Cardiovascular diseases are the leading cause of death worldwide and are not typically diagnosed until the disease has manifested. Endothelial dysfunction is an early, reversible precursor in the irreversible development of cardiovascular diseases and is characterized by a decrease in nitric oxide production. We believe that more reliable and reproducible methods are necessary for the detection of endothelial dysfunction. Both nitric oxide and calcium play important roles in the endothelial function. Here we review different types of molecular sensors used in biological settings. Next, we review the current nitric oxide and calcium sensors available. Finally, we review methods for using both sensors for the detection of endothelial dysfunction. Endothelial dysfunction is the early stage in the development of cardiovascular disease, however, molecular probes for diagnostics of endothelial dysfunction are still underexplored. Here, the authors review the specific nitric oxide and calcium sensors available in the context of detecting endothelial dysfunction. Atherosclerosis is a complex chronic inflammatory disease that develops over time due to chronic endothelial injury. The exact cause of atherosclerosis is unknown, and it often is not diagnosed until the late stages of lipid accumulation leading to plaque formation2. Unfortunately, the number of methods available for the detection of plaques remains limited. One non-invasive method for diagnosis, the cardiac stress test, only detects severely narrowed vessels of greater than 70%3. Still, most existing methods can only detect advanced stages of the disease. To diagnose earlier stages of the disease, and to gain a better understanding of disease progression, early non-invasive methods, such as small molecules sensors, should be used.According to the World Health Organisation, cardiovascular disease is the number one cause of death worldwide4. Other non-invasive methods for diagnosis of diseases involving, especially abnormal blood vessel dilatation, are based on the detection of a signalling molecule called nitric oxide (NO)5 that is present in the exhaled breath9. A variety of reviews on sensing NO in the exhaled breath to detect asthma, especially early diagnosis of exacerbations, and chronic heart failure, have been published16, including reviews of the most recent techniques employed to detect gaseous NO17. However, the current methods for diagnosis are not extremely accurate and little is known about the exact initial causes of ED and disease progression.Some non-invasive methods for evaluating endothelial dysfunction (ED) have been developed in clinical studies. These methods include flow-mediated dilation of the brachial artery, intracoronary Doppler Wire, cardiac magnetic resonance imaging, positron emission tomography, or multi-detector computed tomography20. ED has been linked to other diseases like nonalcoholic fatty liver disease21, diabetes22 and recently, SAR-CoV-2 (Covid-19)24. Endothelial cells regulate blood vessel homoeostasis through their production of NO as a vasodilator26. The generation of NO starts with increasing Ca2+ concentration in the endothelial cell cytosol. In several works, it has been shown that in the case of ED Ca2+ intracellular concentration still fluctuates27 while NO releasing by the cells has stopped28. These two obstacles make Ca2+ and NO possible targets for ED diagnostic tools through simultaneous sensing of both analytes. Unfortunately, in vivo imaging has multiple limitations such as biocompatibility, cell bilayer penetration by molecules, intracellular targeting, light penetration, and signal-to-noise ratio, so new molecular probes and sensing methods must be developed30. This review provides an overview of recent developments in the modelling of ED, in the sensing of NO and calcium cations in biologically relevant conditions, and it gives an outlook for future research.ED can be defined as the earliest reversible precursor disease stage in the irreversible development of atherosclerosis31. Therefore, animal models may be seen as more realistic for testing new diagnostic tools, but they also have limitations for studying human disease progression32. Next to animal models, in vitro methods may further help to determine disease mechanisms, estimate disease risk levels, and help establish viable treatment options33. In vitro modelling started with 2D cell monocultures, which is a cheap and simple way to determine how cells react to different factors.  More complex 2D cultures also exist that allow for the co-culture of more than one cell type. Usually, modelling atherosclerosis and ED involves the co-culture of endothelial and smooth muscle cells. Some methods of indirect 2D cell culture are using conditioned medium, microcarriers and bilayers or Transwell/Boyden chambers33. However, 2D cell cultures do not replicate the in vivo 3D environment and cannot replicate the complex structure, extracellular matrices and cell-to-cell interactions present in animal models or humans34.Since the exact causes and dynamics of ED are not known, in vivo and in vitro models have been developed to study disease progression. Both in vivo and in vitro models have different benefits and drawbacks Fig. . Animal 35. Current methods for co-culturing endothelial cells and smooth muscle cells in a 3D environment include the use of a scaffold or a gel for anchoring or separating the different cells33. To replicate a blood vessel with physiological shear stress, bioreactors with a pump have been used to grow the cells in a tube-like fashion and model how the cells react under flow37. Microfluidic devices have been developed to culture cells in 3D and under shear. These devices are often manufactured from PDMS or another plastic34. Some microfluidic devices allow for the growth of endothelial tubes in a high-throughput manner, allowing for the study of endothelial cell interactions with immune cells, like monocytes38 or a co-culture of endothelial cells and podocytes to create a human glomerular filtration model39.3D methods allow cells to form structures that do not involve animal experiments but are more representative of what is found in vivo40. Another consideration within a given species, is the cell type that is being used. Often human umbilical vein endothelial cells (HUVECs) are used in in vitro studies; however, these cells might not be the best for modelling an arterial disease since they are vein cells from the umbilical cord and have different expression of genes and respond differently to environmental cues compared to artery endothelial cells41. Primary cells from human patients are an excellent source of cells for modelling diseases. However, these cells have to be used at a low passage number to maintain physiological relevance and often there is high donor-to-donor variability33. Finally, the culture substrate or vessel is often ridged in 2D culture and does not mimic the environment found in vivo34.Although in vitro models of ED are indispensable for optimizing ED diagnostic tools, these models have several limitations that need to be considered. The origin of the cells used in these studies needs to be considered carefully: cells from porcine, mouse or rat have been used in vitro, and the disease mechanism may vary between species, as also seen in vivo. For instance, mouse models have been used to study the immune system, shedding light on antibody synthesis and T cells. However, due to differences in immune systems, most immunological research done on mice does not translate directly to humans42. Menon et al. created a microfluidic stenosis model to look specifically at the leucocyte to endothelial interactions43. Junaid et al. used the MIMETAS OrganoPlate® to look at the metabolic response of blood vessels to TNFα, which contributes the vascular stiffness in atherosclerosis44. For all these studies, the microfluidic systems still had a hard time mimicking the in vivo environment in regard to shear stress and flow patterns. In vitro models struggle to fully recapitulate the dynamic in vivo environment and maintain the simplicity that can be consistent and reproducible. While great progress has been made in developing in vitro models for atherosclerosis and ED, each model still has its limitations, and a lot is still unknown about the disease’s progression and prevention.However, new in vitro models are continuously being developed to help better study these diseases. Zhang et al. developed tissue engineered blood vessels using primary human endothelial colony forming cells, human coronary smooth muscle cells and human neonatal dermal fibroblasts with the addition of human monocytes to model early stage atherosclerosis2+ sensors, used either in a cell-free context, in cells as an end-point assay or in living cells for constant monitoring of both analytes.Recently, a lot of efforts have been made to develop molecular sensors that can be incorporated into cells for timely and spatially resolved monitoring of a selection of biomolecules in living cells. In this review, we further highlight NO and Ca45. One of the factors leading to an increase in NO production is the change in cytosolic Ca2+ inside the ECs. This change in intracellular Ca2+ is due to influxes of extracellular Ca2+ into the cytosol and the release of Ca2+ from calcium ion reserves inside the endothelial cells46. The cytosolic Ca2+ binds to calmodulin (CaM), which activates an enzyme called endothelial nitric oxide synthase (eNOS)47. eNOS is coupled to tetrahydrobiopterin (BH4), which stabilizes the active dimeric form of the enzyme . NO is a known factor that can cause the smooth muscle cells to undergo EDH. However, the change in intracellular Ca2+ can also lead to EDH without the synthesis of NO25. Félétou nicely summarized the role of intracellular Ca2+ on EDH and ED in his review article25.Vascular tone is directly related to the amount of blood vessel constriction relative to the maximal dilation. In diseased or atherosclerotic vessels, there is a decrease in vascular tone. Hyperpolarization of the endothelium and smooth muscle cells, through increases in intracellular Ca2+ in the endothelium and smooth muscle cells, understanding the dynamics of these molecules as a function of time is crucial. As previously discussed, NO production decreased in cells with ED. For example, Balbatum et al. used electrodes to trace NO in individual healthy and diseased single endothelial cells51. Figure 51. The dynamics of the evolution of Ca2+ concentration differs from that of the concentration of NO. Even unstimulated endothelial cells have changes in Ca2+ level, typically referred to as Ca2+ “puff” (intra-cellular) or Ca2+ “waves” (inter-cellular)52. Burdyga et al. illustrated the changes in Ca2+ concentration  using a calcium-dependent fluorophore and showed how the changes in [Ca2+] in a cell vary depending on location  cannot. Spatial resolution and intracellular localization are also difficult to achieve for such electrodes, the tip of which remains big compared with cells. Extensive efforts have hence been made in the past decades to develop molecular probes for sensing, i.e., molecules that modulate their optical or magnetic properties in the presence of a particular biological analyte. Such sensor molecules are mostly used for intra-cellular sensing using optical microscopy; however, they can also be used to functionalize classical electronic devices to improve selectivity; NO-sensitive chemoresistors, for example, utilize the specific affinity of metalloporphyrins or corroles to NO for detecting NO in exhaled breath or gas mixtures7. In comparison with conventional electrodes, molecular probes offer the advantage of being tiny, easily operated, highly sensitive, and potentially very selective, which offers quick optical readouts with minimal invasiveness for the cells. In addition, they can measure the concentration of an analyte inside the cell, and they can be adapted for probing a wide range of bioanalytes, such as NO or O2, but also radicals , metal ions , proteins, etc. On the other hand, the large number of published molecular probes has made it increasingly difficult to select one for a particular application, and the marketed selectivity might be challenged in the complicated environment of a cell . Every probe has its advantages and disadvantages, which we shortly discuss below. Therefore, several critical properties should be considered for selecting molecular sensors for detecting ED. In this part, we discuss general properties such as solubility, working wavelength, brightness, toxicity, intracellular targeting, and photostability, and in parts “Nitric oxide sensors” and “Calcium chemosensors” we will focus on NO and Ca2+ probes, respectively.As mentioned above, electrodes sensitive to molecular gases offer after calibration direct reading of gas concentrations in a medium, and this is in a time-dependent fashion. Such electrodes exist for several gases, notably for NO, CO, OP). Both properties directly influence the cellular uptake and intracellular localization of the probe, which in turn influences how it may interact with its targeted analyte. For example, hydrophobic probes may target membranes, while water-soluble ones may end up in the cytosol. Another example is that positively charged hydrophobic cationic species might end up in the mitochondria, which have a negatively charged membrane, while negatively charged molecules may end up in the acidic environment of the lysosome. However, many details of the molecular formula of a probe matter, so general rules are difficult to be drawn. More specific intracellular targeting of fluorescent probes can be obtained by modifying the probe with a functional group that “tags” the probe for specific transport to the organelle of interest. For example, mitochondria targeting has been achieved by covalent modification with a triphenylphosphonium group or by quaternized pyridines55, while morpholine is often used as a lysosome-targeting group56.Small molecular probes are characterized by their solubility and hydrophilicity  of the spectrum, are of higher interest. They allow for low autofluorescence interferences, deeper tissue penetration properties (for in vivo imaging), and minimal photodamage to biological samples, which are critical in particular for long timescale observations. Therefore, NIR fluorescent probes are often considered the best choice when it comes to the detection of biologically relevant analytes such as Ca2+, NO, superoxide, etc. On the other hand, many NIR dyes require extended polyaromatic structures, which may lead to high hydrophobicity, problematic aggregation, and off-toxicity. Also, NIR light is not visible to the naked eye or conventional cameras, thus NIR probes often require special techniques or equipment to visualize the signal. Conventional NIR emitters also suffer from large non-radiative decay rates and low quantum yields61. The narrow optical energy gap in the NIR region results in more non-radiative processes from the first excited state (S1) to the ground state (S0) to occur as stated by the energy gap law62. Newer strategies that consist in employing intermolecular charge-transfer and charge-transfer aggregates via nonadiabatic coupling suppression in organic emitters have been developed recently to overcome these energy-gap law related limitation of NIR emitters63. Finally, when looking at different analytes at the same time, it is critical to have several probes that can be excited and detected in different regions of the spectrum, allowing multicolour imaging65. Overall, this domain of research is very active, and many specific molecular probes have been developed based on blue, green, red, far-red, or NIR fluorophores67.In principle, all visible wavelengths are eligible for intracellular fluorescent probes. However, fluorescent probes with both absorption and emission wavelengths in the spectral range of 600–900 nmε) and its luminescence quantum yield68. Since most excitation and emission light is lost due to light scattering and absorption inside the cellular environment, the brightness of a probe is a very important parameter. The higher the brightness, the higher the signal-to-noise ratio will be during imaging, therefore allowing deeper light penetration and deeper tissue imaging. Moreover, contrast is also critical for imaging, so the molecular probe should show large differences in brightness between the on and off states of the probe.The luminescence brightness of a molecular probe is defined as the product of its molar absorption coefficient at the excitation wavelength , thus leading to a full loss of the emission properties of the exogenous probe. Photostability varies greatly with the probe. For example, probes with linear extended conjugation like cyanine dyes, are more prone to photobleaching than cyclic aromatic systems like porphyrins, aza-BODIPYs, etc.0 absorbs a photon, and one electron in the HOMO orbital jumps into a higher energy level , resulting in a singlet excited state S1. From the LUMO, it will relax back to the HOMO either by emitting energy in the form of a photon, or by non-radiative decay. The PET quenching mechanism  in the immediate vicinity of the dye group. One electron of the PET donor group may be transferred, in the excited state S1 of the dye, into the lowest-energy singly occupied orbital (SOMO) of the excited state. The electron in the resulting highest-energy SOMO orbital from the photo-reduced dye, relaxes back into QO, recovering the ground state S0 of the sensor molecule. In this non-radiative decay process, the photon energy is lost into molecular vibrations, and the photon emission observed in the unquenched dye (a) does not take place: the emission is quenched by the PET donor. In many Ca2+- or NO-sensing molecules, the PET quenching mechanism takes place in the unbound state of the probe, which usually contains high-energy, nitrogen-based electron donor groups in their structure . Therefore, the sensor’s emission is low in the absence of an analyte.Box Fig. Box Fig.  differs Upon binding of the analyte to the chelate, PET quenching is prevented by the interaction between the calcium ion or NO molecule, and the electron-rich functional group of the probe. This interaction dramatically lowers the energy level of the QO orbital, thus preventing PET quenching to occur Box Fig. . TherefoBox 1 PET sensingPhotoinduced electron transfer, or PET, is a mechanism where the light emission of a dye in its excited state is quenched by an electron donor, thus affording little or no emission179. When PET “quenches” the emission, the dye molecule absorbs a photon, but the resulting excited state returns to the ground state either without the emission of a visible photon, or with reduced emission intensity. The reduced emission intensity typically occurs because other photophysical processes, called non-radiative processes, are faster than photon emission. When such a quenching mechanism is cancelled, for example upon the interaction of the PET donor with a calcium ion or NO, the dye molecules recover their ability to emit a photon. The number of emitted photons, divided by the number of photons absorbed by a given number of dye molecules, is called the emission quantum yield. The difference between the energy of the emitted photon, and that corresponding to the absorption maximum of the dye, is called the Stoke shift; it is usually given in eV.Box Fig. 1 Mechanism of photoinduced electron transfer.a Fluorescence emission in a fluorescent molecular probe (blue sphere). PET quenching of the fluorescence emission of a molecular probe for Ca2+ ions b in the absence of Ca2+  and c in the presence of Ca2+ (brown sphere). PET photo-electron transfer.72. It was a great surprise for the scientific community to realize that a radical molecule like NO was of biological relevance, as radicals were considered to be toxic species. The repercussions and implications of this finding were later recognized with the Nobel Prize in Physiology and Medicine (1998), awarded jointly to Robert F. Furchgott, Louis J. Ignarro and Ferid Murad, “for their discoveries concerning NO as a signalling molecule in the cardiovascular system”73. Chemically, NO is a diatomic free radical molecule that forms a gas at 1 atm and 25 °C. It has one unpaired electron in an antibonding singly occupied molecular orbital (SOMO). Because NO has a low dipole moment (0.159 D)74, it forms relatively weak intermolecular interactions. In comparison to other radicals, NO radical is relatively stable, as it does not dimerize easily. NO is also both hydrophobic (solubility in water is only 1.94 mmol L−1 at 25 °C) and hydrophilic (its water–octanol partition coefficient log Po/w is 0.74)76. The diffusion of NO through cell membranes is hence very rapid, characterized by permeability coefficients from 18 to 73 cm s−1 for lipid membranes79. Unlike other free radicals, such as superoxide O2•– or the hydroxy radical OH•, NO is a poor oxidant and a poor reducing agent under physiological conditions83. Additionally, NO reacts very specifically towards biological targets like oxyhemoglobin, methemoglobin, soluble guanylate cyclase (sGC), cytochrome c oxidase (CcOx), and more conductors or transparent supports for external electronic or photometric NO-detection devices based on absorption changes. They can also be directly added into living systems to realize intra-cellular NO detection based on bioimaging of the local changes in optical emission of the dye due to its interaction with NO. In fact, bioimaging techniques have made life easier for biologists to carry out in vitro and in vivo studies on NO. It has always been difficult to design a sensing device suitable for direct, rapid, non-invasive, and timely and spatially resolved, detection of NO in living systems. The combination of bioimaging techniques with NO-sensitive molecular probes is, on the other hand, ideal for that purpose, because it enables in situ, in vitro and in vivo observations that are direct consequences of the presence of NO in tissues and cells91. Fluorescence imaging has numerous advantages over other NO imaging techniques such as chemiluminescence or electron paramagnetic resonance spectroscopy: it is extremely sensitive, selective, spatiotemporally resolved, and easy to realized experimentally, using optical microscopes that are readily available in most laboratories93. As the fluorescence signal and localization of molecular probes can be drastically modulated by chemical modifications, sensors relying on light of different power and wavelengths, and localizing in different regions of cells, can be prepared. NO-sensing fluorescent probes may even provide dynamic information concerning the localization and concentration of NO molecules. For example, Fig. 1, a NO-sensitive molecular probe targeted to the endoplasmic reticulum (ER). It is capable of imaging the level of exogenous and endogenous NO in ER of a living cell during tunicamycin-induced ER stress94. In many of these studies, interdisciplinary collaboration between chemists, biologists and medical doctors, is necessary, to refine the properties of the molecular probes necessary for diagnostic applications.Over the past two decades, many techniques have been developed to detect NO in tissue or exhaled breath95 it has been determined mostly using NO-specific electrodes. This rather low value asks for highly sensitive molecular probes capable of reacting with minute amounts of NO. Moreover, NO is typically short-lived in a biological environment, with a half-life of 1–3 s97. This intrinsic instability also sets kinetic requirements for NO detection by molecular probes: basically, the probe should react quickly upon generation of its substrate, before NO degrades by reaction with other molecules. Of course, low toxicity, a suitable range of physiological pH, and selectivity concerning other interfering molecules are other properties that need to be considered when talking about molecular probes for NO sensing.NO exerts its biological effects via the direct and reversible interaction with specific targets, for example, soluble guanylate cyclase (sGC) or through the generation of secondary reactive nitrogen species  during the measurements. This biofouling resists the usage of such sensors in the blood  and tissue . Even though strategies to reduce biofouling by passive protection of sensors using membranes like Nafion and polyurethanes have been proposed, biofouling still results in poor reproducibility and sub-optimal analytical performance102. Another disadvantage of these sensors is that the oxidation or reduction of many other electroactive molecules PPD sensors103) reduces sensor selectivity and therefore accuracy. A detailed review of various electrochemical NO sensors is discussed by Privett et al.102. Additionally, a few of the porphyrin-based electrochemical sensors developed by Malinski and Taha84 and Vergnani et al.104 have been used to measure NO release from healthy and diseased endothelial cells or in tissues.Electrochemical NO sensors are small electrodes that allow direct sensing of the NO level. They have been used both in vitro and in vivo. Enhanced sensitivity of on-site measurements, as well as rapid response (at least 100 ms), are among the major advantages of direct electrochemical measurements. The most commonly used material for the working electrode of these sensors includes platinum and its alloys, carbon fibre, and gold105. In the absence of NO, the fluorescence of the fluorophore is quenched by photoinduced electron transfer (PET). PET is due to the high energy of the electrons in the free amine group, which allows this electron to relax in the lowest SOMO orbital of the probe’s excited state, followed by relaxation of the electron in the high-energy SOMO or the probe’s excited state, back into the temporarily empty free amine orbital. In the presence of dioxygen, NO and the phenylenediamine probe react chemically to form an electron-poor triazole ring. The non-bonding electron pairs of this triazole ring are much lower in energy, as a consequence of which they cannot quench the excited state of the nearby fluorophore. In phenylenediamine-based molecular probes for NO the PET quenching process is blocked upon forming the triazole ring in the presence of NO, which enhances fluorescence emission, compared to the diamine, thereby allowing NO sensing108. The nature of the fluorophore allows for fine-tuning the excitation and emission properties of the sensor; the type and size of the linker separating the fluorophore from the diamine moiety influences the difference in brightness between the on and off states; and the overall molecular properties of the probe, such as its charge, hydrophobicity, or molecular shape, influence its cellular uptake and intracellular localization.Molecular probes potentially offer sub-cellular selective detection of an analyte, high selectivity, and the possibility to combine NO sensing with other molecular probes. Regarding NO sensing, many molecular probes, such as those developed by the Nagano group, are organic fluorophores conjugated to an ortho-phenylenediamino groupo-phenylenediamino group, which differ by the nature of the fluorophore and its localization inside the cell. Amine-based NO sensors are prone to interference from protonation107. The pH-dependence of bodipy-amine NO sensors was discussed in detail by the Nagano group wherein at pH 7 and above, the fluorescence was quenched due to the accelerated PET process. At high pH, the triazole loses a hydrogen atom and forms the triazolate with a higher HOMO energy turning on the PET process which was turned off in triazole due to the lower-lying HOMO energy compared to that in the amine-functionalized fluorophore. According to the Rehn–Weller equation, the free energy change of the PET process is determined by both the electron-donating ability of reactive triazole sites and by the reduction potential and the excitation energy of the fluorophore. Therefore, modification of the fluorophore to change the reduction potential and the excitation energy can facilitate the synthesis of a pH-independent amino-based NO sensor109. Other organic functional groups employed as the NO recognition site include dihydropyridine (Hantzsch ester)111 and aromatic secondary amines114. Though these diamine probes are by far the ones that have been mostly used in the literature, they do not directly detect NO, but an intermediate N2O3 is a nitrosating agent for diamine as shown in Eq. –fluorescein (FL) complexes, where the FL molecule comprises an 8-aminoquinaldine ligand attached to the 40 position of the fluorescein xanthene ring. The formation of a 1:1 CuII:probe complex (CuFL) resulted in a dim fluorescent species. Reaction with NO generated a bright nitrosylated fluorophore FL-NO. The reaction is presumably occurring through a NO-mediated reduction of Cu(II) to Cu(I) followed by nitrosylation of FL and finally dissociation of Cu(I) halide as shown in Fig. 119. Sensors based on other metal ions, including Co(II), Fe(II), Ru(II) and Rh(II), have also been developed recently120. Other metal-based sensors use colour changes upon axial binding of NO to Fe(II) porphyrins and Fe(III) corroles . In the presence of NO, the fluorescence is recoveredles Fig. ; this mi123. Their smaller size makes them ideal for interfacing non-invasively with the plasma membrane, peptides126, and cholesterol derivatives128. Nanoparticles also possess various optoelectronic properties  to serve NO sensing at the membrane. On the other hand, the tunability of circulation time and clearance properties in animal models makes them good candidates for NO sensing in vivo129. For example, the Li group developed an upconversion nanoparticle (UCNP)-based NO probe for the ratiometric measurement of NO in cells130. The probe consists of a UCNP core surrounded by a shell of mesoporous SiO2, loaded with rhodamine B-derived molecules (the NO-reactive molecule). NO sensing is achieved via luminescence resonance energy transfer (LRET) between rhodamine B and the UCNP. Reaction with NO shows a recovery of the strong absorption of rhodamine B at 500–600 nm. Quantitative measurement of NO was achieved through the intensity ratio of rhodamine B emission to upconverted emission from the UCNP, I656/I540. The Da-Wei Li group developed gold nanoparticles modified with o-phenylenediamine (OPD)- based NO sensor to detect the level of the endogenous NO in murine macrophages (RAW 264.7)131. In presence of NO, OPD reacts with NO to form a triazole, resulting in SERS variations of the AuNPs/OPD nanoprobes. The intensity ratio, I789/I585 was used as indicator for NO level, as the peak at 789 cm−1 was strongly dependent on the NO-triggered reaction whereas the peak at 585 cm−1 was nearly unrelated to the presence of NO. The Li group also developed Au-Ag alloy/porous-SiO2 core/shell nanoparticle-based NO probe for ratiometric analysis of NO in HeLa cells132. A 3,4-dia-minobenzene-thiol (DABT) was self-assembled on the Au-Ag alloy nanoparticle’s surface to get the NO probe and a strong scattering peak at 804 cm−1 appeared after treatment with NO. The researchers also showed nitrogen oxides did not interfere with the sulfhydryl group in Au-S bonds in the probe indicating good stability and excellent selectivity of the SERS nanoprobe. The Huang group reported a red-emitting Fe(III)-bound dithiocarbamate functionalized CdSe-ZnS quantum dot (QD) NO sensor showed a subsequent increase in QD photoluminescence upon NO exposure due to decreased FRET from the QD donor133. The NO probe showed a limit of detection (LOD) for NO at ~3.0 µM. Liu et al. developed a modified hyperbranched polyether (mHP) nanospheres with fluorescent CdSe QDs encapsulation for NO detection and release134. The QDs-mHP-NO nanosphere releases NO molecules when placed in aqueous buffer resulting in an increase in QD photoluminescence, whereas introducing NO to the QDs-mHP nanosphere increases the fluorescence quenching. The nanoprobe also showed high selectivity in the presence of other ROS and RNS like H2O2, ClO−, ONOO−, NO2− and NO3−. Other nanoparticle-based systems for NO sensing developed recently include probes based on Ce-based metal−organic framework135, aminoguanidine hydrochloride and citric acid-based carbon-dots136, 4-amino)methyl)benzoic acid functionalized CdTe/CdS/ZnS quantum dots137, electrochemically active gold nanoparticle-modified tungsten oxide (WO3) nanoflakes138, or Au nanoparticles that undergo azide–alkyne click chemistry-mediated aggregation-based NO sensor139.Recent advances in the field of NO sensing have also shown the development of nanoparticle-based sensor for NO including those based on upconverting nanoparticles, quantum dots, and plasmonics-based nanoparticles. These functional bio-nanomaterials are advantageous over other materials for sensing NO at the plasma membrane141 reported a BODIPY-based NO fluorescent probe ; they hence integrate the NO time evolution. In contrast, inorganic copper-based fluorescent probes and iron-corroles bind reversibly with NO; in principle, they are hence better suited for following in-time NO concentrations.One major aspect that still needs to be addressed in NO sensing is the reversibility and reaction rate of NO with ortho-diamine sensors, considering the time-dependent character of NO production in the endothelium. A  it Fig. . Ideallyyte Fig. , which d−1), a healthy endothelial cell releases NO at the rate of 1200 ± 50 nmol L−1 s−1 with a peak concentration of 430 ± 15 nmol L−1 reached after 600 ± 20 ms. Whereas in a diseased endothelial cell, NO is released at the rate of 460 ± 10 nmol L−1 s−1, and a NO peak at a concentration of 140 ± 15 nmol L−1 was reached after 900 ms51. Similarly, many other agonists like acetylcholine and cicletanine were also used to induce NO release and to study the time evolution. And it was observed that the rate of NO release and NO peak concentration varied with different agonists. The peak NO concentration achieved in the presence of calcium ionophore (595 ± 30 nM), acetylcholine (390 ± 20 nM) and cicletanine (160 ± 8 nM) are all different. Moreover, the kinetics of NO release after stimulation with acetylcholine are distinctively different from those observed after stimulation of NO release by calcium ionophore147. Further studies for the concentration of NO released by normal endothelial cells from different parts of the aorta of rats indicated that the dynamics of NO release differs significantly in different locations of the cells within the aorta  channels. Thus, for healthy EC, cytosolic Ca2+ level reaches ~1 μM in the presence of shear stress. Unfortunately, we were not able to find published values of Ca2+ concentration in dysfunctional EC, so only hypotheses can be made. According to Du et al.27 the Ca2+ influx in the cytosol of ECs, even in the case of ED, is not blocked. Hence, we can suggest that Ca2+ concentration in the cytosol of EC, even in the case of ED, still increases in the presence of shear stress. Thus, for further ED sensor design, it might be reasonable to consider that Ca2+ levels also increase up to 1 µM in the cytosol of dysfunctional EC upon external stimulus.Ca2+ chemosensors can be divided into two parts, a Ca2+-chelating moiety and a luminophore—often a fluorophore. The calcium-binding ligand is mostly characterized by its selectivity towards other biologically relevant metal ions , and its sensitivity to Ca2+, in which for a range of Ca2+ concentrations, the luminescence will vary. The sensitivity might be expressed as a ratio ΔF/F0, where ΔF is a difference in the emission of the complex CaL (Fmax or min) vs. free sensor L (F0):Usually, Ca2+ ions to chelates is usually considered as being fast. Hence, calcium molecular probes are usually considered as being under thermodynamic equilibrium. As a consequence, the main parameters describing analyte binding to the sensor is its association constant Ka (in M−1) or dissociation constant Kd = 1/Ka (in M). The sensitivity of Ca2+-binding chemosensors is strongly dependent on Kd. Kd is defined by Eq. 2+ concentration for which half of the chelate is bound to Ca2+. Additionally, an increase in Ca2+ concentration typically induces NO production. Thus, a perfect calcium chemosensor should not disrupt the biological process of NO production, and its intracellular concentration should remain low, compared to biological Ca2+ concentrations. From simple Kd calculations 197. Maximizing the difference in dissociation constant of the sensor, Kd, between the larger Ca2+ ion on the one hand, and the smaller Mg2+ and Zn2+ ions on the other hand, is critical for calcium sensing in biology.Another important characteristic of a Cao-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid 193, bichromophore 21194, anthraquinone-modified calix[4]arene 22162, the chemosensor 23163. Unfortunately, proper selectivity does not guarantee good sensitivity, and the calcium-binding Kd of these sensors are not low enough for in vitro investigations.Other chemical structures have been described that are selective enough for calcium biosensing purposes. A selection is shown in Fig. 24 and 25 sensors have Kd values for Ca2+ of 309 and 69 nM, respectively164. However, their aza-crown ether structure makes them suitable for different cations, and the chemosensors are not very selective. The sensor 26, reported by Kim et al.165 has a Kd value of 19 nM and was shown to be selective towards Ca2+. The sensor 27, presented by Kacmaz et al.195 and 28, reported by Saleh et al.166 are sensitive enough to be used in biology. Unfortunately, the selectivity of both sensors was tested in the presence of only 10 µM of Na+, Mg2+ and Zn2+ for 27 and 20 µM for the chemosensor 28, which are lower than their levels in the cytosol. From these data, we cannot conclude whether or not these compounds are selective enough to be used for Ca2+ imaging in biology. Overall, since BAPTA is one of the best Ca2+ chelators available in the literature in terms of binding strength and selectivity, new Ca2+ chemosensors with BAPTA as the chelating ligand appear regularly170  and infrared  regions of the spectrum are sometimes called the “first” and “second” therapeutic windows, respectively159. They are preferred for designing calcium probes, although they are also more difficult to design (and sometimes to synthesize) than probes excited at lower wavelengths. With the light of higher energies , the risk of causing damage to the cells during imaging, for example via a photodynamic effect (see above), is higher. Such phototoxicity often limits observation under a microscope for a long time160. Many of the chemosensors mentioned above have an emission wavelength, λem, in the green area (~485–550 nm)168  is preferred, which incites research towards new calcium probes. For example, sensor 32  provides excitation (λex) and emission (λem) wavelengths of 546 nm and 569 nm, respectively169 while 33170 has an even higher λex = 620 nm and λem > 650 nm. Chemosensors such as 34171 absorb and emit red light  intensometric, (ii) ratiometric or (iii) pseudo-ratiometric chemosensors174. The type of sensing influences the relation between the light intensity and the Ca02+ concentration to be measured  and the fluorescence intensity are known. Ca02+ is given by Eq. Fmax is the maximal fluorescence intensity of [CaL] complex, F0 the fluorescence intensity of the Ca2+-free sensor, and F the fluorescence intensity signal at a given Ca02+ concentration.First, the fluorescence intensity of intensometric sensors is proportional to the Caon Figs.  and S1a.L0 and F. In principle, the sensor intracellular concentration in vitro needs to be calibrated, but such calibration is usually impossible, as cellular uptake and localization cannot be quantified easily, while the emission of the probe depends on both concentrations in the bound complex ([CaL]) and the free ligand ([L]).However, often it is hard to know 2+ addition. In an in vitro imaging experiment, one can usually directly determine the ratio between the two bands, which allows one to derive the ratio between the bound complex ([CaL]) and the free ligand ([L]) concentrations175. With so-called ratiometric probes  the intensity of the Ca2+-independent emission, the local probe concentration can be obtained, and by comparing this emission intensity to that of the Ca2+-dependent band the true local Ca2+ ions concentration can be calculated. In other words, in pseudo-ratiometric sensors, Ca02+ is independent of L0 . This kind of sensing has been used a lot for dioxygen-sensing for example, which is often based on dynamic quenching of the triplet excited states of the probe by O179. Similarly to NO sensing, in calcium sensors such as BAPTA-based derivatives shown in Fig. In the previous paragraphs, we discussed the question of the binding strength and selectivity of the calcium-binding chelating part and the photophysical properties of the fluorophore attached to the chelate. Usually, these different characteristics combine into calcium sensing via PET sensing see Box 179. SimiF/F0 ratio leads to a larger difference between the emission of the Ca2+-free ligand L and that of the CaL complex. The detection of a particular Ca2+ concentration, both in vitro and in vivo, becomes more precise due to the increased signal-to-noise ratio. PET can also be used to switch off the light emission in the presence of the analyte. Those chemosensors are called “turned-off” chemosensors  sensors are usually preferable in particular because they offer the possibility to follow the dynamics of Ca2+ concentration changes, and because of their higher signal-to-noise ratio.Such a sensing mechanism corresponds to a “turned-on” sensor, where the emission is recovered in the presence of the analyte. From Eq. , it is c80 Table  show emi2+ fluxes in healthy and diseased EC, one could argue that not only the thermodynamics of the calcium-binding process but also the timescale for Ca2+ binding and unbinding, hence the kinetics of the binding equilibrium, should be considered in the design of molecular probe for calcium sensing in ED diagnostics. If the Ca2+ concentration in the cell cycles vs. time, it might be important to measure the variations of the Ca2+ concentration with a probe that offers faster binding compared to the biological time variations of the Ca2+ concentration. Indeed, such biological variations of [Ca2+] might change drastically during the cycle of a healthy or diseased EC. Unfortunately, the time during which Ca2+ concentration increases, how long its maximal concentration plateau persists, and how long it takes to decrease again to the resting state, are poorly known in EC. These kinetics are usually not considered in existing diagnostic tools. As for NO sensing, studies aimed at following in time calcium concentrations inside endothelial cells, are rare. A few kinetic studies with BAPTA-based sensors have been published in the 1980s and 1990s182. For example, it was shown that 38 (Fura-2) has a second-order rate constant for Ca2+ binding of kon = 6.2 × 108 M−1 s−1 as defined by Eq. (181.ron and roff are the rates (in M s−1) of the association or dissociation reactions; kon is an association rate constant (in M−1 s−1) and koff is a dissociation rate constant (in s−1).Considering the time-dependent evolution of Cad by Eq. 181.\docu2+ concentration increase in EC cytosol after stimuli using molecular drugs183. It was found that the concentration increases dramatically in 7–10 s after the drug addition to the cell media. Minute time scales were used in other studies185 to show the dependence of Ca2+ concentration vs. time. Today we know that Ca2+ cytosolic concentration changes within seconds or even milliseconds52. Unfortunately, kinetic studies have not become a standard characterization routine for modern calcium sensors, so very little information is available in the literature about the most recent calcium probes.Later this sensor was used for the measurement of CaBox 2 What is a dissociation constant of an ideal calcium sensor for endothelial dysfunction?2+]o = 1 µM, and if after the addition of a calcium ion sensor L at a concentration L0 we would like to keep the Ca2+ concentration after binding to the sensor mostly undisturbed, e.g., [Ca2+] = 0.99 µM, then the concentration of the sensor-calcium complex CaL should be 0.01 µM (1%). Let us consider here two sensors L1 and L2 with different Kd, Kd1 = 0.1 µM and Kd2 = 10 µM. To calculate the sensor concentration, we need to add to the cell to detect 1 µM of calcium ions, one should consider the following simple calculation:If in a cell the initial calcium ion concentration is  and [NO], more precise kinetic studies will be necessary. On the other hand, understanding the interdependence between NO and Ca2+ release in EC may make use of the concept of logic gates introduced decades ago by de Silva et al.188. In this approach, the evolution of the emission intensity of a molecular probe with experimental conditions can be re-interpreted in the form of a logic table by defining an emission intensity threshold Et: when the emission intensity of the solution is higher than this threshold, the output of the logic gate is 1, and when it is lower than this threshold, for example due to some form of quenching, then the output of the logic gate is 0. The input of such a logic gate is also interpreted in terms of concentrations of the different analytes to sense. For example, when the concentration of a first analyte is higher than a threshold C1,t, then the first input is considered 1, and when it is lower, it is 0. For the second analyte a second threshold Ct2, is defined that may be different from Ct1,, and that also defined a second input as 0 or 1 for a second analyte. The response of such a system to the concentrations of both analytes in solution can be interpreted in the form of a logic table  and NO as well, while in diseased EC calcium is high, too (probably around 1 μM), but NO remains low. In the simplest approach, if sensors for Ca2+ and NO would be available that are not toxic to endothelial cells, and would respond to biologically relevant concentrations of both analytes, then an ideal ED sensor would be a molecule (or a supramolecular system) that shows the logical table shown in Table 2+ is high and NO low, and in all other cases, the response should be 0. This table is a simple functional table that does not consider where Ca2+ and NO are detected. NO is membrane permeable, so the NO molecular probe may be either in blood, inside the EC or SMC, or at their surface. By contrast, Ca2+ trafficking is strongly regulated as Ca2+ ions are impermeable to biological membranes, so calcium molecular probes should be selectively taken up into the cytosol of endothelial cells. In addition, such a table corresponds to the simplest approach where the response of both sensors is essentially time-independent or provide a time-averaged response to Ca2+ ions and NO. A more sophisticated sensing platform may give a response that combines time-dependent sensing of the concentration of both analytes. Ideally, such probes should targeted ECs, or at least be taken up preferentially (or more quickly) by EC. Overall, it appears that although a wide range of molecular probes for NO and calcium ions have been designed and prepared in the past, we are still far from an integrated diagnostic platform that would provide by a simple reading of a fluorescence signal, whether a given set of EC or SMC show signs of ED or not. Preparing such a sensing system remains a daunting challenge that will require active collaboration between synthetic chemists, photochemists, and biologists.For example, when comparing healthy and diseased endothelial cells, it appears that in healthy cells CaPeer Review FileSupplementary Information


IKZF1 mutation in AML specifically is extremely limited. In a previous work, we described the distribution pattern of IKZF1 mutation in AML, but its clinical impact has remained undefined due to the limited number of cases. Herein, we attempt to answer this question in one relatively large cohort covering 522 newly diagnosed AML patients. A total of 26 IKZF1 mutations were found in 20 AML patients . This condition has a young median age of onset of morbidity (P = 0.032). The baseline characteristics of IKZF1-mutated and wild-type patients were comparable. IKZF1 mutation showed significant co-occurrences with CEBPA (P < 0.001), SF3B1 (P < 0.001), and CSF3R (P = 0.005) mutations, and it was mutually exclusive with NPM1 mutation (P = 0.033). Although IKZF1-mutated AML was more preferably classified into the intermediate-risk group (P = 0.004), it showed one inferior complete remission rate (P = 0.032). AML with high burden of IKZF1 mutation  showed relatively short overall survival period (P = 0.012), and it was an independent factor for the increased risk of death . In subgroup analysis, our results showed that IKZF1 mutation conferred poor therapeutic response and prognosis for SF3B1-mutated AML (P = 0.0017). We believe this work improves our knowledge of IKZF1 mutation.Genetic heterogeneity poses a great challenge to the understanding and management of acute myeloid leukemia (AML). Knowledge of the The online version contains supplementary material available at 10.1186/s40164-023-00398-y. IKZF1 mutation is one rare but recurrent alteration in AML. In a previous work, we described its distribution pattern in AML [IKZF1 mutation on AML remains undefined. We here address this issue in a cohort of 522 newly diagnosed AML patients . Missense mutation preferred to localize at the exon 5 (91.67%), which mainly influences the DNA binding of IKZF1. A total of 35.7% of nonsense and frame-shift mutations were found to disrupt the DNA-binding domain and caused loss of the dimerization domain, while 64.3% of them only disrupted the dimerization domain  showed significantly poorer OS than those with low VAF or IKZF1WT, but the RFS did show any statistically significant difference   was more common than non-CEBPAbZIP−inf mutation (16.7%). IKZF1 mutation conferred a relatively low CR in the CEBPAWT/non-CEBPAbZIP−inf−MUT group, but not in the CEBPAbZIP−inf−MUT group . This may be because patients were of different races or it may be because of differences in sequencing depth. IKZF1 deletion, caused by -7/monosomy 7, was detected in 3.20% of our patients. Unlike in ALL [IKZF1 mutation and deletion were equally dominant in AML [IKZF1 mutations, and it almost affected the DNA-binding domain in AML, while its DNA-binding domain and dimerization domain involvement was relatively balanced in ALL [IKZF1 aberration conferred poor prognosis in ALL [IKZF1 mutation predicted poor OS in AML because IKZF1 mutation with VAF < 10% accounted for 35% of all IKZF1MUT patients, and IKZF1 mutation contributed less to disease than other mutations did in this group of patients. CEBPA mutation was the most common co-mutation that occurred alongside IKZF1 mutation in AML [Compared with foreign cohorts  Frequent mutations with more than 10 counts in our cohort were showed. (B) The relationship between mutations was analyzed, concurrent and mutually-exclusive mutations were indicated. (C) The concurrent or mutually-exclusive mutations for common rearrangements in AML were exhibited.Additional file 2: Fig S2. The prognostic role of CSF3R, CEBPAbZIP-inf, or SF3B1 mutation in AML. (A-B) The prognostic role of CSF3R mutation in AML, and OS (A) as well as PFS (B) were showed. (C-D) The OS (C) and PFS (D) of patients with CEBPAWT plus non-CEBPAbZIP-inf mutation or CEBPAbZIP-inf mutation in AML. (E-F) The OS (E) and RFS (F) of SF3B1WT and SF3B1MUT groups in our AML cohort.Additional file 3: Fig S3. The prognostic role of IKZF1 mutation in the specific genetic AML subtype. (A-B) The influence of IKZF1 mutation on the OS (A) and PFS (B) of CSF3R-mutated AML. (C-D) The influence of IKZF1 mutation on the prognosis of the CEBPA-mutated AML was studied, and the OS (A) as well as RFS (B) of CEBPAWT plus non-CEBPAbZIP-inf-MUT and CEBPAbZIP-inf-MUT groups with or without IKZF1 mutation were showed.Additional file 4: Table S1. IKZF1-mutated AML patients in our cohort.Additional file 5: Table S2. The CR rate of AML with different burdens of IKZF1 mutation.Additional file 6: Table S3. Univariate and multivariate analysis for overall survival duration.Additional file 7: Table S4. The influence of IKZF1 mutation on AML with different CSF3R-mutated status.Additional file 8: Table S5. The influence of IKZF1 mutation on AML with different CEBPA-mutated status.Additional file 9: Table S6. The influence of IKZF1 mutation on AML with different SF3B1-mutated status.


N-ethylmaleimide sensitive factor Attachment protein REceptor), the Ca2+-sensor synaptotagmin, the activator/regulator complexin, and other factors. Here, we review the interactions between the SNARE complex and synaptotagmin, with focus on the so-called primary interface between synaptotagmin and the SNARE complex that has been validated in terms of its physiological relevance. We discuss several other but less validated interfaces as well, including the so-called tripartite interface, and we discuss the pros and cons for these possible alternative interfaces. We also present new molecular dynamics simulations of the tripartite interface and new data of an inhibitor of the primary interface in a reconstituted system of synaptic vesicle fusion.Synaptic neurotransmitter release is mediated by an orchestra of presynaptic proteins that precisely control and trigger fusion between synaptic vesicles and the neuron terminal at the active zone upon the arrival of an action potential. Critical to this process are the neuronal SNAREs (Soluble  Because the extracellular Ca2+ concentration is much higher than the basal cytoplasmic concentration, Ca2+ will flow into the cytoplasm. In turn, Ca2+ will trigger fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane in less than a millisecond.2 Upon fusion, neurotransmitter molecules are released into the synaptic cleft, and then bind to receptors that are in the postsynaptic membrane.Synaptic transmission between pre- and postsynaptic neurons occurs when the presynaptic neuron terminal is temporarily depolarized upon an action potential, opening voltage-gated CaSoluble N-ethylmaleimide sensitive factor Attachment protein REceptor), the Ca2+-sensor synaptotagmin, the activator/regulator complexin, the assembly factors Munc18 , and Munc13   . However6 A major focus is the so-called primary interface between synaptotagmin and the SNARE complex  on the synaptic vesicle, and syntaxin-1A and Synaptosomal-Associated Protein, 25kDa (SNAP-25) on the plasma membrane initially form a trans SNARE complex, with the transmembrane domains of synaptobrevin-2 and syntaxin-1A in the synaptic and plasma membranes, respectively. During fusion, the SNARE complex completely zippers into the fully assembled cis SNARE complex where the soluble core consists of a parallel, four α-helix bundle.9In the following, we briefly summarize some of the key findings about neuronal SNAREs – for a recent comprehensive reviews, see refs. 10 Single-molecule optical and magnetic trap pulling experiments suggest that the free energy that is released by the zippering of one SNARE complex is approximately 36 kBT.12 This estimated free energy is somewhat less than the energy that is required to overcome the hydration-force barrier for the formation of a lipid stalk13; however, this should be viewed only as a qualitative comparison because the exact free energies depend on the particular membrane composition and molecular environment. In this context, we note that at least two synaptobrevin-2 molecules, and presumably-two SNARE complexes, are required for fast Ca2+-triggered exocytosis.14The assembly of the SNARE complex is thought to provide the energy necessary for membrane fusion.8. Briefly, Syts constitute an evolutionary conserved family of proteins15 that are composed of an N-terminal single transmembrane-spanning domain, a variable juxtamembrane linker, and two C-terminal cytoplasmic Ca2+-binding C2 domains, termed C2A and C2B, respectively.16 Syts differ in their cellular expression and subcellular localization patterns17 in addition to the fusion kinetics they endow. Several Syt isoforms  reside on the synaptic vesicles and are vital for synchronous Ca2+-triggered synaptic vesicle fusion20; these isoforms are strictly coupled to an action potential. In contrast, Syt7 mediates asynchronous release,22 and it is loosely coupled to an action potential.In the following, we briefly summarize some of the key molecular and functional properties of synaptotagmins (Syts) – for comprehensive recent reviews, see refs.2+-dependent and Ca2+-independent manners.32 Syt1 function and membrane binding is specific to Ca2+ binding to the C2 domains: Mg2+ does not trigger synaptic vesicle fusion,33 while other divalent cations such Sr2+ or lanthanides trigger limited synaptic vesicle fusion with significantly different kinetics and possibly either through Syt-independent or through SNARE-independent mechanisms.37Syt1 interacts with anionic membranes and SNARE complexes in both Ca2+-triggered proteoliposome lipid mixing38 and full fusion.40 However, Ca2+-triggered fusion with this minimal system is relatively inefficient. More complete reconstitutions greatly increase the efficiency and synchrony of Ca2+-triggered fusion.42 Syt1 has been implied as an activating factor upon Ca2+-binding, for example, by bending membranes45 or bridging membranes.48 However, such an activating role does not explain the effect of certain dominant negative mutants of Syt1 that abolish evoked release in the background of endogenous wildtype Syt1.51 Moreover, genetic deletion of Syt increased the frequency of spontaneous release in Drosophila,53 and a similar phenotype was observed upon deletion of Syt1 in mouse neurons.54 Additionally, expression of a dominant negative Syt1 mutant also increased spontaneous release in mouse neurons in a Ca2+-dependent fashion,4 suggesting that Syt1 also has inhibiting roles at resting Ca2+ concentration and that a Ca2+-sensor other than Syt1 is important for spontaneous release.SNAREs and Syt1 alone are sufficient to promote Ca8. Briefly, Cpx consists of four domains: The N-terminal domain is important for activation of synchronous Ca2+-triggered release in murine neurons58; the accessory domain regulates evoked and spontaneous release57; the central domain is required for all functions of Cpx1 and it binds with high affinity (~10 nM) to the neuronal ternary SNARE complex61; and the C-terminal domain is involved in vesicle priming and binds to anionic membranes in a curvature sensitive fashion.64In the following, we briefly summarize some of the key findings about complexin  – for comprehensive recent reviews, see refs. 2+-triggered amplitude and synchrony of proteoliposome fusion, and it also suppresses Ca2+-independent single vesicle fusion (content mixing).65 By varying the Cpx1 concentration, these single-vesicle studies suggest that the regulatory effect on Ca2+-independent fusion and the facilitating role on Ca2+-triggered fusion are governed by distinct molecular mechanisms involving different subsets of the four domains of Cpx1. Similarly, genetic experiments in Drosophila also suggest distinct mechanisms for activation of fast synchronous release and regulation of spontaneous release.67In conjunction with neuronal SNAREs and Syt1, Cpx1 increases the Ca2+-sensor for triggered fusion, these molecules cooperate to reduce or prevent Ca2+-independent fusion, and they provide the framework for fast triggered fusion. The molecular basis of this cooperation has been elusive until relatively recently. In the following, in order of validated physiological relevance, we summarize the interactions that have been characterized at the atomic level.Because SNAREs alone mediate constitutive fusion, and Syts are the Ca3 .4 For the first structures of this SNARE–Syt1 complex , covalently linked chimeras of the components of the complex were used.3 Subsequently, the inclusion of Cpx1 and truncation of the 23 C-terminal residues of the cytoplasmic domain of synaptobrevin-2 alleviated the need for such linkers .4The so-called primary interface between the SNARE complex and the Syt1 C2B domain was first discovered by X-ray crystallography at atomic resolution3 . This in2 that produces a complementary pattern of charge-charge interactions .4 Fully solvated molecular dynamics simulations suggest that the primary interface is energetically stable up to 1-microsecond, implying off-rates smaller than 10−6/sec. The five 1-microsecond simulations resulted in variations around the crystal structure without major conformational changes or dissociation events68  experiments with a different set of mutations of the polybasic region (Lys326Ala/Lys327Ala) suggested a KD of 3.3 μM4, although there are some caveats with ITC experiments on this system, as discussed below. Consistent with other studies,3 mutation of two arginine residues involved in the primary interface  SNAREs, a low-resolution cryo-EM structure of the primary complex on lipid nanotubes suggests that membrane interactions with the polybasic region can co-exist with the primary interface.69 Molecular dynamics simulations further support the interaction of the primary-interface complex with an anionic membrane.70 Moreover, in these simulations, one of the residues that is critical for the primary interface (Arg 398) formed a more extensive interaction with a negative pocket formed by residues Glu55, Gln58, and Glu62 of SNAP-25A. A similar interaction is formed in five, 1-μsec molecular dynamics simulations of the primary interface alone and in the absence of membranes.68Based on the surface charge distribution of the primary complex, it was predicted to simultaneously interact with anionic membranes through the polybasic region of the Syt1 C2B domain  and the 2+-triggered fusion, but they had no detectable effects on Ca2+-independent (spontaneous) fusion and had varying effects on vesicle association.3 Physiological support for the primary interface came from experiments in neuronal cultures of conditional Syt1 knockout mice. These experiments revealed that these mutations of the C2B domain of Syt1 also disrupted evoked release, while a subset of the mutations unclamped spontaneous release. All mutations resulted in facilitation during high-frequency stimulation compared to depression for the wildtype cultures.3The primary interface was tested with a single-vesicle fusion assay that included neuronal SNAREs, Syt1, and Cpx1. Mutations of both Syt1 C2B and SNAP-25A were designed that were expected to disrupt the primary interface based on the crystal structures. All mutations disrupted Ca71 The three mutations were individually tested in SNAP-25 knockout mice that were rescued with wildtype or mutant SNAP-25B. The three mutations all result in substantially lowered eIPSC amplitudes and rise slopes; in other words, less Ca2+-triggered neurotransmitter release and less synchrony.72 Similar results were obtained when interface variants were overexpressed in wildtype neurons.More physiological support came from disease-related mutations of SNAP-25A that are at the primary interface  was rescued by several Syt1 C2B mutations; some localize to the primary interface in Drosophila.73 Consistent with this observation, the quintuple mutant of Syt1 C2B that disrupts the primary interface mildly rescued IPSCs, although EPSCs were not rescued using a different dominant negative mutant  in mouse neuronal cultures.4 These different dominant negative effects may be related to the different type of Ca2+ binding site mutation  or differences in species (mouse vs Drosophila).Additional physiological support for the primary interface came from CaIn summary, the physiological relevance of the primary interface for synaptic neurotransmitter release has been validated by a variety of studies.4 revealed another interface in addition to the primary interface where a second crystal-symmetry-related Syt1 molecule interacts with the SNARE–Cpx1 side of the complex . A shape and charge complementarity exists between the molecules involved in the SNARE–Cpx1–Syt1 tripartite interface , and it may suggest possible supramolecular arrangements.74 However, considering that the tripartite interface is presumably weaker than the primary interface, it may have been influenced by crystal packing. For example, the tripartite interface could represent an ensemble of conformations, and the crystallization may have favored one conformation out of this ensemble. Using the same protocols and programs that were used for the simulation of the primary interface,68 we tested this by performing five, 1 μsec molecular dynamics simulations of the tripartite interface 71 . The wil4 Two sets of Syt1 C2B mutations were designed that were expected to disrupt binding based on the crystal structure of the tripartite interface . Such a low affinity would be consistent with the molecular dynamics simulations of the tripartite interface that suggest relatively low stability of the tripartite interface on the 1-microsecond timescale . The TGQQ mutant also rescued evoked IPSC amplitude and mIPSC frequency, whereas the LLQQ mutant of Syt1 failed to rescue synaptic release. These results were mirrored by experiments with a dominant negative Ca2+-binding mutant of Syt1 . While Syt1WT in cultured WT neurons induced no phenotype, Syt1DA expression reduced the amplitudes of both evoked inhibitory postsynaptic currents IPSCs and evoked excitatory postsynaptic currents, and increased the frequencies of both miniature IPSCs and miniature EPSCs.4 The dominant-negative activity of Syt1DA was strongly eliminated by the LLQQ mutant, whereas the TGQQ mutant of Syt1DA was still dominant although slightly less strong than Syt1DA itself.Thus far, the strongest support for the tripartite interface comes from several electrophysiology experiments. Syt1 and its two mutants, again LLQQ and TGQQ, were separately expressed in cultured cortical neurons derived from double mutant mice harboring Syt1 conditional and Syt7 constitutive KO alleles.WT expression, the Cpx1/2 DKD partially decreased the evoked IPSC and EPSC amplitudes and increased mIPSC and mEPSC frequencies, whereas in WT neurons with exogenous Syt1DA expression, the Cpx1/2 DKD partially reversed the massive dominant-negative effect of Syt1DA.4 As a result, synaptic responses in Cpx1/2 DKD neurons were identical in neurons with Syt1WT and Syt1DA expression. Considering the milder effect of Cpx1/2 DKD than expression of Syt1DA, this result cannot be explained by saturation due to overexpression. Rather, the dominant negative effect of the Syt1DA mutant requires Cpx, in support of the tripartite interface since Cpx is involved in the tripartite interface. However, an alternative explanation6 suggested that Cpx can function in conjunction with the primary interface alone by keeping the membranes apart prior to putative dissociation of the primary interface upon Ca2+ binding.The tripartite interface was further tested by experiments with Cpx1/2 double knockdown (DKD) in WT neurons. In WT neurons with exogenous Syt12+-triggered release in PC12 cells,77 and increased spontaneous release, the release probability of the readily releasable pool (RRP), and the probability of synchronous release in primary cortical neuron cultures.78 Interestingly, studies with hippocampal Syt1 knockout cultures did not result in phenotypes with this mutant79; the differences between these studies are unclear. In any case, as observed in negative stain images of reconstituted Syt1 C2A-C2B fragments, this mutation also disrupts Syt1 ring formation. However, it is still uncertain if and when such rings might occur prior to formation of prefusion synaptic complexes. In summary, this phenotype of the Phe349 mutation could be explained by disruption of such rings or by disruption of the tripartite interface, or both.Further potential physiological support for the tripartite interface comes from Syt1 C2B Phe349  which is2+, and some of these interactions are enhanced by PIP2.30 As mentioned above, some of these interactions enhance the primary interface in the absence of Ca2+ by simultaneous binding of basic regions of the C2B domain and the SNARE complex to an anionic membrane.6The C2B domain contains many charged residues consisting of clusters of basic residues on the surface of the β-sandwich, including so-called polybasic region . These r2+, the C2B domain can also interact with the SNARE complex via another region that is distinct from both the primary and tripartite interfaces as determined by solution NMR experiments with paramagnetic relaxation enhancement (PRE) labels in the presence of a chaotropic agent.23 These experiments revealed a broad ensemble of structures where the polybasic region of the C2B domain binds to the acidic residues on the SNARE complex. The strong disruption of C2AB binding to the SNARE complex caused by mutations in the polybasic region could be consistent with this ensemble and correlated with the effects of these four mutations on neurotransmitter release in electrophysiology experiments in neuronal cultures.5 However, subsequent systematic studies of Syt1–SNARE and Syt1–lipid interactions using nanodiscs showed that the electrophysiological data are better explained by the effects of the mutations on Ca2+-dependent binding of Syt1 to PIP2-containing membranes.6 In the presence of Ca2+, Syt1 C2B binds to PIP2-containing membranes with higher affinity than to SNARE complex,6 suggesting that the Ca2+-dependent interaction in solution between the polybasic region of the Syt1 C2B domain and the SNARE complex is probably not physiologically relevant.In the absence of membranes, and in both the absence and presence of Ca2+.80 These interactions cannot be explained with a single binding mode, and because each one of these regions of Syt1 contains basic residues, and the SNARE complex has abundant negatively charged regions. The relevance of these interactions is still unclear, although it is possible that upon Ca2+ influx, binding of Syt1 to the C- terminus of the SNARE complex releases the inhibition of neurotransmitter release caused by the complexin accessory helix76.In addition to the interactions between the SNARE complex and Syt1 C2B discussed above, there are additional possible interactions between acidic residues at the C-terminal end of the SNARE complex and the polybasic region and other regions of Syt1 C2B as observed by solution NMR experiments with PRE labels in the absence of Cai.e., full length proteins and membranes). Elucidating the molecular architecture of these protein complexes in a Ca2+-free prefusion state in the presence of membranes is an important next step to fully elucidate the mechanism of Ca2+-triggered exocytosis. Single vesicle fusion experiments and low resolution cryo-electron microscopy suggested the existence of stable point contacts between vesicles with reconstituted SNAREs, Syt, and Cpx in the absence of Ca2+,40 although the resolution of these EM studies was insufficient to visualize the nature of these point contacts. Nevertheless, injection of Ca2+ resulted in rapid fusion of most of these vesicle associations. Cryo-electron tomography studies of similar reconstituted vesicles produced first glimpses of the morphologies of these contacts in the Ca2+-free state and suggested that membranes are kept apart (>30 Å) by protein complexes situated between membranes.81 Again, addition of Ca2+ resulted in disappearance of most of these contacts and fused vesicles. More recently, a cryo-ET data set of synaptic protein complexes was collected in their native environment between isolated synaptic vesicles (ISVs) and synthetic vesicles that mimic the plasma membrane82 . Thus, new technologies had to be developed to analyze such cryo-ET data sets.82 3D signal permutation was performed as an intervening step between subtomogram extraction using suitable masks and subsequent 3D classification and refinement. This 3D signal permutation, combined with feature-guided alignment, produced strong densities for three class average maps at the intermembrane density locations, and each of these intermembrane densities were morphologically distinct from one another. Work is in progress to collect additional tomograms to obtain higher resolution class averages.Subtomogram averaging is exceedingly difficult due to the relatively small size of SNAREs, Cpx1 and Syt1  in addition to the relatively low abundance of fully assembled presynaptic complexes at a given synaptic terminal. Moreover, analyses are hindered by a crowded molecular environment and the non-discrete nature of the particles  was used for several further studies.The primary interface involves residues on SNAP-25A and syntaxin-1A that interact with the Syt1 C2B domain. Of these interactions, a major part is the N-terminal SNAP-25A SNARE helix (~residues 37–58). One would therefore expect that a peptide fragment of SNAP-25A that includes residues from this region would act as a competitive inhibitor of the primary interface by binding to Syt1 C2B. However, this peptide would normally be unstructured, hindering its potential inhibition activity and prone to degradation. Thus, hydrocarbon staples were introduced,68 In contrast, the unstapled peptide is much more variable and rapidly moves away from the starting conformation. To corroborate this notion, fluorescence anisotropy experiments between Cy3-labeled SP9 and Syt1-C2B were performed, and suggested that SP9 binds to Syt1-C2B with a KD of 23 μM.68 In contrast, the unstapled peptide did not result in observable binding. Moreover, the “quintuple” mutant of Syt1 C2B (Arg281Ala/Glu295Ala/Tyr338Trp/Arg398Ala/Arg399Ala) that disrupts the primary interface3 showed only weak binding consistent with the disruption of salt bridges involving residues Arg281 and Glu295  upon pair association  from mouse brain homogenates, acceptor vesicles that contain Munc18-1–syntaxin-1A complexes, along with Cpx1, Munc13, N-ethylmaleimide-sensitive factor (NSF), αSNAP, and an ATP regeneration system . All comely rare , C). Theociation . SP9 didn events , D), sug2+, Ca2+-triggered fusion events  were monitored and analyzed . In tcroscope . Additiod fusion  with a pd fusion .3 Moreover, it is relatively conserved for other Ca2+ triggered exocytosis processes. For example, it is highly conserved for homologues involved in airway epithelial mucin hypersecretion,68 syntaxin-3, SNAP-23, VAMP8, and Syt-2. The residues that are at or near the primary interface are identical for Syt1/Syt2 except for Val292Cys and identical for SNAP-25A/SNAP-23 except for Lys40Glu, Leu47Ile, and Val48Thr. SP9 binds to both Syt1 C2B and Syt2 C2B with a similar dissociation constant (Kd) of 24 μM and 35 μM, respectively.68 When 10 μM SP9 was added in reconstitution assay for mucin hypersecretion, the Ca2+-triggered fusion amplitude, the cumulative fusion probability, and the synchronization were strongly inhibited, and the continuous (as opposed to immediate) Ca2+-independent fusion probability was only moderately reduced compared to the Ca2+-triggered amplitude and cumulative fusion probabilities. Moreover, SP9 had no effect on vesicle association.The primary interface between Syt1 and neuronal SNAREs is conserved among all species, and it is highly conserved among Syt1, Syt2, and Syt9, all known to be involved in fast synchronous release.2+-triggered membrane fusion in reconstituted systems of neuronal exocytosis and airway mucin hypersecretion, but it does not affect spontaneous or baseline release/secretion. From a mechanistic perspective, these findings have further solidified the critical role of the conserved primary interface for Ca2+-triggered membrane fusion.Taken together, SP9 inhibits Ca2+-triggered exocytosis inside cells , SP9 was conjugated to cell penetrating sequences (CPPs). Peptides were applied to primary human airway epithelial cells.68 Conjugation of SP9 with either penetratin (PEN) or TAT CPPs resulted in substantial peptide uptake into secretory airway cells. Treatment with CPP-conjugated TAT-SP9-Cy3 or PEN-SP9-Cy3 reduced ATP-stimulated, Ca2+-triggered MUC5AC. Importantly, baseline secretion was not affected by any of the peptides in the IL-13 (metaplastic) cultures, consistent with the absence of an effect of Syt2 deletion on baseline secretion in mice.88 The efficacy of CPP-conjugated SP9 was also tested in mice.68 Short-term treatment of mice with aerosolized PEN-SP9-Cy3 resulted in substantial peptide uptake into distal airway epithelial cells, and reduced methacholine-stimulated, Ca2+-triggered mucin secretion and airway mucus occlusion, whereas the non-stapled PEN-P9-Cy3 peptide did not exhibit an inhibitory effect. This peptide is expected to be a lead towards a new therapeutic to combat acute exacerbation of airway disease .To enable disruption of Ca2+-binding to the C2 domains of Syts, the inhibition is released, and fusion is triggered on a fast time scale, most likely by the action of SNAREs and possibly also by an active role of Syts upon Ca2+ binding. However, the details of these processes that occur upon Ca2+-triggering remain to be uncovered.Recent atomic resolution structures , low-res42 Briefly, synthetic plasma membrane mimic (PM) vesicles containing the self-quenching dye, sulforhodamine B, reconstituted syntaxin-1A, and SNAP-25A were incubated in an “SM conversion” solution containing, 20 nM NSF, 50 nM α-SNAP, 2 μM Munc18, 1 mM MgCl, 1 mM ATP, 5 U/mL Creatine Kinase, and 15 U/mL Creatine phosphate to convert the starting complex from syntaxin-1A–SNAP-25A to syntaxin-1A–Munc18-1 (SM) containing vesicles. These SM vesicles are then tethered to the imaging surface of custom-made microfluidic slides via biotin-peg-neutravidin-biotin-PE interactions. Isolated synaptic vesicles (ISVs) were purified from whole mouse brain homogenate, similar to previous methods89; however in contrast to previous isolations, vesicles here were gently competitively eluted using a small peptide corresponding to the antibody epitope. ISVs were then incubated in 1:1000 anti-synaptophysin-Alexa645 overnight followed by 2hr dialysis in a 300KD-cutoff dialysis cassette to remove unbound antibody. We then added SM conversion mixture proteins to the same final concentration as above and additionally included soluble accessory proteins with final concentrations of 500 nM C1C2BMUNC2C, 1 mM SNAP-25A, and 1 mM Cpx1.The fusion assay has been described previously.2C, SNAP-25A, and Cpx1. After wash, imaging was resumed, and after 30 frames (6 seconds), we injected the same buffer solution supplemented with 50 mM Ca2+ and free Alexa647 dye to track Ca2+ arrival. Fusion assay results were analyzed with a Matlab using custom scripts (available in the Zenodo repository https://zenodo.org/record/7159049.) Imaging data are deposited in the Dryad repository https://doi.org/10.5061/dryad.280gb5mss.SM vesicles were used to find an appropriate imaging area, after which ISVs are added to the imaging chamber, and vesicle-vesicle association is monitored for 1 minute. Following association, the chamber was washed with 40 column volumes (200 mL) of buffer containing the SM conversion components as well as C1C2BMUNC90 were used with a non-bonded cutoff of 11 Å. Five independent 1-μsec simulations were performed using different initial random number seeds. Trajectories of the molecular dynamics simulations of the tripartite interface are deposited in the Dryad repository https://doi.org/10.5061/dryad.f1vhhmh15.The molecular dynamics simulations of the tripartite interface described in this work  were carPyMol session file for MD simulation of the primary interfacePyMol session file for MD simulation of the tripartite interface


Astropecten aranciacus) and its dynamic changes upon insemination. The results have shown that the altered seawater pH strongly affected the sperm-induced Ca2+ response and the polyspermy rate. When immature starfish oocytes were stimulated with 1-MA in acidic or alkaline seawater, the maturation process displayed a strong dependency on pH in terms of the dynamic structural changes of the cortical F-actin. The resulting alteration of the actin cytoskeleton, in turn, affected the pattern of Ca2+ signals at fertilization and sperm penetration.In starfish, the addition of the hormone 1-methyladenine (1-MA) to immature oocytes  arrested at the prophase of the first meiotic division induces meiosis resumption (maturation), which makes the mature eggs able to respond to the sperm with a normal fertilization response. The optimal fertilizability achieved during the maturation process results from the exquisite structural reorganization of the actin cytoskeleton in the cortex and cytoplasm induced by the maturing hormone. In this report, we have investigated the influence of acidic and alkaline seawater on the structure of the cortical F-actin network of immature oocytes of the starfish ( The landmark discovery of 1-methyladenine (1-MA) as the hormone inducing the resumption of the meiotic cycle (maturation) of immature starfish (GV-stage) oocytes arrested at the first prophase of the meiotic division has allowed profound in vitro studies on the structural and biochemical changes that immature oocytes must undergo to reach the state of optimum fertilizability following the maturation process . It has 2+ signals in the cytoplasm and nucleus and at different stages of the maturation process /F0, where F represents the average fluorescence level of the entire egg and F0 the baseline fluorescence, the overall Ca2+ signals were quantified for each moment, and Frel was expressed as RFU (relative fluorescence unit) for plotting the Ca2+ trajectories. Applying the formula Finst = [Ft−F(t−1)]/F(t−1), the instantaneous increment of the Ca2+ level was analyzed to locate the specific area of transient Ca2+ increase. The values of Ca2+ signals were obtained from three independent experiments (N) using three different females, and the number of eggs (n) being analyzed for each condition is specified in the Results. To visualize the actin cytoskeleton, 50 µM (pipette concentration in methanol) of the fluorescent F-actin probe AlexaFluor 568-phalloidin  was microinjected into GV-stage oocytes and the unfertilized mature eggs in two independent experiments (N = 2), using two different females. To study the effect of the altered pH of the seawater on the morpho-functionality of the jelly coat triggering the sperm acrosome reaction, living immature oocytes and eggs matured with 1-MA in seawater at various pH conditions were incubated with 25 µM of the synthetic fluorescent polyamine BPA-C8-Cy5 to label the extracellular matrix (File S1). The distinct excitation and emission spectra of BPA-C8-Cy5 and Alexa568-phallodin enabled us to label the extracellular matrix and F-actin simultaneously. The fluorescent polyamine BPA-C8-Cy5 also visualized the VL, and the sperm acrosomal processes during insemination in acidic or alkaline seawater of immature oocytes and mature eggs. Alexa568-phalloidin, on the other hand, allowed us to monitor the sequential changes of the cortical F-actin in the same oocyte/egg. The acrosomal processes in A. aranciacus sperm were visualized by diluting sperm in seawater at different pH containing the fluorescent polyamine before insemination in given conditions. The signals of the fluorescent probes from the samples were detected with a Leica TCS SP8X confocal laser scanning microscope equipped with a white light laser and hybrid detectors using the Lightning deconvolution mode . The number of eggs examined for each condition (n) is specified in the Results.Intact immature and maturing oocytes were microinjected using an air-pressure transjector . To monitor intracellular CaA. aranciacus oocytes and eggs were counted in the cytoplasm of fertilized eggs 10–15 min after insemination using a cooled CCD (Charge-Coupled Device) camera  mounted on a Zeiss Axiovert 200 microscope with a Plan-Neofluar 40X/0.75 objective with a UV fluorescent lamp. The number of fertilized eggs examined (n) for each condition in three or four independent experiments (N) using three different females is shown in the Results and Diluted sperm were stained with 5 µM Hoechst-33342  for 30 s before insemination. The labeled sperm nuclei incorporated into p < 0.05 was considered statistically significant. For ANOVA results showing p < 0.05, the statistical significance of the difference between the two groups was assessed by Tukey’s post hoc tests. The two groups of data showing significant differences from each other were marked with symbols indicating the p values in the figure legends. For each experimental condition, 5 or 6 oocytes were used, and they were from the same animal. Now, of course, the same experiment was repeated 2–4 times (N) utilizing the oocytes from as many (N) animals. Thus, N refers to the number of replicates. On the other hand, the numerical data for Ca2+ signal and other values were pooled together for statistical Analysis by using all the oocytes treated in the same way. This number of oocytes was referred to with ‘n’.The numerical MetaMorph data were compiled and analyzed with Excel (Microsoft Office 2010) and reported as mean ± standard deviation (SD) in all cases in this manuscript. Oneway ANOVA was performed through Prism 5.00 (GraphPad Software), and Astropecten aranciacus, immature oocytes isolated from the ovary in natural seawater  are arrested at the prophase of meiosis I. At this stage of the maturation process, they are penetrated by multiple sperm upon insemination (A. aranciacus immature oocyte isolated from the ovary contains the large nucleus (GV) at the animal pole. The oocyte is surrounded by the jelly coat (JC) to which follicle cells (FC) adhere (In the starfish mination  and fail) adhere A. The mi) adhere C to the ) adhere E. The biA. aranciacus immature oocytes in NSW at pH 6.8 for 20 min apparently did not remove the JC surrounding the oocytes  were penetrated by a more significant number of sperm as a result of the increased receptivity to the sperm, which is ascribable to the pH-dependent structural changes of the oocyte cortex . That thA. aranciacus egg matured with 1-MA for 70 min which is the time for this species to achieve the optimal physiological conditions necessary to respond correctly to the fertilizing sperm. The hormonal stimulation induces the disassembly of the nuclear envelope of the GV and the intermixing of the nuclear components with the cytoplasm [At variance with immature oocytes that permit entry of multiple sperm, mature eggs obtained by incubation of the same oocytes in the presence of 1-MA exhibit a normal fertilization response with monospermy due to the restructuration of the oocyte cortex occurring a few minutes after hormonal stimulation and around the time of GVBD ,12,33. Fytoplasm . InseminA. aranciacus) are stimulated with 1-MA to induce maturation in acidic seawater (pH 6.8), the transmitted light microscopic images of GVBD and the elevation of the fertilization envelope , GVBD did not occur, as shown in the transmitted light image . The conlization ,20,22,27Since GVBD did not take place in the oocytes stimulated with 1-MA in seawater at pH 9, the GV-stage oocytes were first treated for 50 min with 1-MA in NSW (pH 8.1) to allow the breakdown of the nuclear envelope and then transferred to seawater at pH 9 for 20 min . A maturp < 0.01). Since the organization of the F-actin structures in the oocyte cortex that are formed to incorporate sperm is quite different in the oocytes inseminated in acidic seawater (p < 0.01), as shown in the fluorescent image of To test whether the lower or higher pH of seawater could affect the polyspermy, characteristic of immature starfish oocytes, GV-stage oocytes were incubated for 20 min in NSW , 6.8 (acidic), or 9.0  and subsequently inseminated with Hoechst 33342-stained sperm . The numseawater C, as comseawater F, the reA. aranciacus oocytes incubated with 1-MA for 70 min in NSW (pH 8.1) led to monospermic fertilization in most cases (48 out of 60 eggs). In the remaining mature eggs, the number of sperm counted was two or three per egg to make the average 1.23 ± 0.25 (n = 60). When oocytes were induced to undergo maturation with 1-MA in acidic seawater (pH 6.8) (p < 0.01 in comparison with the control NSW pH 8.1), which is also indicated by the visualization of one fertilized egg with seven sperm inside  is dependent on the structural organization of the oocyte cortex. At this stage of the maturation process (prophase I), the immature oocytes have longer microvilli [2+ response at fertilization, which is quite different from that in mature and overmatured eggs [Previous studies from our laboratory showed that the sperm-induced Cacrovilli ,21 and mred eggs .2+ due to the interaction of two sperm with the oocyte plasma membrane (arrowheads). These Ca2+ releases are followed by a subitaneous increase in Ca2+ at the periphery of the oocyte  that lasts only a few seconds and is followed by two Ca2+ waves that converge and propagate to the opposite pole in about two minutes, as indicated by the pseudocolor image of the relative fluorescence analysis (n = 13). The graph in 2+ release was 22.5 ± 7.7 s. The Ca2+ waves reached an amplitude of 0.37 ± 0.04 RFU in 358.7 ± 36.3 s. At variance with the control, oocytes inseminated in acidic seawater (pH 6.8) showed a Ca2+ response only in 4 out of 13 oocytes with a much longer delay . The peak amplitude of the Ca2+ wave was also significantly lower than in the control  as compared to the control  , even if the sequence of occurrence of the initial Ca2+ spots and CF was similar to that of the control, striking differences were observed in the pattern of Ca2+ release and the decline as shown in the pseudocolor images  and that the higher Ca2+ increase  induced by the fusion of multiple Ca2+ waves  than in the control.r images C–E. The 2+ response (the CF), which takes place simultaneously at the periphery of the egg cortex as a result of the activation of the L-type Ca2+ channels promoting Ca2+ influx [2+ wave propagating to the opposite pole   with 1-MA at a final concentration of 10 µM experience the first Ca+ influx . The pseite pole A. The gr 16.1 s) A,E.2+ response, optimal physiological conditions of the eggs are also required for a typical separation of the vitelline layer, which will form the FE seen in 2+ response and the number of sperm entries, all the eggs matured in acidic seawater (n = 13) responded to sperm stimulation by elevating their intracellular Ca2+ with a CF, which was followed by a Ca2+ wave  which was then followed by a CF and a series of Ca2+ waves as a result of the fusion of multiple sperm transducing several Ca2+ signals in 8 eggs out of 14. The Ca2+ waves then converged and propagated faster to the opposite pole  than the control . The analysis of the relative fluorescence changes has also indicated that 3 out of 14 eggs did not elicit any CF. Furthermore, the Ca2+ peak amplitude was also affected when eggs were matured in alkaline seawater , as shown in the graph of 2+ response was highlighted in eggs inseminated after being matured for 50 min in NSW (pH 8.1) and 20 min in seawater at pH 9 (n = 12). While the Ca2+ wave always followed the CF as in control in A, its amplitude was significantly lower than that of the control , as shown in the graph in D (light brown color) and histogram in On the other hand, when GV-stage oocytes were induced to mature in seawater at pH 9, they all failed to undergo GVBD. As aforementioned, 70 min after the addition of 1-MA, a GV with an abnormally elliptical shape was still visible A. Upon id in NSW . The rel2+ increase and sperm incorporation [2+ signaling and actin cytoskeletal remodeling that take place in the eggs after fertilization [Eggs of marine invertebrates have been broadly used in laboratories to study fertilization because the eggs are spawned freely into the sea, where they are immediately mixed with the spermatozoa. Thus, the fertilization process and the following developmental changes of the embryo can be easily observed in the petri dish. It has been known that the structural organization of the cortical F-actin in the oocytes and eggs profoundly impacts how they respond to the fertilizing sperm with Caporation ,34,35,42lization ,46.2+ signaling pattern upon insemination, sperm incorporation, and cortical granule exocytosis in some cases. The results are summarized in 2+ changes upon their fusion [In this communication, by using starfish as the experimental model, we have surveyed the effect of acidic and alkaline seawater not only on fertilization, but also on meiotic maturation. To begin with, the structural organization of the GV-stage oocytes and mature eggs are strikingly different in terms of the actin cytoskeleton (including microvilli) and the distribution of cortical granules and vesicles ,40. Becar fusion ,28.A. aranciacus in natural seawater (pH 8.1) are prone to polyspermy due to their structural organization of the oocyte surface and cortex [2+ responses with a pattern of Ca2+ release  did not make much difference to the morphology of the JC of immature oocytes, as judged by the fluorescent labeling by BPA-C8-Cy5 compare C, and di2+ response was not affected, indicating a standard functionality of the JC but the propagation of the Ca2+ wave and sperm entry were compromised due to the alteration of the cortical F-actin dynamics , and sea urchin eggs [2+ signaling and the subsequent F-actin reorganization necessary for cleavage [The advantage of designing controlled experimental conditions in the laboratory to study the effect of the altered seawater pH on the physiology of the fertilization process of marine eggs is of great importance. Indeed understanding how changes in the pH values of seawater affect the structural organization and dynamics of the actin cytoskeleton of the egg cortex may shed light on the molecular mechanisms regulating the F-actin-dependent Cahin eggs ,39,40 afcleavage ,39. Thes2+ response and penetration by only one sperm. The oocyte maturation process following the addition of 1-MA brought about by the intermixing of the nucleoplasm with the cytoplasm ensures the F-actin-dependent reorganization of the cortex of the mature egg for a normal fertilization response. In line with this, immature oocytes are prone to polyspermic fertilization. The results of this contribution have shown that the acidic or alkaline seawater pH reduced or increased polyspermy rates, respectively, and the altered Ca2+ responses at fertilization. Altered pH also affects the acrosome reaction of the sperm and the structure and dynamics of the actin filaments in the oocyte cortex. Morphological modifications of the cortical F-actin network of eggs matured in seawater at different pHs influencing the sperm-induced fertilization response were also observed. The results have highlighted the strict interdependence between the seawater pH and the sperm-induced Ca2+ signals in immature oocytes and mature eggs, which reflect the different organization of the cortical F-actin in their two maturation stages.In starfish, it has been well known that the breakdown of the immature oocytes’ large nucleus  induced by hormonal stimulation must occur to make the mature egg ready to be successfully fertilized with normal Ca