons for the axonal theta were identified through cross correlation. Functionally, sparse axonal theta phase and amplitude correlated with target subregional spiking and more strongly with burst length. These results suggest that theta voltage oscillations in axons may contribute to activation of slow voltage-gated calcium channels to drive stronger synaptic release of transmitter to coordinate hippocampal activity between subregions. We propose that theta oscillations are controlled by specific ion channels distinct from those that generate spikes, a multiplex coding mechanism for inter-regional communication with implications for routing, executive control, disease states and artificial neural networks.

Malaria-causing parasites from the Plasmodium genus possess a mitochondrion that is essential across all life-cycle stages and highly divergent from its hosts, making it a suitable drug target. Transport of metabolites across the inner membrane of this metabolically active organelle is mediated by mitochondrial carrier (MC) proteins. Among these, the apicomplexan-specific MC1 (AMC1) stands out due to its likely essential function and limited conservation even within the Apicomplexa phylum. Bioinformatics and structural predictions reveal that P. falciparum AMC1 (PfAMC1) lacks canonical gating residues and shows a distorted membrane barrel, suggesting it may not function as a transporter. Using two independent mutant parasite lines, we demonstrate that PfAMC1 localises to the periphery of mitochondria, which exhibit increased dispersion and rounding during male gametogenesis, when the C-terminus is modified. Additionally, we identify the mammalian MTCH2, the function of which is still debated, as a potential structural homologue of PfAMC1, opening new avenues for research. Our findings emphasize the unique role of PfAMC1 in mitochondrial dynamics and lay the groundwork for further exploration of its molecular mechanism.

Enteropathogenic Escherichia coli (EPEC) is a major bacterial enteropathogen causing infectious diarrhea among children in developing countries. Here, we found that EPEC induced isolated Ca2+ responses in epithelial cells, triggered by extracellular ATP (eATP). These responses were dependent on type III secretion (T3S) and down-regulated by the bacterial secreted protease EspC, consistent with eATP released by the T3S translocon pore-forming activity in host membranes. By performing high speed Ca2+ imaging, we uncovered that at the onset of infection, low eATP levels triggered Ca2+-responses involving the whole cell but showing the small amplitude and fast kinetics usually associated with local Ca2+ responses. The findings, supported by theoretical modeling, evocate a conceptual shift whereby low amounts of inositol 1, 4, 5-trisphosphate (IP3) induced by low eATP levels and subsequent moderate Ca2+ release enable the fast coordination of IP3 receptor cluster activation throughout the cell. Importantly, these yet undescribed coordinated fast responses occurred over prolonged time periods and defined a cell state with dampened activation of the pro-inflammatory transcriptional activator NF-kB associated with a decrease in its Ca2+-dependent O-linked {beta}-N-acetylglucosamine modification.

Calcium dynamics controls learning and memory. Changes in calcium-induced calcium release (CICR), which is caused by opening ryanodine receptors (RyR) located on endoplasmic reticulum (ER) membrane, have been implicated in neurodegenerative disorders, such as Alzheimers disease (AD), manifesting with disruptions in calcium homeostasis and signaling. Calmodulin, one of the most abundant proteins in the brain, inhibits RyR2, expressed in the dendrites of hippocampal CA1 neurons, with several reported consequences: relieving of this inhibition is responsible for heart failure, and enhancing calmodulin to RyR binding [1] alleviates cell loss and AD-like neuronal hyperexcitability. To investigate the role of calmodulin in aging and AD, we built a sophisticated reaction-diffusion model of a dendritic branch with ER. We showed that relieving inhibition of RyR2 by calmodulin increased spatial and temporal spread of calcium transients in the dendrite. This effect was also visible in a model of old age, where disinhibition of RyR2 increased spatial spread of calcium transients by a factor of 2, and disinhibition of RyR2 combined with increased concentration of calcium buffering molecules increased duration of calcium transients, likely contributing to the deficits in learning and memory observed in old age. Lower activation of plasma membrane calcium ATPase (PMCA), which is also activated by calmodulin and inhibited by {beta}-Amyloid oligomers, and not RyR2 disinhibition, led to the increase resting intracellular calcium concentration observed in AD. Overall, our research demonstrates that changes in calmodulin that are associated with AD and aging, by regulation of RyR2 and PMCA, underlie changes in calcium dynamics that cause deficits in learning and memory. Author summaryCalcium dynamics controls learning and memory. In neurons calcium dynamics is regulated by a complex system including calcium-permeable channels and extrusion pumps. The components of this system are regulated by calmodulin, which is enriched in the brain. We show that relieving calmodulin inhibition of calcium permeable channels, which accompanies aging, decreases spatial and temporal specificity of calcium release contributing to the deficits in learning and memory observed in old age. In contrast, calcium extrusion pumps that are activated by calmodulin, are likely responsible for increased resting intracellular calcium concentration observed in Alzheimers disease. In consequence, a novel role emerges for calmodulin, which in the brain is primarily considered a fast-acting calcium buffering molecule, as an important regulator in neuronal calcium dynamics underlying pathology.

Mitochondrial membrane potential ({Delta}{Psi}m) is central to ATP production, ion homeostasis, and cell survival, reflecting the functional state of the inner mitochondrial membrane and oxidative phosphorylation. Accurate assessment of {Delta}{Psi}m is therefore essential for understanding mitochondrial physiology and dysfunction in health, ageing, and disease. Lipophilic cationic fluorescent dyes, such as TMRM and TMRE, are widely used to monitor {Delta}{Psi}m in live cells, enabling high-temporal-resolution imaging of both steady-state membrane potential and dynamic fluctuations. Beyond stable bioenergetic measurements, live-cell imaging reveals transient, reversible depolarisation events, known as mitochondrial "flickers." These events, observed across multiple cell types and imaging platforms, are often associated with brief openings of the mitochondrial permeability transition pore (mPTP) and may represent regulated mitochondrial excitability, rather than irreversible damage. While excessive or synchronised depolarisations may signal mitochondrial injury, transient flickers are increasingly viewed as potential signalling mechanisms within the mitochondrial network. This work discusses methodological considerations for {Delta}{Psi}m imaging, the biological significance of mitochondrial flickers, and the importance of distinguishing physiological events from probe- and light-induced artefacts, highlighting the emerging concept of mitochondria as dynamic and communicative bioenergetic networks.

The action potential (AP) is thought to be generated at the axon initial segment and to faithfully propagate along the axon. However, data from both invertebrate and mammalian systems show that the axon is an underappreciated locus of activity modulation and neuronal computation. We assessed axonal AP propagation in neocortical parvalbumin-expressing interneurons (PVINs) during prolonged, high-frequency activity through paired whole-cell somatic and axon-attached patch clamp recordings in acute brain slices from mouse and human. We found that PV-IN axonal AP propagation remains robust during prolonged activity at moderate frequencies, such as during the entrainment to PV-IN firing patterns recorded in awake, behaving mice in vivo. However, prolonged, high-frequency activity during evoked trains of APs and during seizure-like events resulted in changes in the waveform of the axonal (but not somatic) AP, at least in part due to intrinsic use-dependent mechanisms. This use-dependent decrement in the axonal AP waveform is associated with decreases in calcium influx at PV-IN boutons and subsequent PV-IN-mediated synaptic transmission, indicating this phenomenon may lead to a dissociation between somatic and axonal excitability that could shape PV-IN contributions to circuit dynamics during periods of high activity.

Congenital heart defects frequently arise from alterations in the elongation of the cardiac outflow tract (OFT). Proper elongation of the OFT depends on the coordinated deployment of progenitor cells from the second heart field (SHF) and on dynamic interactions with the extracellular matrix (ECM). Among ECM components, fibronectin (Fn1) and tenascin-C (TnC) have emerged as key regulators of cardiac morphogenesis. Studies in mouse embryos have shown that mesodermal Fn1 is required to maintain proper TnC localization within SHF cells. To study heart development, mammalian models are challenging to use because of their in utero development. This limitation highlights the need for alternative models with external development, where direct observation is possible; however, in these systems, the cellular organization of the SHF and the dynamics of its ECM environment remain poorly characterized Here, we investigated the cellular and extracellular architecture of SHF cells localized to the dorsal pericardial wall (DPW) during heart development in Xenopus laevis. We show that SHF cells undergo a stage-dependent transition from a predominantly monolayered organization at NF35 to a multilayered structure at NF42. This transition is accompanied by dynamic remodeling of the ECM, characterized by increased expression of Fn1, TnC, and Collagen I (ColI) and by redistribution of ECM components within the DPW. Functional experiments revealed that depletion of Fn1 disrupts cardiac morphogenesis, leading to shortening of the OFT and reduced ventricular size. Moreover, loss of Fn1 decreases TnC and ColI levels and alters the spatial organization of TnC within the DPW, indicating that Fn1 is required for proper ECM assembly within the SHF cells. These findings identify Fn1 as a key regulator of ECM assembly within the DPW and highlight how ECM remodeling contributes to the organization of SHF progenitor cells during OFT elongation. Altogether, we demonstrated that Xenopus laevis is a powerful model for studying ECM-driven mechanisms of cardiac morphogenesis.

Sparsely active granule cells in the dentate gyrus (DG) project onto CA3 pyramidal neurons through mossy fibers (MF), forming a feedforward network that ensures even similar inputs activate distinct and minimally overlapping populations of CA3 neurons. This process, known as pattern separation, is a key computational feature at this synapse. However, such sparse activity and connectivity increase the risk of information loss, as multiple presynaptic inputs are typically required to elicit a postsynaptic action potential. Interestingly, MF synapses exhibit robust short-term plasticity (STP), enabling a dynamic increase in release probability during brief bursts of presynaptic activity. This mechanism ensures that a short, high-frequency burst from a single granule cell can reliably generate a spike in its postsynaptic CA3 target. Unlike other hippocampal synapses, MF boutons are large, with multiple active zones, each coupled to a cluster of voltage-dependent calcium channels (VDCCs). MF boutons also possess a large readily releasable pool of vesicles. The functional consequences of this unusual synaptic design remain largely unexplored. In fact, the MF synapse is often depicted as a synapse with multiple sites, each behaving as an independent transmission line, analogous to several CA3 boutons contacting a single CA1 dendrite. We developed a physiologically realistic spatial model of the MF bouton to investigate how its peculiar structural and functional properties affect synaptic signaling and plasticity. Contrary to earlier assumptions, our computational model revealed, release sites are not independent transmission units. Crosstalk between calcium domains is necessary for the observed strong STP and for the timely activation of CA3 neurons. VDCCs in the MF bouton are only loosely coupled to active zones, and the distance between active zones is relatively large. In addition to the synaptic design and the known role of calbindin-D28k and synaptotagmin-7 in STP, we find that loose coupling of VDCCs to active zones and large inter-AZ distances are crucial. These features keep the basal release probability low, and their combined action is required to generate the facilitation that triggers postsynaptic action potentials in response to bursts filtering out non-informative dentate activity, and provides a strong rationale for the mossy fibers synaptic architecture.