Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis

In flowering plants, pollen tubes are guided into ovules by multiple attractants from female gametophytes to release paired sperm cells for double fertilization. It has been well-established that Ca²⁺ gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca²⁺ channels in pollen tube tips are core components that regulate Ca²⁺ gradients by mediating and regulating external Ca²⁺ influx. Therefore, Ca²⁺ channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca²⁺ channels essential for pollen tube guidance. Here, we report that the point mutations R491Q or R578K in cyclic nucleotide-gated channel 18 (CNGC18) resulted in abnormal Ca²⁺ gradients and strong pollen tube guidance defects by impairing the activation of CNGC18 in Arabidopsis. The pollen tube guidance defects of cngc18-17 (R491Q) and of the transfer DNA (T-DNA) insertion mutant cngc18-1 (+/−) were completely rescued by CNGC18. Furthermore, domain-swapping experiments showed that CNGC18’s transmembrane domains are indispensable for pollen tube guidance. Additionally, we found that, among eight Ca²⁺ channels (including six CNGCs and two glutamate receptor-like channels), CNGC18 was the only one essential for pollen tube guidance. Thus, CNGC18 is the long-sought essential Ca²⁺ channel for pollen tube guidance in Arabidopsis.Pollen tubes deliver paired sperm cells into ovules for double fertilization, and signaling communication between pollen tubes and female reproductive tissues is required to ensure the delivery of sperm cells into the ovules (1). Pollen tube guidance is governed by both female sporophytic and gametophytic tissues (2, 3) and can be separated into two categories: preovular guidance and ovular guidance (1). For preovular guidance, diverse signaling molecules from female sporophytic tissues have been identified, including the transmitting tissue-specific (TTS) glycoprotein in tobacco (4), γ-amino butyric acid (GABA) in Arabidopsis (5), and chemocyanin and the lipid transfer protein SCA in Lilium longiflorum (6, 7). For ovular pollen tube guidance, female gametophytes secrete small peptides as attractants, including LUREs in Torenia fournieri (8) and Arabidopsis (9) and ZmEA1 in maize (10, 11). Synergid cells, central cells, egg cells, and egg apparatus are all involved in pollen tube guidance, probably by secreting different attractants (9–15). Additionally, nitric oxide (NO) and phytosulfokine peptides have also been implicated in both preovular and ovular pollen tube guidance (16–18). Thus, pollen tubes could be guided by diverse attractants in a single plant species.Ca²⁺ gradients at pollen tube tips are essential for both tip growth and pollen tube guidance (19–27). Spatial modification of the Ca²⁺ gradients leads to the reorientation of pollen tube growth in vitro (28, 29). The Ca²⁺ gradients were significantly increased in pollen tubes attracted to the micropyles by synergid cells in vivo, compared with those not attracted by ovules (30). Therefore, the Ca²⁺ gradients in pollen tube tips are essential for pollen tube guidance. The Ca²⁺ gradients result from external Ca²⁺ influx, which is mainly mediated by plasma membrane Ca²⁺ channels in pollen tube tips. Thus, the Ca²⁺ channels are the key components for regulating the Ca²⁺ gradients and are consequently essential for pollen tube guidance. Using electrophysiological techniques, inward Ca²⁺ currents were observed in both pollen grain and pollen tube protoplasts (31–36), supporting the presence of plasma membrane Ca²⁺ channels in pollen tube tips. Recently, a number of candidate Ca²⁺ channels were identified in pollen tubes, including six cyclic nucleotide-gated channels (CNGCs) and two glutamate receptor-like channels (GLRs) in Arabidopsis (37–40). Three of these eight channels, namely CNGC18, GLR1.2, and GLR3.7, were characterized as Ca²⁺-permeable channels (40, 41) whereas the ion selectivity of the other five CNGCs has not been characterized. We hypothesized that the Ca²⁺ channel essential for pollen tube guidance could be among these eight channels.In this research, we first characterized the remaining five CNGCs as Ca²⁺ channels. We further found that CNGC18, out of the eight Ca²⁺ channels, was the only one essential for pollen tube guidance in Arabidopsis and that its transmembrane domains were indispensable for pollen tube guidance.     

Calmodulin extracts the Ras family protein RalA from lipid bilayers by engagement with two membrane-targeting motifs

RalA is a small GTPase and a member of the Ras family. This molecular switch is activated downstream of Ras and is widely implicated in tumor formation and growth. Previous work has shown that the ubiquitous Ca²⁺-sensor calmodulin (CaM) binds to small GTPases such as RalA and K-Ras4B, but a lack of structural information has obscured the functional consequences of these interactions. Here, we have investigated the binding of CaM to RalA and found that CaM interacts exclusively with the C terminus of RalA, which is lipidated with a prenyl group in vivo to aid membrane attachment. Biophysical and structural analyses show that the two RalA membrane-targeting motifs (the prenyl anchor and the polybasic motif) are engaged by distinct lobes of CaM and that CaM binding leads to removal of RalA from its membrane environment. The structure of this complex, along with a biophysical investigation into membrane removal, provides a framework with which to understand how CaM regulates the function of RalA and sheds light on the interaction of CaM with other small GTPases, including K-Ras4B.

RalA and RalB are members of the Ras superfamily of small GTPases, plasma membrane-associated molecular switches that regulate signal transduction affecting a plethora of cellular processes. Acting as one of the principal branches of the Ras signaling network, recruitment of a Ral-specific guanine exchange factor (RalGEF) promotes activation of RalA/B. Despite being less well studied than the MAPK and PI3K pathways, activation of RalGEFs is sufficient to induce Ras-driven transformation of human cells (1), and the inhibition of RalGEF disrupts colony formation in Ras-driven human cancer cell lines (2). It has also been reported previously that the RalGEF signaling pathway is crucial in the development of bone metastasis originating from pancreatic cancer in mice (3), and skin carcinoma mouse models deficient in RalGEF show decreased tumor size and number (4). Together, these findings indicate critical roles for both RalA and RalB in tumor formation and cancer progression, suggesting that it is important to expand our knowledge of their signaling roles and regulation.RalA and RalB share 82% sequence identity in their G-domains (guanine nucleotide-binding domain) and are almost identical structurally (5). Both proteins contain two switch regions, the conformations of which are sensitive to the bound nucleotide, allowing downstream effectors to select the active form of the protein. The effector binding sequences of RalA/B are identical, and it is therefore surprising that they display functional divergence in vivo, mediating distinct cellular effects in both normal cells and in cancer settings (6–10). Most of the sequence diversity between the Ral isoforms comes from the aptly named hypervariable region (HVR) located at the C terminus. HVRs are short, intrinsically disordered regions found in all Ras and Rho family proteins, which have recently come under scrutiny for their ability to interact with membranes to regulate and modify signaling output of the G domain [reviewed by Cornish et al. (11)]. Some of the HVRs may have a propensity for secondary structure formation, such as the K-Ras4B HVR, which is α-helical under certain circumstances (12).The C-terminal “CaaX box” motif (C = Cys, a = aliphatic, X = any residue) is the recognition sequence for isoprenylation of small GTPases, which facilitates their attachment to membranes. The C terminus of Ral proteins is recognized by GGTase-I (13), which adds a geranylgeranyl moiety to the Cys sidechain. The proteins are further processed by removal of the “aaX” motif and methylation of the new C-terminal carboxyl of the prenylcysteine (14). A secondary membrane attachment signal comes from positively charged Lys and Arg sidechains within the HVR, which interact with negatively charged phospholipid headgroups in the membrane bilayer. More than just a simple membrane anchor, the HVRs of RalA/B contain Ser residues that can be differentially phosphorylated in vivo. Ser194 of RalA is an Aurora kinase A target, phosphorylation of which has been shown to facilitate relocation to the mitochondrial membrane and binding to the effector RLIP76 (15).CaM (calmodulin) is a ubiquitous calcium sensor that regulates a multitude of partners. It is a small (16.7 kDa), pseudosymmetrical protein with two lobes (16), each comprising two EF-hand motifs. Upon calcium binding, the EF hands reorient to expose methionine-rich hydrophobic pockets that engage target proteins (17). The unusually high proportion of methionine residues, in conjunction with a flexible linker between the two lobes, confers extensive binding plasticity (18), allowing considerable sequence and structural diversity in CaM-interacting proteins. Binding often involves CaM wrapping around a positively charged helix in its target that contains hydrophobic anchors at defined positions in the sequence. These anchor residues dock into the hydrophobic pockets of the two CaM lobes. Alongside this canonical “wrap-around” mechanism, CaM also employs a variety of known noncanonical binding modes, which result in more extended conformations (19).CaM has been shown to interact with the HVRs of RalA and RalB in a Ca²⁺-dependent manner (19–21). It also binds the related small GTPase K-Ras4B in an interaction that involves burial of its C-terminal isoprenyl (farnesyl) group. Despite the interest in this interaction, there is no structure of K-Ras4B in complex with CaM. Although early data indicated that binding of K-Ras4B was nucleotide dependent, it is now thought that CaM binds to the prenylated C-terminal K-Ras4B HVR and that the nucleotide only controls accessibility of that region rather than binding directly to CaM (21). Biophysical data from one group indicated that two K-Ras4B molecules can bind to a single CaM, with the C-lobe of CaM binding around 10 times more tightly than the N-lobe (21). Another study used NMR titrations to show that the K-Ras4B protein is not necessary and that farnesyl compounds themselves are able to bind CaM (22). This was supported by a structure of a complex of CaM with farnesylated, methylated Cys, which binds exclusively to the C-lobe of CaM, in line with its higher affinity for that part of the CaM protein.The interaction between RalA and CaM has not been studied as extensively, and there are conflicts in the literature as to the number of CaM-binding sites on Ral (20, 23) and the cellular consequences of the interaction. One study found that the interaction with CaM stimulated the GTPase “off-switch” of RalA (24), but another report showed that CaM binding caused RalA activation (20). Previous work has alluded to the importance of the prenyl anchor in the interaction (25), although there is a lack of biophysical data to corroborate and explain this observation. An in-depth structural and biophysical analysis of the RalA–CaM complex would allow an assessment of the potential functional consequences of this interaction. In this investigation, using maleimide-conjugated prenyl mimics, we sought to elucidate the molecular basis for the interaction between RalA and CaM to better understand its role in vivo. We establish that the binding motif for CaM is within the RalA-HVR and demonstrate the importance of the prenyl anchor for high affinity binding. We have solved the first structure of CaM in complex with a lipid-modified HVR, which shows that the N-lobe of CaM encases the prenyl group whereas the C-lobe interacts with key hydrophobic residues of the HVR. Furthermore, we show that CaM is able to extract RalA from the surface of a lipid membrane and propose a stepwise temporal order for the mechanism. Other small GTPases suspected of interaction with CaM, such as RalB, Rac1, Cdc42 (26), and Rap1A (27), have features in common with RalA, including a polybasic motif and prenyl moieties at their C termini. The results presented here may therefore provide a framework to understand the interaction of CaM with these proteins.     

Energy landscape views for interplays among folding,binding, and allostery of calmodulin domains

Ligand binding modulates the energy landscape of proteins, thus altering their folding and allosteric conformational dynamics. To investigate such interplay, calmodulin has been a model protein. Despite much attention, fully resolved mechanisms of calmodulin folding/binding have not been elucidated. Here, by constructing a computational model that can integrate folding, binding, and allosteric motions, we studied in-depth folding of isolated calmodulin domains coupled with binding of two calcium ions and associated allosteric conformational changes. First, mechanically pulled simulations revealed coexistence of three distinct conformational states: the unfolded, the closed, and the open states, which is in accord with and augments structural understanding of recent single-molecule experiments. Second, near the denaturation temperature, we found the same three conformational states as well as three distinct binding states: zero, one, and two calcium ion bound states, leading to as many as nine states. Third, in terms of the nine-state representation, we found multiroute folding/binding pathways and shifts in their probabilities with the calcium concentration. At a lower calcium concentration, “combined spontaneous folding and induced fit” occurs, whereas at a higher concentration, “binding-induced folding” dominates. Even without calcium binding, we observed that the folding pathway of calmodulin domains can be modulated by the presence of metastable states. Finally, full-length calmodulin also exhibited an intriguing coupling between two domains when applying tension.Protein folding and conformational dynamics have often been characterized by the energy landscape of proteins (1–5). The energy landscape is dependent on the molecular physiochemistry and thus is modulated by many factors, such as chemical modification and ligand binding. Ligand binding, in turn, is dependent on the conformation of proteins. Thus, folding, binding, and allosteric conformational dynamics are mutually correlated. Despite their obvious correlation in concept, it has been very challenging to characterize how they are indeed coupled for any single proteins. Here, we address, in depth, how these three types of dynamics, folding, binding, and allosteric conformational dynamics, are coupled from the energy landscape perspective for a specific protein, calmodulin (CaM).CaM is a ubiquitous calcium-binding messenger protein involved in signal transduction (6) and, more importantly here, has been a model protein to investigate folding, binding, and allostery. Full-length CaM has two nearly symmetric globular domains connected by a flexible central helix (7, 8). Each domain is composed of paired EF hands containing two Ca²⁺-binding sites (Fig. 1A). Upon binding to Ca²⁺, each CaM domain undergoes substantial conformational change from a closed state to an open state, exposing a hydrophobic patch that can bind with target proteins and regulate downstream processes (9). CaM has been frequently used as a model in studying the folding of multidomain proteins (10, 11), allosteric transitions (12–14), slow conformational dynamics around physiological temperatures (15–18), metal ion binding (19, 20), and correlation between inherent flexibility and protein functions (21, 22). For example, using structure-based coarse-grained (CG) simulations, Chen and Hummer elucidated the coexistence of an unfolded state, a closed state, and an open state around physiological temperatures for the C-terminal domain of CaM (CaM-C) without Ca²⁺ binding (15), which reconciles some seemingly contradictory experimental observations on the slow conformational dynamics of CaM.Open in a separate windowFig. 1.(A) Three-dimensional structure of calmodulin domain at closed [Protein Data Bank (PDB) code: 1cfd] and open states (PDB code:1cll). Calcium ions are represented by yellow spheres. (B) Schematic of coupling among folding, calcium binding, and allosteric motions for the CaM domain. Due to the conformational transitions between open and closed states, in addition to the direct folding pathway (red solid arrow), folding to the most stable state may involve an alternative pathway via a metastable state (green arrow plus blue arrow). The calcium binding can modulate the relative stability of the conformational states and therefore the population of folding pathways. O, C, and U represent open, closed, and unfolded states, respectively.More recently, Rief and coworkers studied the Ca²⁺-dependent folding of CaM based on a new generation technique of single-molecule force spectroscopy, which can probe the reversible folding/unfolding transitions with near equilibrium conditions (10, 23, 24). Their results revealed that at high Ca²⁺ concentrations, the folding pathway of the CaM domain proceeds via a transition state capable of binding Ca²⁺ ions, demonstrating the coupling between Ca²⁺ binding and CaM folding. All these computational and experimental works provided unprecedented understanding of many aspects of the folding and allosteric transitions of CaM. However, a full picture of the coupling among folding, Ca²⁺ binding, and allosteric motions, as schematically shown in Fig. 1B, is still lacking. Particularly, two fundamental issues arising from the allostery and Ca²⁺-binding characteristics of CaM remain elusive: (i) How does the allosteric feature of the energy landscape contribute to the folding complexity? And (ii) how can the folding mechanism of CaM be modulated by Ca²⁺ binding?Motivated by previous computational and experimental studies (15, 23), in this work we investigated the folding coupled with Ca²⁺ binding and allosteric motions of the isolated CaM domains as well as the full-length CaM. To do so, we first integrated computational tools developed for folding, ligand binding, and allosteric motions together. The proposed CG protein model was used for the subsequent series of molecular dynamics (MD) simulations. First, corresponding to Rief’s experiments, we performed MD simulations of isolated CaM domains with pretensions, which gave consistent results with the experiments and, in addition, provided the direct structural assignment on the experimentally observed states. Second, at a higher temperature, without pretension we performed reversible folding/unfolding simulations for a wide range of Ca²⁺ concentrations. The conformational and ligand-binding energy landscape revealed as many as nine distinctive states. Then, we analyzed the binding-coupled folding reactions in terms of the nine states, finding multiple routes and their modulation by Ca²⁺ concentrations. Interestingly, as the Ca²⁺ concentration increases, the CaM domain folding mechanism switches from “combined spontaneous folding and induced fit” to “binding-induced folding,” which accords with the scenario deduced from single-molecule force spectroscopy experiments. Finally, the effects of tension on the conformational fluctuations of the full-length CaM are discussed.     

PMCA2 regulates HER2 protein kinase localization and signaling and promotes HER2-mediated breast cancer

In the lactating mammary gland, the plasma membrane calcium ATPase2 (PMCA2) transports milk calcium. Its expression is activated in breast cancers, where high tumor levels predict increased mortality. We find that PMCA2 expression correlates with HER2 levels in breast cancers and that PMCA2 interacts with HER2 in specific actin-rich membrane domains. Knocking down PMCA2 increases intracellular calcium, disrupts interactions between HER2 and HSP-90, inhibits HER2 signaling, and results in internalization and degradation of HER2. Manipulating PMCA2 levels regulates the growth of breast cancer cells, and knocking out PMCA2 inhibits the formation of tumors in mouse mammary tumor virus (MMTV)-Neu mice. These data reveal previously unappreciated molecular interactions regulating HER2 localization, membrane retention, and signaling, as well as the ability of HER2 to generate breast tumors, suggesting that interactions between PMCA2 and HER2 may represent therapeutic targets for breast cancer.Plasma membrane calcium ATPases (PMCAs) are a family of ion pumps that transport calcium out of cells and maintain low resting intracellular calcium levels (1–3). PMCA2 (gene symbol Atp2b2) is highly expressed in the apical membrane of mammary epithelial cells only during lactation, where it has been shown to transport calcium into milk (4–6). After weaning, PMCA2 expression rapidly decreases, contributing to the initiation of programmed cell death and mammary gland involution (7, 8). PMCA2 is also expressed in breast cancers (8–10), and high levels of tumor PMCA2 expression predict increased mortality in patients (8).Approximately 25–30% of invasive breast cancers overexpress human epidermal growth factor receptor 2 (HER2) as a result of amplification of the ERBB2 kinase gene (11–13), and overexpression of HER2 causes breast tumors in mouse mammary tumor virus (MMTV)-Neu transgenic mice (14). HER2 functions as a heterodimer with other ERBB family members, most commonly pairing with EGFR or human epidermal growth factor receptor 3 (HER3) in breast cancers (11, 13). For reasons that remain poorly understood, in contrast to other ERBB family members, which are internalized and degraded after stimulation, HER2 remains on the cell surface and continues to signal for prolonged periods (12, 15).In this study, we describe a previously unrecognized function for PMCA2: supporting active HER2 signaling and HER2-mediated tumor formation. Our data suggest that PMCA2 interacts with HER2 within specific membrane domains and is required for HER2 expression, membrane retention, and signaling.     

Time-resolved DEER EPR and solid-state NMR afford kinetic and structural elucidation of substrate binding to Ca2+-ligated calmodulin

Recent advances in rapid mixing and freeze quenching have opened the path for time-resolved electron paramagnetic resonance (EPR)-based double electron-electron resonance (DEER) and solid-state NMR of protein–substrate interactions. DEER, in conjunction with phase memory time filtering to quantitatively extract species populations, permits monitoring time-dependent probability distance distributions between pairs of spin labels, while solid-state NMR provides quantitative residue-specific information on the appearance of structural order and the development of intermolecular contacts between substrate and protein. Here, we demonstrate the power of these combined approaches to unravel the kinetic and structural pathways in the binding of the intrinsically disordered peptide substrate (M13) derived from myosin light-chain kinase to the universal eukaryotic calcium regulator, calmodulin. Global kinetic analysis of the data reveals coupled folding and binding of the peptide associated with large spatial rearrangements of the two domains of calmodulin. The initial binding events involve a bifurcating pathway in which the M13 peptide associates via either its N- or C-terminal regions with the C- or N-terminal domains, respectively, of calmodulin/4Ca²⁺ to yield two extended “encounter” complexes, states A and A*, without conformational ordering of M13. State A is immediately converted to the final compact complex, state C, on a timescale τ ≤ 600 μs. State A*, however, only reaches the final complex via a collapsed intermediate B (τ ∼ 1.5 to 2.5 ms), in which the peptide is only partially ordered and not all intermolecular contacts are formed. State B then undergoes a relatively slow (τ ∼ 7 to 18 ms) conformational rearrangement to state C.

Calmodulin (CaM) is a universal eukaryotic calcium sensor that plays a central role in calcium signaling (1). Binding of two Ca²⁺ ions per CaM domain exposes methionine-rich hydrophobic patches that prime the system for high-affinity binding to a wide range of protein partners (2). Free calcium–loaded calmodulin (CaM/4Ca²⁺) is predominantly extended (3–5), although sparsely populated, highly transient compact states are sampled (6, 7), but clamps down upon target substrates like two hands capturing a rope in the final complex (8–11) (Fig. 1). Concomitant conformational changes involve the transition of a largely helical interdomain linker to a long flexible loop and the adoption of a helical conformation by the intrinsically disordered substrate. Although CaM has been the subject of extensive biophysical studies (12–16), current structural knowledge is limited to calcium-free, calcium-loaded, and calcium-loaded/peptide-bound states as separate entities, with little experimental information about the molecular mechanisms that connect the latter two states. For example, it is not known whether substrates bind first to the N-terminal domain (NTD) or C-terminal domain (CTD) of CaM/4Ca²⁺, whether identifiable intermediate states exist, whether there is a single predominant pathway for complex formation, or at what stage in the process the CaM binding regions of target protein substrates become conformationally ordered. To address this knowledge gap, we have performed time-resolved electron paramagnetic resonance (EPR)-based double electron-electron resonance (DEER) and solid-state (ss) NMR studies that jointly elucidate the process of CaM/4Ca²⁺–peptide complex formation in quantitative kinetic and structural terms. This work relies on three technological advances: 1) rapid mixing and freeze quenching to sequentially trap the state of the reaction mixture on the millisecond timescale (17–24), thereby permitting time-resolved DEER EPR and ssNMR measurements of protein–substrate interactions; 2) the application of phase-memory time (T_m) filtering (25) to quantitatively extract species populations from DEER data (26) so that time-dependent probability distance distributions between pairs of spin labels can be monitored; and 3) quantitative analysis of ssNMR ¹³C-¹³C correlation spectra to provide residue-specific information on the appearance of structural order and the development of intermolecular contacts between substrate and protein (21, 23).Open in a separate windowFig. 1.Schematic overview of the time-resolved DEER EPR and ssNMR experiments. The predominant extended conformation of free CaM/4Ca²⁺ seen in the crystal structure is shown in Upper Left (3); the locations of the R1 nitroxide spin labels (transparent red spheres) at A17C–R1 and A128C–R1 in the NTD (blue) and CTD (purple), respectively, and of the relevant methionine residues (gray balls) are indicated. The intrinsically disordered M13 peptide, comprising the CaM binding site of skMLCK, is shown in Upper Right, with ¹³C-labeled residues in the N- and C-terminal halves of the peptides shown as blue and mauve balls, respectively. The structure of the final CaM/4Ca²⁺–M13 complex (8, 9) is shown in Lower, with the helical M13 peptide in red. The reaction time is controlled by the flow rate through the mixer and the flight distance from the mixer nozzle to the spinning copper disk cooled to 77K.     

Conserved properties of individual Ca2+-binding sites in calmodulin

Calmodulin (CaM) is a Ca²⁺-sensing protein that is highly conserved and ubiquitous in eukaryotes. In humans it is a locus of life-threatening cardiomyopathies. The primary function of CaM is to transduce Ca²⁺ concentration into cellular signals by binding to a wide range of target proteins in a Ca²⁺-dependent manner. We do not fully understand how CaM performs its role as a high-fidelity signal transducer for more than 300 target proteins, but diversity among its four Ca²⁺-binding sites, called EF-hands, may contribute to CaM’s functional versatility. We therefore looked at the conservation of CaM sequences over deep evolutionary time, focusing primarily on the four EF-hand motifs. Expanding on previous work, we found that CaM evolves slowly but that its evolutionary rate is substantially faster in fungi. We also found that the four EF-hands have distinguishing biophysical and structural properties that span eukaryotes. These results suggest that all eukaryotes require CaM to decode Ca²⁺ signals using four specialized EF-hands, each with specific, conserved traits. In addition, we provide an extensive map of sites associated with target proteins and with human disease and correlate these with evolutionary sequence diversity. Our comprehensive evolutionary analysis provides a basis for understanding the sequence space associated with CaM function and should help guide future work on the relationship between structure, function, and disease.Eukaryotes use Ca²⁺ in numerous intracellular signaling pathways. Calmodulin (CaM) is a highly versatile Ca²⁺ signaling protein that is essential for at least dozens of cellular processes in eukaryotic cells. In humans it binds to more than 300 targets (1–3). Humans have three genes that encode identical CaM proteins, but mutations in just one of the three copies can cause disease (4–8), as can altered gene expression (9). Although CaM has been extensively studied, many details about its function are still poorly understood. The high evolutionary conservation along with the wide range of targets brings up the question of how a single Ca²⁺-binding protein displays both selectivity and flexibility in the context of its various signaling pathways.CaM binds Ca²⁺ at four, nonidentical sites that contain the structural motif called an EF-hand (10, 11), each of which contains an acidic Ca²⁺-coordinating loop, or “EF-loop” (Fig. 1A). The EF-loop spans 12 amino acids and provides at least six oxygen atoms for coordinating Ca²⁺ (12). The coordinating oxygen atoms are provided by the side chains at the first, third, fifth, and 12th positions of the EF-loop, and an oxygen from a main chain carbonyl group is provided at the seventh position (10). Water molecules participate in the Ca² coordination geometry (13). CaM functions as a sensor over a broad range of Ca²⁺ signals that vary in amplitude, duration, and location. Although biophysical and evolutionary sequence studies have resulted in a general understanding of the bulk properties of EF-hand–binding sites, the implications of differences in Ca²⁺ affinity among the four EF-hands deserves a thorough investigation.Open in a separate windowFig. 1.(A) Example of a Ca²⁺-bound EF-hand structure from PDBID 1CLL. A cartoon of an EF-hand peptide chain threads through a semitransparent representation of its molecular surface. The surface is the interface between molecular atoms and solvent rendered in PyMOL. Only atoms nearest the Ca²⁺ are shown and are depicted as spheres—green for Ca²⁺ and red for oxygens. A Ca²⁺-coordinating water is depicted as a semitransparent red sphere. Helices are gray, and the EF-loop is tan. (B) Maximum likelihood branch lengths of CaM and tubulin constrained to match the species tree in Torruella et al. (40). This tree covers much of eukaryotic diversity. Holozoa and Holomycota include animals and fungi, respectively, and their closely related protist lineages. SARPAE is described in the text. Both proteins are highly constrained, but whereas tubulin’s rate has been fairly consistent across eukaryotes, CaM underwent a dramatic speed-up in Ascomycete fungi, which include the model system S. cerevisiae.Previous reports showed that the large family of EF-hand proteins likely arose from a founder protein with a single EF-hand in the most recent common ancestor of all extant eukaryotes (11, 14–18). Different EF-hand–containing proteins bind Ca²⁺ with different affinities, suggesting that a protein with multiple EF-hands, such as CaM, may bind Ca²⁺ with a different affinity at each site (19–28). It has therefore been suggested that CaM’s four sites display different affinities and perhaps cooperativity (29, 30). We therefore hypothesized that CaM’s four, nonidentical loops may generate some of their functional flexibility by binding Ca²⁺ using different physical properties and explored whether such differences could be discerned in the evolutionary record.Evolutionary analyses can provide mechanistic insight into how CaM is used as a Ca²⁺ sensor across eukaryotes. Prior work showed that the protein sequence of CaM is evolving at a faster pace in fungal species (11, 31–33), reflecting the fact that although CaM is essential in Saccharomyces cerevisiae, the cells can survive with all four EF-hands ablated (34). However, previous evolutionary studies focused on a small subset of eukaryotes, either because few sequences were available at the time of publication or because the study was focused on a particular lineage. The vast expansion of taxonomic coverage in sequence databases, and the recent availability of new NMR and X-ray crystal structures of CaM, therefore demands a more comprehensive analysis. Unfortunately, CaM is a small, ancient, and highly conserved protein and therefore does not contain enough information to infer phylogenetic tree topologies. Kretsinger and Nakayama and coworkers (11, 16, 17, 35), for instance, found little correspondence between phylogenies inferred from protein, DNA, or intron–exon structure.To overcome this hurdle, we used a variety of techniques to explore sequence and structural conservation in CaM across eukaryotes. Our approach allows us to address several key questions: (i) How fast is CaM diverging in different phyla? (ii) How does the function of a site, or its association with disease, correlate with sequence conservation? (iii) What properties of the EF-hands are conserved over deep evolutionary time, and how might this correspond to functional plasticity?     

Thermodynamics of formation of coffinite,USiO4

Coffinite, USiO₄, is an important U(IV) mineral, but its thermodynamic properties are not well-constrained. In this work, two different coffinite samples were synthesized under hydrothermal conditions and purified from a mixture of products. The enthalpy of formation was obtained by high-temperature oxide melt solution calorimetry. Coffinite is energetically metastable with respect to a mixture of UO₂ (uraninite) and SiO₂ (quartz) by 25.6 ± 3.9 kJ/mol. Its standard enthalpy of formation from the elements at 25 °C is −1,970.0 ± 4.2 kJ/mol. Decomposition of the two samples was characterized by X-ray diffraction and by thermogravimetry and differential scanning calorimetry coupled with mass spectrometric analysis of evolved gases. Coffinite slowly decomposes to U₃O₈ and SiO₂ starting around 450 °C in air and thus has poor thermal stability in the ambient environment. The energetic metastability explains why coffinite cannot be synthesized directly from uraninite and quartz but can be made by low-temperature precipitation in aqueous and hydrothermal environments. These thermochemical constraints are in accord with observations of the occurrence of coffinite in nature and are relevant to spent nuclear fuel corrosion.In many countries with nuclear energy programs, spent nuclear fuel (SNF) and/or vitrified high-level radioactive waste will be disposed in an underground geological repository. Demonstrating the long-term (10⁶–10⁹ y) safety of such a repository system is a major challenge. The potential release of radionuclides into the environment strongly depends on the availability of water and the subsequent corrosion of the waste form as well as the formation of secondary phases, which control the radionuclide solubility. Coffinite (1), USiO₄, is expected to be an important alteration product of SNF in contact with silica-enriched groundwater under reducing conditions (2–8). It is also found, accompanied by thorium orthosilicate and uranothorite, in igneous and metamorphic rocks and ore minerals from uranium and thorium sedimentary deposits (2, 4, 5, 8–16). Under reducing conditions in the repository system, the uranium solubility (very low) in aqueous solutions is typically derived from the solubility product of UO₂. Stable U(IV) minerals, which could form as secondary phases, would impart lower uranium solubility to such systems. Thus, knowledge of coffinite thermodynamics is needed to constrain the solubility of U(IV) in natural environments and would be useful in repository assessment.In natural uranium deposits such as Oklo (Gabon) (4, 7, 11, 12, 14, 17, 18) and Cigar Lake (Canada) (5, 13, 15), coffinite has been suggested to coexist with uraninite, based on electron probe microanalysis (EPMA) (4, 5, 7, 11, 13, 17, 19, 20) and transmission electron microscopy (TEM) (8, 15). However, it is not clear whether such apparent replacement of uraninite by a coffinite-like phase is a direct solid-state process or occurs mediated by dissolution and reprecipitation.The precipitation of USiO₄ as a secondary phase should be favored in contact with silica-rich groundwater (21) [silica concentration >10⁻⁴ mol/L (22, 23)]. Natural coffinite samples are often fine-grained (4, 5, 8, 11, 13, 15, 24), due to the long exposure to alpha-decay event irradiation (4, 6, 25, 26) and are associated with other minerals and organic matter (6, 8, 12, 18, 27, 28). Hence the determination of accurate thermodynamic data from natural samples is not straightforward. However, the synthesis of pure coffinite also has challenges. It appears not to form by reacting the oxides under dry high-temperature conditions (24, 29). Synthesis from aqueous solutions usually produces UO₂ and amorphous SiO₂ impurities, with coffinite sometimes being only a minor phase (24, 30–35). It is not clear whether these difficulties arise from kinetic factors (slow reaction rates) or reflect intrinsic thermodynamic instability (33). Thus, there are only a few reported estimates of thermodynamic properties of coffinite (22, 36–40) and some of them are inconsistent. To resolve these uncertainties, we directly investigated the energetics of synthetic coffinite by high-temperature oxide melt solution calorimetry to obtain a reliable enthalpy of formation and explored its thermal decomposition.     

In vivo genetic dissection of O2-evoked cGMP dynamics in a Caenorhabditis elegans gas sensor

cGMP signaling is widespread in the nervous system. However, it has proved difficult to visualize and genetically probe endogenously evoked cGMP dynamics in neurons in vivo. Here, we combine cGMP and Ca²⁺ biosensors to image and dissect a cGMP signaling network in a Caenorhabditis elegans oxygen-sensing neuron. We show that a rise in O₂ can evoke a tonic increase in cGMP that requires an atypical O₂-binding soluble guanylate cyclase and that is sustained until oxygen levels fall. Increased cGMP leads to a sustained Ca²⁺ response in the neuron that depends on cGMP-gated ion channels. Elevated levels of cGMP and Ca²⁺ stimulate competing negative feedback loops that shape cGMP dynamics. Ca²⁺-dependent negative feedback loops, including activation of phosphodiesterase-1 (PDE-1), dampen the rise of cGMP. A different negative feedback loop, mediated by phosphodiesterase-2 (PDE-2) and stimulated by cGMP-dependent kinase (PKG), unexpectedly promotes cGMP accumulation following a rise in O₂, apparently by keeping in check gating of cGMP channels and limiting activation of Ca²⁺-dependent negative feedback loops. Simultaneous imaging of Ca²⁺ and cGMP suggests that cGMP levels can rise close to cGMP channels while falling elsewhere. O₂-evoked cGMP and Ca²⁺ responses are highly reproducible when the same neuron in an individual animal is stimulated repeatedly, suggesting that cGMP transduction has high intrinsic reliability. However, responses vary substantially across individuals, despite animals being genetically identical and similarly reared. This variability may reflect stochastic differences in expression of cGMP signaling components. Our work provides in vivo insights into the architecture of neuronal cGMP signaling.The second messenger cyclic guanosine monophosphate (cGMP) regulates a range of physiological processes. In nervous systems, it can transduce sensory inputs (1) and modulate neuronal excitability and learning (2) and is implicated in control of mood and cognition (3). Precise regulation of cGMP levels ([cGMP]) is thought critical for these functions. This importance has prompted development of genetically encoded cGMP indicators, with the goal of visualizing cGMP dynamics with high temporal and spatial resolution (4, 5). Although these sensors have been used to image pharmacologically evoked changes in cGMP in cultured cells or tissue slices (6–10), endogenous cGMP dynamics have not been visualized and functionally dissected in vivo in any nervous system (4, 5).Local [cGMP] reflects the net activity of guanylate cyclases (GCs) that synthesize cGMP (11) and phosphodiesterases (PDEs) that degrade it (12, 13). Mammals have several families of GCs (14, 15) and eight families of cGMP PDEs (16), each with distinct regulatory properties. Different PDE types are often coexpressed, but little is known about how they work together. cGMP signaling alters cell physiology by controlling cGMP-dependent protein kinases (PKG) (17, 18), cGMP-gated channels (CNGC) (19), and cGMP-regulated PDEs (12). These cGMP effectors can also feed back to control cGMP dynamics.cGMP is a major second messenger in Caenorhabditis elegans, implicated in the function of a third of its sensory neurons, including thermosensory, olfactory, gustatory, and O₂-sensing neurons (20). Genetic and behavioral studies suggest that cGMP mediates sensory transduction in many of these neurons (21–25). Despite this pervasiveness, cGMP has not been visualized in any C. elegans cell: genetic inferences about its roles in signal transduction are untested, and we have no mechanistic insights into cGMP signaling dynamics and feedback control. Consistent with the prominence of cGMP signaling in the nematode, the C. elegans genome encodes 34 GCs (26), six PDE genes, at least one PKG (24, 27, 28), and six CNGC subunits (19, 21, 22, 29–31).Here, we use cGMP and Ca²⁺ sensors to visualize and dissect cGMP signaling dynamics in a C. elegans O₂ sensor. We image single and double mutants defective in a soluble guanylate cyclase (sGC), CNGC subunits, PDE-1, PDE-2, and PKG. Our results reveal a signaling network of interwoven checks and balances. Counterintuitively, cGMP activation of PDE-2 promotes cGMP accumulation by controlling gating of CNGC and limiting Ca²⁺-mediated negative feedback, including activation of PDE-1. We show that cGMP signal transduction is highly reliable when the same individual is stimulated repeatedly but, surprisingly, is highly variable across genetically identical, similarly reared animals. Finally, simultaneous imaging of O₂-evoked cGMP and Ca²⁺ responses suggests that cGMP dynamics can differ in distinct subcellular compartments of a C. elegans neuron, consistent with the existence of cGMP nanodomains.     

Calcium levels in the Golgi complex regulate clustering and apical sorting of GPI-APs in polarized epithelial cells

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are lipid-associated luminal secretory cargoes selectively sorted to the apical surface of the epithelia where they reside and play diverse vital functions. Cholesterol-dependent clustering of GPI-APs in the Golgi is the key step driving their apical sorting and their further plasma membrane organization and activity; however, the specific machinery involved in this Golgi event is still poorly understood. In this study, we show that the formation of GPI-AP homoclusters (made of single GPI-AP species) in the Golgi relies directly on the levels of calcium within cisternae. We further demonstrate that the TGN calcium/manganese pump, SPCA1, which regulates the calcium concentration within the Golgi, and Cab45, a calcium-binding luminal Golgi resident protein, are essential for the formation of GPI-AP homoclusters in the Golgi and for their subsequent apical sorting. Down-regulation of SPCA1 or Cab45 in polarized epithelial cells impairs the oligomerization of GPI-APs in the Golgi complex and leads to their missorting to the basolateral surface. Overall, our data reveal an unexpected role for calcium in the mechanism of GPI-AP apical sorting in polarized epithelial cells and identify the molecular machinery involved in the clustering of GPI-APs in the Golgi.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are localized on the apical surface of most epithelia, where they exert their physiological functions, which are regulated by their spatiotemporal compartmentalization.In polarized epithelial cells, the organization of GPI-APs at the apical surface is driven by the mechanism of apical sorting, which relies on the formation of GPI-AP homoclusters in the Golgi apparatus (1, 2). GPI-AP homoclusters (containing a single GPI-AP species) form uniquely in the Golgi apparatus of fully polarized cells (and not in nonpolarized cells) in a cholesterol-dependent manner (1, 3, 4). Once formed, GPI-AP homoclusters become insensitive to cholesterol depletion, suggesting that protein–protein interactions stabilize them (1, 2). At the apical membrane, newly arrived homoclusters coalesce into heteroclusters (containing at least two different GPI-AP species) that are sensitive to cholesterol depletion (1). Of importance, in the absence of homoclustering in the Golgi (e.g., in nonpolarized epithelial cells), GPI-APs remain in the form of monomers and dimers and do not cluster at the cell surface (1, 5). Thus, the organization of GPI-APs at the apical plasma membrane of polarized cells strictly depends on clustering mechanisms in the Golgi apparatus allowing their apical sorting. This is different from what was shown in fibroblasts where clustering of GPI-APs occurs from monomer condensation at the plasma membrane, indicating that distinct mechanisms regulate GPI-AP clustering in polarized epithelial cells and fibroblasts (1, 6, 7). Furthermore, in polarized epithelial cells, the spatial organization of clusters also appears to regulate the biological activity of the proteins (1) so that GPI-APs are fully functional only when properly sorted to the apical surface and less active in the case of missorting to the basolateral domain (1, 8, 9). Understanding the mechanism of GPI-AP apical sorting in the Golgi apparatus is therefore crucial to decipher their organization at the plasma membrane and the regulation of their activity. The determinants for protein apical sorting have been difficult to uncover compared to the ones for basolateral sorting (10–14). Besides a role of cholesterol, the molecular factors regulating the clustering-based mechanism of GPI-AP sorting in polarized epithelial cells are unknown. Here, we analyzed the possible role of the actin cytoskeleton and of calcium levels in the Golgi. The actin cytoskeleton is not only critical for the maintenance of the Golgi structure and its mechanical properties but also provides the structural support favoring carrier biogenesis (15–18). The Golgi exit of various cargoes is altered in cells treated with drugs either depolymerizing or stabilizing actin filaments (19, 20), and the post-Golgi trafficking is affected either by the knockdown of the expression of some actin-binding proteins, which regulate actin dynamics, or by the overexpression of their mutants (12, 21–23), all together revealing the critical role of actin dynamics for protein trafficking. Only few studies have shown the involvement of actin remodeling proteins in polarized trafficking, mostly in selectively mediating the apical and basolateral trafficking of transmembrane proteins [refs. 24–26; and reviewed in ref. 27]; thus, it remains unclear whether actin filaments play a role in protein sorting in polarized cells.On the other hand, the Golgi apparatus exhibits high calcium levels that have been revealed to be essential for protein processing and the sorting of some secreted soluble proteins in nonpolarized cells (28–31). Moreover, a functional interplay between the actin cytoskeleton and Golgi calcium in modulating protein sorting in nonpolarized cells has been shown (22).In this study, we report that in epithelial cells, actin perturbation does not impair GPI-AP clustering capacity in the Golgi and therefore their apical sorting. In contrast, we found that the Golgi organization of GPI-APs is drastically perturbed upon calcium depletion and that the amount of calcium in the Golgi cisternae is critical for the formation of GPI-AP homoclusters. We further show that the TGN calcium/manganese pump, SPCA1 (secretory pathway Ca(2+)-ATPase pump type 1), which controls the Golgi calcium concentration (32), and Cab45, a calcium-binding luminal Golgi resident protein previously described to be involved in the sorting of a subset of soluble cargoes (33, 34), are essential for the formation of GPI-APs homoclusters in the Golgi and for their subsequent apical sorting. Indeed, down-regulation of SPCA1 or Cab45 expression impairs the oligomerization of GPI-APs in the Golgi complex and leads to their missorting to the basolateral surface but does not affect apical or basolateral transmembrane proteins. Overall, our data reveal an unexpected role for calcium in the mechanism of GPI-AP apical sorting in polarized epithelial cells and identify the molecular machinery involved in the clustering of GPI-APs in the Golgi.     

Two-dimensional electronic–vibrational sum frequency spectroscopy for interactions of electronic and nuclear motions at interfaces

Interactions of electronic and vibrational degrees of freedom are essential for understanding excited-states relaxation pathways of molecular systems at interfaces and surfaces. Here, we present the development of interface-specific two-dimensional electronic–vibrational sum frequency generation (2D-EVSFG) spectroscopy for electronic–vibrational couplings for excited states at interfaces and surfaces. We demonstrate this 2D-EVSFG technique by investigating photoexcited interface-active (E)-4-((4-(dihexylamino) phenyl)diazinyl)-1-methylpyridin-1- lum (AP3) molecules at the air–water interface as an example. Our 2D-EVSFG experiments show strong vibronic couplings of interfacial AP3 molecules upon photoexcitation and subsequent relaxation of a locally excited (LE) state. Time-dependent 2D-EVSFG experiments indicate that the relaxation of the LE state, S₂, is strongly coupled with two high-frequency modes of 1,529.1 and 1,568.1 cm⁻¹. Quantum chemistry calculations further verify that the strong vibronic couplings of the two vibrations promote the transition from the S₂ state to the lower excited state S₁. We believe that this development of 2D-EVSFG opens up an avenue of understanding excited-state dynamics related to interfaces and surfaces.

Electronic and vibrational degrees of freedom are the most important physical quantities in molecular systems at interfaces and surfaces. Knowledge of interactions between electronic and vibrational motions, namely electronic–vibrational couplings, is essential to understanding excited-states relaxation pathways of molecular systems at interfaces and surfaces. Many excited-states relaxation processes occur at interfaces and surfaces, including charge transfer, energy transfer, proton transfer, proton-coupled electron transfer, configurational dynamics, and so on (1–11). These relaxation processes are intimately related to the electronic–vibrational couplings at interfaces and surfaces. Strong electronic–vibrational couplings could promote nonadiabatic evolution of excited potential energy and thus, facilitate chemical reactions or intramolecular structural changes of interfacial molecules (10, 12, 13). Furthermore, these interactions of electronic and vibrational degrees of freedom are subject to solvent environments (e.g., interfaces/surfaces with a restricted environment of unique physical and chemical properties) (9, 14, 15). Despite the importance of interactions of electronic and vibrational motions, little is known about excited-state electronic–vibrational couplings at interfaces and surfaces.Interface-specific electronic and vibrational spectroscopies enable us to characterize the electronic and vibrational structures separately. As interface-specific tools, second-order electronic sum frequency generation (ESFG) and vibrational sum frequency generation (VSFG) spectroscopies have been utilized for investigating molecular structure, orientational configurations, chemical reactions, chirality, static potential, environmental issues, and biological systems at interfaces and surfaces (16–52). Recently, structural dynamics at interfaces and surfaces have been explored using time-resolved ESFG and time-resolved VSFG with a visible pump or an infrared (IR) pump thanks to the development of ultrafast lasers (6–9, 13–15, 49, 53–61). Doubly resonant sum frequency generation (SFG) has been demonstrated to probe both electronic and vibration transitions of interfacial molecular monolayer (15, 62–71). This frequency-domain two-dimensional (2D) interface/surface spectroscopy could provide information regarding electronic–vibrational coupling of interfacial molecules. However, contributions from excited states are too weak to be probed due to large damping rates of vibrational states in excited states (62, 63). As such, the frequency-domain doubly resonant SFG is used only for electronic–vibrational coupling of electronic ground states. Ultrafast interface-specific electronic–vibrational spectroscopy could allow us to gain insights into how specific nuclear motions drive the relaxation of electronic excited states. Therefore, development of interface-specific electronic–vibrational spectroscopy for excited states is needed.In this work, we integrate the specificity of interfaces and surfaces into the capabilities of ultrafast 2D spectroscopy for dynamical electronic–vibrational couplings in excited states of molecules; 2D interface-specific spectroscopies are analogous to those 2D spectra in bulk that spread the information contained in a pump−probe spectrum over two frequency axes. Thus, one can better interpret congested one-dimensional signals. Two-dimensional vibrational sum frequency generation (2D-VSFG) spectroscopy was demonstrated a few year ago (72–74). Furthermore, heterodyne 2D-VSFG spectroscopy using middle infrared (mid-IR) pulse shaping and noncollinear geometry 2D-VSFG experiments have also been developed to study vibrational structures and dynamics at interfaces (31, 75–78). Recently, two-dimensional electronic sum frequency generation (2D-ESFG) spectroscopy has also been demonstrated for surfaces and interfaces (79). On the other hand, bulk two-dimensional electronic–vibrational (2D-EV) spectroscopy has been extensively used to investigate the electronic relaxation and energy transfer dynamics of molecules, biological systems, and nanomaterials (80–90). The 2D-EV technique not only provides electronic and vibrational interactions between excitons or different excited electronic states of systems but also, identifies fast nonradiative transitions through nuclear motions in molecules, aggregations, and nanomaterials. However, an interface-specific technique for two-dimensional electronic–vibrational sum frequency generation (2D-EVSFG) spectroscopy has yet to be developed.Here, we present the development of 2D-EVSFG spectroscopy for the couplings of electronic and nucleic motions at interfaces and surfaces. The purpose of developing 2D-EVSFG spectroscopy is to bridge the gap between the visible and IR regions to reveal how structural dynamics for photoexcited electronic states are coupled with vibrations at interfaces and surfaces. As an example, we applied this 2D-EVSFG experimental method to time evolution of electronic–vibrational couplings at excited states of interface-active molecules at the air–water interface.     

Direct single-molecule observation of calcium-dependent misfolding in human neuronal calcium sensor-1

Neurodegenerative disorders are strongly linked to protein misfolding, and crucial to their explication is a detailed understanding of the underlying structural rearrangements and pathways that govern the formation of misfolded states. Here we use single-molecule optical tweezers to monitor misfolding reactions of the human neuronal calcium sensor-1, a multispecific EF-hand protein involved in neurotransmitter release and linked to severe neurological diseases. We directly observed two misfolding trajectories leading to distinct kinetically trapped misfolded conformations. Both trajectories originate from an on-pathway intermediate state and compete with native folding in a calcium-dependent manner. The relative probability of the different trajectories could be affected by modulating the relaxation rate of applied force, demonstrating an unprecedented real-time control over the free-energy landscape of a protein. Constant-force experiments in combination with hidden Markov analysis revealed the free-energy landscape of the misfolding transitions under both physiological and pathological calcium concentrations. Remarkably for a calcium sensor, we found that higher calcium concentrations increased the lifetimes of the misfolded conformations, slowing productive folding to the native state. We propose a rugged, multidimensional energy landscape for neuronal calcium sensor-1 and speculate on a direct link between protein misfolding and calcium dysregulation that could play a role in neurodegeneration.Most proteins have evolved to fold rapidly into a specific and functional 3D structure immediately after translation from the ribosome. The folding process is, however, not adequately efficient to prevent the occurrence of misfolded states in vivo (1), especially in the case of larger multidomain proteins which comprise roughly 75% of the human proteome (2, 3). Normally, to tackle and destroy these unproductive structures, cells are equipped with competent clean-up machinery, such as chaperones, proteasomes, and unfoldases (4). If misfolding cannot be ameliorated, these nonnative states accumulate in the cell to form aggregates with potential pathophysiological consequences (5).The emerging view that protein misfolding is a common phenomenon in living cells is still largely unsubstantiated, as detecting and characterizing misfolded states has been experimentally challenging (2, 6). The mechanistic details that have accumulated over the last decades on misfolding have mostly come from studies on the resulting oligomeric structures and amyloid formation (1), whereas our understanding of the structural rearrangements and pathways leading to precursory misfolded states is still highly incomplete. Importantly, the formation of prefibrillar monomeric and oligomeric misfolded states is, contrary to amyloids, reversible and thus these states provide a potential target for drug design.Sparse populations and their associated weak signals limit the use of traditional bulk methods for monitoring the early events of misfolding, and relatively few systems have been studied in detail (7–13). Now, single-molecule force spectroscopy techniques, such as optical tweezers, enable detection of rare alternative folding pathways and short-lived misfolded states by direct mechanical manipulation (14–19). Although aggregation requires more than one molecule, nonnative structural rearrangements within a single molecule only report on monomeric misfolded states. Recent works have exploited these properties to study misfolding of well-known disease-related proteins, such as the prion protein, as well as proteins not generally associated with misfolding, such as the EF-hand calcium sensor calmodulin (CaM) (20–22).The EF-hand superfamily of calcium sensors is responsible for translating changing levels of intracellular Ca²⁺ concentration into a biochemical signal through conformational changes that allow them to interact with an array of binding targets (23). The subfamily of neuronal calcium sensors (NCS) is mostly expressed in neurons and currently includes 15 members (24, 25). Neuronal calcium sensor-1 (NCS-1) is the most ancient member of this family (Fig. 1A), and it has been functionally associated with cognitive processes, such as learning and memory (26, 27), and with a number of cellular processes such as neurotransmitter release (28, 29), and regulation of ion channels, and G protein coupled receptors (GPCRs) (24, 30), including the dopamine receptor D2 (31). NCS-1 has also been linked to serious neurodegenerative disorders including schizophrenia, bipolar disorder (BD) (32), and autism (33, 34). However, the dysfunctions of NCS-1 are poorly characterized on the molecular level, and whether they involve altered functional profiles or loss of function due to formation of misfolded states is currently unknown.Open in a separate windowFig. 1.Misfolding pathways of NCS-1. (A) The NMR structure of NCS-1 (PDB 2LCP), with the N domain (EF1/EF2) depicted in gray and the C domain (EF3/EF4) in blue. Black spheres represent Ca²⁺ ions. EF1 does not bind Ca²⁺ because of a conserved cysteine-proline mutation (35). (B) Sketch of the experimental setup. NCS-1 was tethered between functionalized beads via DNA handles and stretched and relaxed by moving the pipette relative to the optical trap (16, 62). (C) Native folding pathway of NCS-1. After being mechanically stretched and unfolded (red trace), NCS-1 refolds upon relaxation of the applied force into its native state via two intermediate states, I1 and I2. Dashed lines are worm-like-chain fits to the data. Color-coded arrows indicate the pulling/relaxing directions. (D) Misfolding pathways of NCS-1. During refolding, NCS-1 sometimes follows alternative pathways leading to misfolded states M1 (blue) or M2 (green), which are less compact than the native state (red). Dashed lines are worm-like-chain fits to the data. (E) Rescue of the native state of NCS-1. During relaxation (black), the molecule misfolded. During stretching (red), nonnative contacts of the misfolded conformation are progressively broken, until the molecule can find its native folding pathway (“rescue transition”). (F) Fraction of folding pathways leading to either M1 or M2 as a function of Ca²⁺ concentration and relaxation speed. Higher Ca²⁺ concentrations and relaxation speeds facilitate NCS-1 misfolding. Error bars indicate SEs of mean. At least five different molecules were used for each calcium concentration.Because only a few systems have been studied experimentally, little is known about folding and/or misfolding mechanisms of members of the EF-hand superfamily (21, 35–37). The extensively studied CaM has been shown on the single-molecule level to frequently visit misfolded states that slow down the overall folding rate of the protein (21). The physiological consequences of CaM misfolding have not yet been explored. NCS-1 shares modest sequence homology with CaM, mostly within and around the calcium binding sites (24). Similar to CaM, NCS-1 contains four EF hands organized in two EF domains (Fig. 1A) yet it exhibits a larger number of interdomain contacts (38), a feature that has been suggested to increase the probability of misfolding in proteins (2). The formation of misfolded states along the folding pathway of NCS-1 may have important consequences with regards to its function as a calcium sensor and might also play a role in disease pathologies.Using optical tweezers, we have recently characterized the native folding pathway of NCS-1 (39). Here we use a similar experimental approach to monitor individual NCS-1 molecules as they populate nonnative misfolded states in real time. We identified two misfolding trajectories leading to two distinct misfolded conformations, characterized by different extensions and different pathways on the energy landscape. Both misfolding pathways originated from a partially folded on-pathway intermediate state, and they competed with native folding. The occupancy probability of both misfolded states could be controlled by modulating either the relaxation rate of the applied force and/or the calcium concentration. Remarkably for a calcium sensor, higher calcium concentrations, even within physiologically relevant conditions, lead to an increased probability of NCS-1 misfolding.     

The spontaneous emergence of conventions: An experimental study of cultural evolution

How do shared conventions emerge in complex decentralized social systems? This question engages fields as diverse as linguistics, sociology, and cognitive science. Previous empirical attempts to solve this puzzle all presuppose that formal or informal institutions, such as incentives for global agreement, coordinated leadership, or aggregated information about the population, are needed to facilitate a solution. Evolutionary theories of social conventions, by contrast, hypothesize that such institutions are not necessary in order for social conventions to form. However, empirical tests of this hypothesis have been hindered by the difficulties of evaluating the real-time creation of new collective behaviors in large decentralized populations. Here, we present experimental results—replicated at several scales—that demonstrate the spontaneous creation of universally adopted social conventions and show how simple changes in a population’s network structure can direct the dynamics of norm formation, driving human populations with no ambition for large scale coordination to rapidly evolve shared social conventions.Social conventions are the foundation for social and economic life (1–7), However, it remains a central question in the social, behavioral, and cognitive sciences to understand how these patterns of collective behavior can emerge from seemingly arbitrary initial conditions (2–4, 8, 9). Large populations frequently manage to coordinate on shared conventions despite a continuously evolving stream of alternatives to choose from and no a priori differences in the expected value of the options (1, 3, 4, 10). For instance, populations are able to produce linguistic conventions on accepted names for children and pets (11), on common names for colors (12), and on popular terms for novel cultural artifacts, such as referring to junk email as “SPAM” (13, 14). Similarly, economic conventions, such as bartering systems (2), beliefs about fairness (3), and consensus regarding the exchangeability of goods and services (15), emerge with clear and widespread agreement within economic communities yet vary broadly across them (3, 16).Prominent theories of social conventions suggest that institutional mechanisms—such as centralized authority (14), incentives for collective agreement (15), social leadership (16), or aggregated information (17)—can explain global coordination. However, these theories do not explain whether, or how, it is possible for conventions to emerge when social institutions are not already in place to guide the process. A compelling alternative approach comes from theories of social evolution (2, 18–20). Social evolutionary theories maintain that networks of locally interacting individuals can spontaneously self-organize to produce global coordination (21, 22). Although there is widespread interest in this approach to social norms (6, 7, 14, 18, 23–26), the complexity of the social process has prevented systematic empirical insight into the thesis that these local dynamics are sufficient to explain universally adopted conventions (27, 28).Several difficulties have limited prior empirical research in this area. The most notable of these limitations is scale. Although compelling experiments have successfully shown the creation of new social conventions in dyadic and small group interactions (29–31), the results in small group settings can be qualitatively different from the dynamics in larger groups (Model), indicating that small group experiments are insufficient for demonstrating whether or how new conventions endogenously form in larger populations (32, 33). Important progress on this issue has been made using network-based laboratory experiments on larger groups (15, 24). However, this research has been restricted to studying coordination among players presented with two or three options with known payoffs. Natural convention formation, by contrast, is significantly complicated by the capacity of individuals to continuously innovate, which endogenously expands the “ecology” of alternatives under evaluation (23, 29, 31). Moreover, prior experimental studies have typically assumed the existence of either an explicit reward for universal coordination (15) or a mechanism that aggregates and reports the collective state of the population (17, 24), which has made it impossible to evaluate the hypothesis that global coordination is the result of purely local incentives.More recently, data science approaches to studying norms have addressed many of these issues by analyzing behavior change in large online networks (34). However, these observational studies are limited by familiar problems of identification that arise from the inability to eliminate the confounding influences of institutional mechanisms. As a result, previous empirical research has been unable to identify the collective dynamics through which social conventions can spontaneously emerge (8, 34–36).We addressed these issues by adopting a web-based experimental approach. We studied the effects of social network structure on the spontaneous evolution of social conventions in populations without any resources to facilitate global coordination (9, 37). Participants in our study were rewarded for coordinating locally, however they had neither incentives nor information for achieving large scale agreement. Further, to eliminate any preexisting bias in the evolutionary process, we studied the emergence of arbitrary linguistic conventions, in which none of the options had any a priori value or advantage over the others (3, 23). In particular, we considered the prototypical problem of whether purely local interactions can trigger the emergence of a universal naming convention (38, 39).     

Fine-tuning synaptic plasticity by modulation of CaV2.1 channels with Ca2+ sensor proteins

Modulation of P/Q-type Ca²⁺ currents through presynaptic voltage-gated calcium channels (Ca_V2.1) by binding of Ca²⁺/calmodulin contributes to short-term synaptic plasticity. Ca²⁺-binding protein-1 (CaBP1) and Visinin-like protein-2 (VILIP-2) are neurospecific calmodulin-like Ca²⁺ sensor proteins that differentially modulate Ca_V2.1 channels, but how they contribute to short-term synaptic plasticity is unknown. Here, we show that activity-dependent modulation of presynaptic Ca_V2.1 channels by CaBP1 and VILIP-2 has opposing effects on short-term synaptic plasticity in superior cervical ganglion neurons. Expression of CaBP1, which blocks Ca²⁺-dependent facilitation of P/Q-type Ca²⁺ current, markedly reduced facilitation of synaptic transmission. VILIP-2, which blocks Ca²⁺-dependent inactivation of P/Q-type Ca²⁺ current, reduced synaptic depression and increased facilitation under conditions of high release probability. These results demonstrate that activity-dependent regulation of presynaptic Ca_V2.1 channels by differentially expressed Ca²⁺ sensor proteins can fine-tune synaptic responses to trains of action potentials and thereby contribute to the diversity of short-term synaptic plasticity.Neurons fire repetitively in different frequencies and patterns, and activity-dependent alterations in synaptic strength result in diverse forms of short-term synaptic plasticity that are crucial for information processing in the nervous system (1–3). Short-term synaptic plasticity on the time scale of milliseconds to seconds leads to facilitation or depression of synaptic transmission through changes in neurotransmitter release. This form of plasticity is thought to result from residual Ca²⁺ that builds up in synapses during repetitive action potentials and binds to a Ca²⁺ sensor distinct from the one that evokes neurotransmitter release (1, 2, 4, 5). However, it remains unclear how changes in residual Ca²⁺ cause short-term synaptic plasticity and how neurotransmitter release is regulated to generate distinct patterns of short-term plasticity.In central neurons, voltage-gated calcium (Ca_V2.1) channels are localized in high density in presynaptic active zones where their P/Q-type Ca²⁺ current triggers neurotransmitter release (6–11). Because synaptic transmission is proportional to the third or fourth power of Ca²⁺ entry through presynaptic Ca_V2.1 channels, small changes in Ca²⁺ current have profound effects on synaptic transmission (2, 12). Studies at the calyx of Held synapse have provided important insights into the contribution of presynaptic Ca²⁺ current to short-term synaptic plasticity (13–17). Ca_V2.1 channels are required for synaptic facilitation, and Ca²⁺-dependent facilitation and inactivation of the P/Q-type Ca²⁺ currents are correlated temporally with synaptic facilitation and rapid synaptic depression (13–17).Molecular interactions between Ca²⁺/calmodulin (CaM) and Ca_V2.1 channels induce sequential Ca²⁺-dependent facilitation and inactivation of P/Q-type Ca²⁺ currents in nonneuronal cells (18–21). Facilitation and inactivation of P/Q-type currents are dependent on Ca²⁺/CaM binding to the IQ-like motif (IM) and CaM-binding domain (CBD) of the Ca_V2.1 channel, respectively (20, 21). This bidirectional regulation serves to enhance channel activity in response to short bursts of depolarizations and then to decrease activity in response to long bursts. In synapses of superior cervical ganglion (SCG) neurons expressing exogenous Ca_V2.1 channels, synaptic facilitation is induced by repetitive action potentials, and mutation of the IM and CBD motifs prevents synaptic facilitation and inhibits the rapid phase of synaptic depression (22). Thus, in this model synapse, regulation of presynaptic Ca_V2.1 channels by binding of Ca²⁺/CaM can contribute substantially to the induction of short-term synaptic plasticity by residual Ca²⁺.CaM is expressed ubiquitously, but short-term plasticity has great diversity among synapses, and the potential sources of this diversity are unknown. How could activity-dependent regulation of presynaptic Ca_V2.1 channels contribute to the diversity of short-term synaptic plasticity? CaM is the founding member of a large family of Ca²⁺ sensor (CaS) proteins that are differentially expressed in central neurons (23–25). Two CaS proteins, Ca²⁺-binding protein-1 (CaBP1) and Visinin-like protein-2(VILIP-2), modulate facilitation and inactivation of Ca_V2.1 channels in opposite directions through interaction with the bipartite regulatory site in the C-terminal domain (26, 27), and they have varied expression in different types of central neurons (23, 25, 28). CaBP1 strongly enhances inactivation and prevents facilitation of Ca_V2.1 channel currents, whereas VILIP-2 slows inactivation and enhances facilitation of Ca_V2.1 currents during trains of stimuli (26, 27). Molecular analyses show that the N-terminal myristoylation site and the properties of individual EF-hand motifs in CaBP1 and VILIP-2 determine their differential regulation of Ca_V2.1 channels (27, 29–31). However, the role of CaBP1 and VILIP-2 in the diversity of short-term synaptic plasticity is unknown, and the high density of Ca²⁺ channels and unique Ca²⁺ dynamics at the presynaptic active zone make extrapolation of results from studies in nonneuronal cells uncertain. We addressed this important question directly by expressing CaBP1 and VILIP-2 in presynaptic SCG neurons and analyzing their effects on synaptic plasticity. Our results show that CaM-related CaS proteins can serve as sensitive bidirectional switches that fine-tune the input–output relationships of synapses depending on their profile of activity and thereby maintain the balance of facilitation versus depression by the regulation of presynaptic Ca_V2.1 channels.     

Membrane translocation of TRPC6 channels and endothelial migration are regulated by calmodulin and PI3 kinase activation

Lipid oxidation products, including lysophosphatidylcholine (lysoPC), activate canonical transient receptor potential 6 (TRPC6) channels leading to inhibition of endothelial cell (EC) migration in vitro and delayed EC healing of arterial injuries in vivo. The precise mechanism through which lysoPC activates TRPC6 channels is not known, but calmodulin (CaM) contributes to the regulation of TRPC channels. Using site-directed mutagenesis, cDNAs were generated in which Tyr⁹⁹ or Tyr¹³⁸ of CaM was replaced with Phe, generating mutant CaM, Phe⁹⁹-CaM, or Phe¹³⁸-CaM, respectively. In ECs transiently transfected with pcDNA3.1-myc-His-Phe⁹⁹-CaM, but not in ECs transfected with pcDNA3.1-myc-His-Phe¹³⁸-CaM, the lysoPC-induced TRPC6-CaM dissociation and TRPC6 externalization was disrupted. Also, the lysoPC-induced increase in intracellular calcium concentration was inhibited in ECs transiently transfected with pcDNA3.1-myc-His-Phe⁹⁹-CaM. Blocking phosphorylation of CaM at Tyr⁹⁹ also reduced CaM association with the p85 subunit and subsequent activation of phosphatidylinositol 3-kinase (PI3K). This prevented the increase in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and the translocation of TRPC6 to the cell membrane and reduced the inhibition of EC migration by lysoPC. These findings suggest that lysoPC induces CaM phosphorylation at Tyr⁹⁹ by a Src family kinase and that phosphorylated CaM activates PI3K to produce PIP3, which promotes TRPC6 translocation to the cell membrane.Endothelial cell (EC) migration is required for healing after arterial injuries, such as those that occur with angioplasties. Oxidized low-density lipoprotein and lysophosphatidylcholine (lysoPC), the major lysophospholipid of oxidized low-density lipoprotein, are abundant in plasma and atherosclerotic lesions and inhibit EC migration (1). A brief influx of calcium is required to initiate EC migration (2), but lysoPC causes a prolonged influx of Ca²⁺ that disrupts the cytoskeletal dynamics required for normal EC migration (3, 4). Specifically, lysoPC activates canonical transient receptor potential 6 (TRPC6) channels, as shown by patch clamp recording, with Ca²⁺ influx (3). The increased [Ca²⁺]_i initiates events that result in TRPC5 channel activation (3). The later activation of TRPC5 compared with TRPC6 and the failure of TRPC6 and TRPC5 to coimmunoprecipitate indicates that they do not form a heteromeric complex. The importance of this pathway is found in TRPC6-deficient EC, where lysoPC has little effect on EC migration (3). Furthermore, a high cholesterol diet markedly inhibits endothelial healing in wild-type (WT) mice, but has no effect in TRPC6-deficient (TRPC6^−/−) mice (5). The mechanism of TRPC6 activation by lysoPC is not fully elucidated, limiting the ability to block this important pathway.Calmodulin (CaM), a small, highly conserved, intracellular calcium-binding protein (6), binds to TRPC channels and regulates their activation. TRPC proteins, including TRPC6, possess a C-terminal CaM-binding domain that overlaps with a binding site for the inositol trisphosphate receptor, and CaM and the inositol triphosphate receptor compete for binding at this site (7). Removal of CaM from the common binding site results in activation of TRP3 channels (8). TRPC proteins contain additional binding sites for CaM and other Ca²⁺-binding proteins, indicating a complex regulatory mechanism in response to changes in [Ca²⁺]_i that includes positive and negative regulation of channels (9). In addition to CaM regulating TRPC proteins by direct binding, CaM-dependent kinases activate TRPC channels (10). CaM activity and peptide binding affinity is altered by its phosphorylation state and bound Ca²⁺, and Ca²⁺ can regulate the phosphorylation of CaM (11). LysoPC activates tyrosine kinases, including Src family tyrosine kinases (12), and Src family kinases can phosphorylate CaM (13). The role of CaM and CaM phosphorylation in TRPC6 channel activation or in EC migration is incompletely understood.TRPC6 channel activation generally requires externalization; however, the mechanism of TRPC6 channel translocation to the plasma membrane is not clear. In HEK cells overexpressing TRPC6, stimulation of Gq protein-coupled receptors causes TRPC6 externalization and localization to caveolae or lipid rafts by an exocytotic mechanism (14). In smooth muscle cells, phosphatidylinositol 3-kinase (PI3K) is involved in carbachol-induced TRPC6 externalization (15). The mechanism by which lysoPC induces TRPC6 externalization in EC is unknown.PI3K produces phosphatidylinositol (3,4,5)-trisphosphate (PIP3) from phosphatidylinositol (4,5)-bisphosphate (PIP2) in the inner leaflet of the plasma membrane and participates in numerous intracellular signaling processes, including TRPC6 activation (16). PI3K is composed of a p85 regulatory subunit (p85α, p85β, or p55γ) and a p110 catalytic subunit (p110α, p110β, p110γ, or p110δ), and activity can be influenced by CaM association (17).The purpose of the present study is to explore the underlying mechanism of lysoPC-induced TRPC6 activation. We identify a mechanism in which phosphorylation of CaM at Tyr⁹⁹ plays a key role in lysoPC-induced TRPC6 externalization and inhibition of EC migration.     

Symmetric activation and modulation of the human calcium-sensing receptor

The human extracellular calcium-sensing (CaS) receptor controls plasma Ca²⁺ levels and contributes to nutrient-dependent maintenance and metabolism of diverse organs. Allosteric modulation of the CaS receptor corrects disorders of calcium homeostasis. Here, we report the cryogenic-electron microscopy reconstructions of a near–full-length CaS receptor in the absence and presence of allosteric modulators. Activation of the homodimeric CaS receptor requires a break in the transmembrane 6 (TM6) helix of each subunit, which facilitates the formation of a TM6-mediated homodimer interface and expansion of homodimer interactions. This transformation in TM6 occurs without a positive allosteric modulator. Two modulators with opposite functional roles bind to overlapping sites within the transmembrane domain through common interactions, acting to stabilize distinct rotamer conformations of key residues on the TM6 helix. The positive modulator reinforces TM6 distortion and maximizes subunit contact to enhance receptor activity, while the negative modulator strengthens an intact TM6 to dampen receptor function. In both active and inactive states, the receptor displays symmetrical transmembrane conformations that are consistent with its homodimeric assembly.

Critical to the maintenance of Ca²⁺ homeostasis, the extracellular calcium-sensing (CaS) receptor was the first G protein–coupled receptor (GPCR) discovered to sense ions (1–3). The CaS receptor detects fluctuations in plasma Ca²⁺ at the parathyroid. In response to increases in Ca²⁺, it transmits signals to inhibit the release of parathyroid hormone, in turn preventing further rises in Ca²⁺ concentration (2, 3). In the cortical thick ascending limb of the renal nephron, the CaS receptor is also activated by surges in plasma Ca²⁺ and responds by inhibiting Ca²⁺ reabsorption. The excess urinary calcium excretion arising from CaS receptor activation lowers the plasma Ca²⁺ level. The CaS receptor is implicated in various pathologies associated with hypercalcemia and hypocalcemia (4). It has also been linked to the progression of diseases such as breast and colon cancer, in which the receptor modulates tumor growth (3, 5–7).The CaS receptor senses a diverse array of extracellular stimuli. During normal function, it activates multiple intracellular signaling pathways involving G_q/11, G_i/o, or G_12/13; in tumor cells, it is coupled to G_s (2, 3, 8, 9). In addition to the principal agonist Ca²⁺, the receptor is directly activated by aromatic l-amino acids (10, 11). Other CaS agonists include various divalent and trivalent cations (12), referred to as type I calcimimetics for mimicking the action of Ca²⁺ (13).The activity of the CaS receptor is also subject to allosteric modulation. Positive allosteric modulators (PAMs) are classified as type II calcimimetics for increasing the receptor sensitivity for Ca²⁺ (12–16). The prototypical PAM molecules share a phenylalkylamine structure, including cinacalcet and NPS R-568 (abbreviated as R-568). Cinacalcet was the first drug described to target a GPCR allosterically, and it is used clinically to treat hyperparathyroidism in patients with chronic kidney diseases (15). Negative allosteric modulators (NAMs) of the CaS receptor are referred to as calcilytics for suppressing the receptor response to Ca²⁺ (12–16). Synthetic calcilytics such as NPS-2143 and ronacaleret are also structurally related to phenylalkylamines. Recently, inorganic phosphate has been identified as an inhibitor of the receptor (11, 17).The CaS receptor rests within the class C family of GPCRs and functions as an obligate homodimer. Like other class C GPCRs, each CaS subunit contains a large extracellular domain (ECD) involved in orthosteric ligand binding, a seven-helix transmembrane (TM) domain responsible for G protein coupling, followed by an extended cytoplasmic tail (18–23). The conformations of the CaS ECDs in both the inactive and active states have been determined by X-ray crystallography (11, 24). The ECD structures also revealed how the receptor recognizes various extracellular ligands, including Ca²⁺, the amino acid l-Trp, and inorganic phosphate. Although the role of amino acids is still under debate (25), recent structural studies of full-length CaS receptor further confirmed that Ca²⁺ and amino acids cooperate to activate the receptor (26–28).The TM domain of the CaS receptor harbors the binding sites for PAM and NAM molecules according to previous mutagenesis studies (29–32). Recently reported modulator-bound CaS receptor structures revealed asymmetric TM configurations that are stabilized by PAM molecules binding in different poses within the separate subunits of the homodimer (33). We have determined PAM- and NAM-bound, as well as PAM-free, structures of a near–full-length CaS receptor using cryogenic-electron microscopy (cryo-EM) that display symmetric TM dimers and modulator poses, instead. This finding presents the possibility of receptor activation without requiring asymmetric conformational transition. Our structures also illustrate how distortion of TM6 provides the driving force for receptor activation. Furthermore, the presence of a PAM or NAM stabilizes distinct TM6 helix conformations to promote specific dimer arrangements and differentially modulate receptor function.     

Calcineurin determines toxic versus beneficial responses to α-synuclein

Calcineurin (CN) is a highly conserved Ca²⁺–calmodulin (CaM)-dependent phosphatase that senses Ca²⁺ concentrations and transduces that information into cellular responses. Ca²⁺ homeostasis is disrupted by α-synuclein (α-syn), a small lipid binding protein whose misfolding and accumulation is a pathological hallmark of several neurodegenerative diseases. We report that α-syn, from yeast to neurons, leads to sustained highly elevated levels of cytoplasmic Ca²⁺, thereby activating a CaM-CN cascade that engages substrates that result in toxicity. Surprisingly, complete inhibition of CN also results in toxicity. Limiting the availability of CaM shifts CN''s spectrum of substrates toward protective pathways. Modulating CN or CN''s substrates with highly selective genetic and pharmacological tools (FK506) does the same. FK506 crosses the blood brain barrier, is well tolerated in humans, and is active in neurons and glia. Thus, a tunable response to CN, which has been conserved for a billion years, can be targeted to rebalance the phosphatase’s activities from toxic toward beneficial substrates. These findings have immediate therapeutic implications for synucleinopathies.Cells must tightly regulate Ca²⁺ homeostasis to avoid pathological perturbations and cell death (1). For example, a profound disruption of Ca²⁺ homeostasis is seen in Parkinson disease (PD), the second most common neurodegenerative disorder. Mutations or aberrant expression of α-synuclein (α-syn), a major protein involved in the pathogenesis of PD, can induce Ca²⁺ overload and cell death (2–5). Additional clinical and experimental observations highlight the importance of Ca²⁺ homeostasis in the pathogenesis of PD. Midbrain dopaminergic (DA) neurons that overexpress Ca²⁺-binding proteins, which buffer intracellular Ca²⁺, are characteristically spared from degeneration (6). Patients with hypertension who are treated with the L-type Ca²⁺ channel blocker, isradipine, have a lower incidence of PD (7). Moreover, isradipine protects DA neurons incubated with α-syn fibrils and is protective in animal models of toxin-induced PD (8–10).From yeast to mammals, calcineurin is largely responsible for transducing the signals generated by changes in Ca²⁺ levels (11). Calcineurin (CN) is a calmodulin (CaM)-dependent serine/threonine phosphatase composed of a catalytic subunit (calcineurin A, CNA) and an activating regulatory subunit (calcineurin B, CNB). As intracellular Ca²⁺ levels rise, Ca²⁺ binds to CNB and CaM, another key calcium signaling protein. Together, Ca²⁺-bound CNB and CaM bind CNA, inducing a conformational change that fully activates the phosphatase (11). Signaling through CN plays critical roles in processes ranging from stress response survival in yeast (12) to mammalian development (13).Despite the compelling link between Ca²⁺ homeostasis and PD, we know little about the signaling pathways driven by sustained Ca²⁺ elevations and how they might lead to cell death (4, 5). Yeast provide a powerful model system for such investigations, given their genetic tractability and the remarkable conservation of Ca²⁺-signaling pathways from yeast to humans (14, 15). Moreover, the expression of human α-syn in yeast leads to cellular pathologies directly relevant to neurons and PD, including nitrosative stress (16, 17), defects in vesicle trafficking (18–20), and faulty mitochondrial function (21, 22).     

Single-molecule optofluidic microsensor with interface whispering gallery modes

Label-free sensors are highly desirable for biological analysis and early-stage disease diagnosis. Optical evanescent sensors have shown extraordinary ability in label-free detection, but their potentials have not been fully exploited because of the weak evanescent field tails at the sensing surfaces. Here, we report an ultrasensitive optofluidic biosensor with interface whispering gallery modes in a microbubble cavity. The interface modes feature both the peak of electromagnetic-field intensity at the sensing surface and high-Q factors even in a small-sized cavity, enabling a detection limit as low as 0.3 pg/cm². The sample consumption can be pushed down to 10 pL due to the intrinsically integrated microfluidic channel. Furthermore, detection of single DNA with 8 kDa molecular weight is realized by the plasmonic-enhanced interface mode.

Detecting biological molecules and monitoring their dynamics are of crucial importance in biomedical analysis and disease diagnosis (1, 2). Practical applications generally involve complex biological environments, in which an engineered interface is highly desirable to enable the enrichment, detection, and analysis of specific biomolecules (3). Over the past decades, many techniques on interfacial molecular analysis have been developed, such as lateral flow immunoassay, electrochemical analytical techniques, and optical biosensors (4–7). Among them, optical evanescent microsensors, such as microspheres (8, 9), microtoroids (10–14), and nanowaveguides (15–18), have attracted considerable research interest since they can detect unlabeled molecules and monitor their interactions in real time and in situ with ultrahigh sensitivity, fast response, and miniature footprint.Despite these advantages, potentials of optical evanescent microsensors have not been fully explored. With the peak field intensity confined inside the cavity, these sensors can utilize the weak tail of the evanescent field only on the sensing surface (8, 10–12, 15, 16), thus limiting their sensitivities. Moreover, ultrasmall sample consumption is desired for high-efficiency sensing yet challenging in evanescent microsensors, since they require delicate sample delivery designs such as an additional chamber (9, 12) or a precisely aligned fluidic channel (19, 20). Therefore, an integrated microfluidic platform with ultimate sensitivity is highly demanded.In this work, we demonstrate an ultrasensitive optofluidic microbubble biosensor by exploiting the whispering gallery modes (WGMs) peaked at the interface between the optical resonator and the analyte solution, which are termed as the interface modes. Previously, the microbubble resonator has been widely used for measuring refractive index, biomolecule concentration, and single nanoparticle generally by the mode localized in the liquid core (21–29). Here, we find that the profile of the WGM field can be tuned by varying the wall thickness, and the interface mode emerges when the maximum of the field intensity is drawn onto the interface. Compared with conventional evanescent sensors, the present scheme utilizing interface modes promises maximum sensitivity for interfacial molecular analysis, pushing the detection limit down to 0.3 pg/cm². The scheme is also compatible with the widely adopted techniques to enhance signal- to-noise ratio (SNR) such as plasmonic hybridization (30–33) and frequency tracking (12, 15, 18). As a proof of concept, single-molecule detection is demonstrated with a plasmonic-enhanced interface mode. Naturally integrated into a microfluidic system, the sensor with single-molecule sensitivity exhibits ultrasmall sample consumption down to 10 pL, providing an automatic platform for biomedical analysis.