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.     

Loss of Miro1-directed mitochondrial movement results in a novel murine model for neuron disease

Defective mitochondrial distribution in neurons is proposed to cause ATP depletion and calcium-buffering deficiencies that compromise cell function. However, it is unclear whether aberrant mitochondrial motility and distribution alone are sufficient to cause neurological disease. Calcium-binding mitochondrial Rho (Miro) GTPases attach mitochondria to motor proteins for anterograde and retrograde transport in neurons. Using two new KO mouse models, we demonstrate that Miro1 is essential for development of cranial motor nuclei required for respiratory control and maintenance of upper motor neurons required for ambulation. Neuron-specific loss of Miro1 causes depletion of mitochondria from corticospinal tract axons and progressive neurological deficits mirroring human upper motor neuron disease. Although Miro1-deficient neurons exhibit defects in retrograde axonal mitochondrial transport, mitochondrial respiratory function continues. Moreover, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or mitochondrial calcium buffering. Our findings indicate that defects in mitochondrial motility and distribution are sufficient to cause neurological disease.Motor neuron diseases (MNDs), including ALS and spastic paraplegia (SP), are characterized by the progressive, length-dependent degeneration of motor neurons, leading to muscle atrophy, paralysis, and, in some cases, premature death. There are both inherited and sporadic forms of MNDs, which can affect upper motor neurons, lower motor neurons, or both. Although the molecular and cellular causes of most MNDs are unknown, many are associated with defects in axonal transport of cellular components required for neuron function and maintenance (1–6).A subset of MNDs is associated with impaired mitochondrial respiration and mitochondrial distribution. This observation has led to the hypothesis that neurodegeneration results from defects in mitochondrial motility and distribution, which, in turn, cause subcellular ATP depletion and interfere with mitochondrial calcium ([Ca²⁺]_m) buffering at sites of high synaptic activity (reviewed in ref. 7). It is not known, however, whether mitochondrial motility defects are a primary cause or a secondary consequence of MND progression. In addition, it has been difficult to isolate the primary effect of mitochondrial motility defects in MNDs because most mutations that impair mitochondrial motility in neurons also affect transport of other organelles and vesicles (1, 8–11).In mammals, the movement of neuronal mitochondria between the cell body and the synapse is controlled by adaptors called trafficking kinesin proteins (Trak1 and Trak2) and molecular motors (kinesin heavy chain and dynein), which transport the organelle in the anterograde or retrograde direction along axonal microtubule tracks (7, 12–24). Mitochondrial Rho (Miro) GTPase proteins are critical for transport because they are the only known surface receptors that attach mitochondria to these adaptors and motors (12–15, 18, 25, 26). Miro proteins are tail-anchored in the outer mitochondrial membrane with two GTPase domains and two predicted calcium-binding embryonic fibroblast (EF) hand motifs facing the cytoplasm (12, 13, 25, 27, 28). A recent Miro structure revealed two additional EF hands that were not predicted from the primary sequence (29). Studies in cultured cells suggest that Miro proteins also function as calcium sensors (via their EF hands) to regulate kinesin-mediated mitochondrial “stopping” in axons (15, 16, 26). Miro-mediated movement appears to be inhibited when cytoplasmic calcium is elevated in active synapses, effectively recruiting mitochondria to regions where calcium buffering and energy are needed. Despite this progress, the physiological relevance of these findings has not yet been tested in a mammalian animal model. In addition, mammals ubiquitously express two Miro orthologs, Miro1 and Miro2, which are 60% identical (12, 13). However, the individual roles of Miro1 and Miro2 in neuronal development, maintenance, and survival have no been evaluated.We describe two new mouse models that establish the importance of Miro1-mediated mitochondrial motility and distribution in mammalian neuronal function and maintenance. We show that Miro1 is essential for development/maintenance of specific cranial neurons, function of postmitotic motor neurons, and retrograde mitochondrial motility in axons. Loss of Miro1-directed retrograde mitochondrial transport is sufficient to cause MND phenotypes in mice without abrogating mitochondrial respiratory function. Furthermore, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or [Ca²⁺]_m buffering. These findings have an impact on current models for Miro1 function and introduce a specific and rapidly progressing mouse model for MND.     

From the Cover: Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise

High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca²⁺ release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca²⁺ leak at rest, and depressed force production due to impaired SR Ca²⁺ release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca²⁺-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.It is increasingly clear that regular physical exercise plays a key role in the general well-being, disease prevention, and longevity of humans. Impaired muscle function manifesting as muscle weakness and premature fatigue development are major health problems associated with the normal aging process as well as with numerous common diseases (1). Physical exercise has a fundamental role in preventing and/or reversing these muscle problems, and training also improves the general health status in numerous diseases (2–4). On the other side of the spectrum, excessive muscle use can induce prolonged force depressions, which may set the limit on training tolerance and performance of top athletes (5, 6).Recent studies imply a key role of the sarcoplasmic reticulum (SR) Ca²⁺ release channel, the ryanodine receptor 1 (RyR1), in the reduced muscle strength observed in numerous physiological conditions, such as after strenuous endurance training (6), in situations with prolonged stress (7), and in normal aging (8, 9). Defective RyR1 function is also implied in several pathological states, including generalized inflammatory disorders (10), heart failure (11), and inherited conditions such as malignant hyperthermia (12) and Duchenne muscular dystrophy (13). In many of the above conditions, there is a link between the impaired RyR1 function and modifications induced by reactive oxygen/nitrogen species (ROS) (6, 8, 10, 12, 13). Conversely, altered RyR1 function may also be beneficial by increasing the cytosolic free [Ca²⁺] ([Ca²⁺]_i) at rest, which can stimulate mitochondrial biogenesis and thereby increase fatigue resistance (14–16). Intriguingly, effective antioxidant treatment hampers beneficial adaptations triggered by endurance training (17–19), and this effect might be due to antioxidants preventing ROS-induced modifications of RyR1 (20).A high-intensity interval training (HIIT) session typically consists of a series of brief bursts of vigorous physical exercise separated by periods of rest or low-intensity exercise. A major asset of HIIT is that beneficial adaptations can be obtained with much shorter exercise duration than with traditional endurance training (21–25). HIIT has been shown to effectively stimulate mitochondrial biogenesis in skeletal muscle and increase endurance in untrained and recreationally active healthy subjects (22, 26), whereas positive effects in elite endurance athletes are less clear (21, 27, 28). Moreover, HIIT improves health and physical performance in various pathological conditions, including cardiovascular disease, obesity, and type 2 diabetes (29, 30). Thus, short bouts of vigorous physical exercise trigger intracellular signaling of large enough magnitude and duration to induce extensive beneficial adaptations in skeletal muscle. The initial signaling that triggers these adaptations is not known.In this study, we tested the hypothesis that a single session of HIIT induces ROS-dependent RyR1 modifications. These modifications might cause prolonged force depression due to impaired SR Ca²⁺ release during contractions. Conversely, they may also initiate beneficial muscular adaptations due to increased SR Ca²⁺ leak at rest.     

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones

Ca²⁺ influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)–interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated Ca_V1.3 Ca²⁺ channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca²⁺ imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic Ca_V1.3 Ca²⁺ channels, resulting in reduced synaptic Ca²⁺ influx. Using superresolution microscopy, we found that Ca²⁺ channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca²⁺ current, whereas the apparent Ca²⁺ dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca²⁺ influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca²⁺ channels at IHC AZs and are required for normal hearing.Tens of Ca_V1.3 Ca²⁺ channels are thought to cluster within the active zone (AZ) membrane underneath the presynaptic density of inner hair cells (IHCs) (1–4). They make up the key signaling element, coupling the sound-driven receptor potential to vesicular glutamate release (5–7). The mechanisms governing the number of Ca²⁺ channels at the AZ as well as their spatial organization relative to membrane-tethered vesicles are not well understood. Disrupting the presynaptic scaffold protein Bassoon diminishes the numbers of Ca²⁺ channels and membrane-tethered vesicles at the AZ (2, 8). However, the loss of Bassoon is accompanied by the loss of the entire synaptic ribbon, which makes it challenging to distinguish the direct effects of gene disruption from secondary effects (9).Among the constituents of the cytomatrix of the AZ, RIM1 and RIM2 proteins are prime candidates for the regulation of Ca²⁺ channel clustering and function (10, 11). The family of RIM proteins has seven identified members (RIM1α, RIM1β, RIM2α, RIM2β, RIM2γ, RIM3γ, and RIM4γ) encoded by four genes (RIM1–RIM4). All isoforms contain a C-terminal C₂ domain but differ in the presence of additional domains. RIM1 and RIM2 interact with Ca²⁺ channels, most other proteins of the cytomatrix of the AZ, and synaptic vesicle proteins. They interact directly with the auxiliary β (Ca_Vβ) subunits (12, 13) and pore-forming Ca_Vα subunits (14, 15). In addition, RIMs are indirectly linked to Ca²⁺ channels via RIM-binding protein (14, 16, 17). A regulation of biophysical channel properties has been demonstrated in heterologous expression systems for RIM1 (12) and RIM2 (13).A role of RIM1 and RIM2 in clustering Ca²⁺ channels at the AZ was demonstrated by analysis of RIM1/2-deficient presynaptic terminals of cultured hippocampal neurons (14), auditory neurons in slices (18), and Drosophila neuromuscular junction (19). Because α-RIMs also bind the vesicle-associated protein Ras-related in brain 3 (Rab3) via the N-terminal zinc finger domain (20), they are also good candidates for molecular coupling of Ca²⁺ channels and vesicles (18, 21, 22). Finally, a role of RIMs in priming of vesicles for fusion is the subject of intense research (18, 21–27). RIMs likely contribute to priming via disinhibiting Munc13 (26) and regulating vesicle tethering (27). Here, we studied the expression and function of RIM in IHCs. We combined molecular, morphologic, and physiologic approaches for the analysis of RIM2α knockout mice [RIM2α SKO (28); see Methods] and of hair cell-specific RIM1/2 knockout mice (RIM1/2 cDKO). We demonstrate that RIM2α and RIM2β are present at IHC AZs of hearing mice, positively regulate the number of synaptic Ca_V1.3 Ca²⁺ channels, and are required for normal hearing.     

Rapid stimulus-evoked astrocyte Ca2+ elevations and hemodynamic responses in mouse somatosensory cortex in vivo

Increased neuron and astrocyte activity triggers increased brain blood flow, but controversy exists over whether stimulation-induced changes in astrocyte activity are rapid and widespread enough to contribute to brain blood flow control. Here, we provide evidence for stimulus-evoked Ca²⁺ elevations with rapid onset and short duration in a large proportion of cortical astrocytes in the adult mouse somatosensory cortex. Our improved detection of the fast Ca²⁺ signals is due to a signal-enhancing analysis of the Ca²⁺ activity. The rapid stimulation-evoked Ca²⁺ increases identified in astrocyte somas, processes, and end-feet preceded local vasodilatation. Fast Ca²⁺ responses in both neurons and astrocytes correlated with synaptic activity, but only the astrocytic responses correlated with the hemodynamic shifts. These data establish that a large proportion of cortical astrocytes have brief Ca²⁺ responses with a rapid onset in vivo, fast enough to initiate hemodynamic responses or influence synaptic activity.Brain function emerges from signaling in and between neurons and associated astrocytes, which causes fluctuations in cerebral blood flow (CBF) (1–5). Astrocytes are ideally situated for controlling activity-dependent increases in CBF because they closely associate with synapses and contact blood vessels with their end-feet (1, 6). Whether or not astrocytic Ca²⁺ responses develop often or rapidly enough to account for vascular signals in vivo is still controversial (7–10). Ca²⁺ responses are of interest because intracellular Ca²⁺ is a key messenger in astrocytic communication and because enzymes that synthesize the vasoactive substances responsible for neurovascular coupling are Ca²⁺-dependent (1, 4). Neuronal activity releases glutamate at synapses and activates metabotropic glutamate receptors on astrocytes, and this activation can be monitored by imaging cytosolic Ca²⁺ changes (11). Astrocytic Ca²⁺ responses are often reported to evolve on a slow (seconds) time scale, which is too slow to account for activity-dependent increases in CBF (8, 10, 12, 13). Furthermore, uncaging of Ca²⁺ in astrocytes triggers vascular responses in brain slices through specific Ca²⁺-dependent pathways with a protracted time course (14, 15). More recently, stimulation of single presynaptic neurons in hippocampal slices was shown to evoke fast, brief, local Ca²⁺ elevations in astrocytic processes that were essential for local synaptic functioning in the adult brain (16, 17). This work prompted us to reexamine the characteristics of fast, brief astrocytic Ca²⁺ signals in vivo with special regard to neurovascular coupling, i.e., the association between local increases in neural activity and the concomitant rise in local blood flow, which constitutes the physiological basis for functional neuroimaging.Here, we describe how a previously undescribed method of analysis enabled us to provide evidence for fast Ca²⁺ responses in a main fraction of astrocytes in mouse whisker barrel cortical layers II/III in response to somatosensory stimulation. The astrocytic Ca²⁺ responses were brief enough to be a direct consequence of synaptic excitation and correlated with stimulation-induced hemodynamic responses. Fast Ca²⁺ responses in astrocyte end-feet preceded the onset of dilatation in adjacent vessels by hundreds of milliseconds. This finding might suggest that communication at the gliovascular interface contributes considerably to neurovascular coupling.     

From the Cover: 90Y-daclizumab,an anti-CD25 monoclonal antibody,provided responses in 50% of patients with relapsed Hodgkin’s lymphoma

Despite significant advances in the treatment of Hodgkin’s lymphoma (HL), a significant proportion of patients will not respond or will subsequently relapse. We identified CD25, the IL-2 receptor alpha subunit, as a favorable target for systemic radioimmunotherapy of HL. The scientific basis for the clinical trial was that, although most normal cells with exception of Treg cells do not express CD25, it is expressed by a minority of Reed–Sternberg cells and by most polyclonal T cells rosetting around Reed–Sternberg cells. Forty-six patients with refractory and relapsed HL were evaluated with up to seven i.v. infusions of the radiolabeled anti-CD25 antibody ⁹⁰Y-daclizumab. ⁹⁰Y provides strong β emissions that kill tumor cells at a distance by a crossfire effect. In 46 evaluable HL patients treated with ⁹⁰Y-daclizumab there were 14 complete responses and nine partial responses; 14 patients had stable disease, and nine progressed. Responses were observed both in patients whose Reed–Sternberg cells expressed CD25 and in those whose neoplastic cells were CD25⁻ provided that associated rosetting T cells expressed CD25. As assessed using phosphorylated H2AX (γ-H2AX) as a bioindicator of the effects of radiation exposure, predominantly nonmalignant cells in the tumor microenvironment manifested DNA damage, as reflected by increased expression of γ-H2AX. Toxicities were transient bone-marrow suppression and myelodysplastic syndrome in six patients who had not been evaluated with bone-marrow karyotype analyses before therapy. In conclusion, repeated ⁹⁰Y-daclizumab infusions directed predominantly toward nonmalignant T cells rosetting around Reed–Sternberg cells provided meaningful therapy for select HL patients.Treatment with combination chemotherapy, radiation, and hematopoietic stem cell transplantation has increased the disease-free survival in Hodgkin’s lymphoma (HL) from less than 5% in 1963 to more than 80% at present (1–6). Recently the US Food and Drug Administration approved brentuximab vedotin for the treatment of relapsed HL (7). Furthermore the anti-PD1 agent pembrolizumab has shown promising results in classic HL (8). Nevertheless, a significant fraction of patients do not respond to treatment or subsequently relapse. To date more than 30 different mAb preparations directed toward antigens expressed by malignant Reed–Sternberg cells have been studied (6). These include mAbs linked to drugs or toxins targeting CD25 or CD30 expressed on Reed–Sternberg cells (6–11). Brentuximab vedotin, an anti-CD30 antibody drug conjugate, has induced a significant number of responses in refractory HL (7, 11). Although other antibody immunotoxins have demonstrated some clinical efficacy, they have yielded few complete responses (CRs) (6, 9, 10). An alternative strategy has been to arm mAbs with radionuclides. Radioimmunotherapy using ⁹⁰Y–anti-ferritin and ¹³¹I–anti-CD30 antibodies has resulted in partial (PRs) and CRs in HL (12–15). Deficiencies with these approaches reflect the lack of tumor specificity of ferritin-targeted antibodies and the small number of CD30-expressing Reed–Sternberg cells in the tumor.As an alternative, we identified CD25, the IL-2 receptor alpha subunit (IL-2Rα), as a more favorable target for systemic radioimmunotherapy of HL (16–22). The scientific rationale is that, with the exception of Treg cells, CD25 is not expressed by normal resting lymphoid cells, but it is expressed on both a minority of Reed–Sternberg cells and, critically, on T cells rosetting around Reed–Sternberg cells in HL (6, 23, 24). ⁹⁰Y, an energetic β particle emitter with a mean tissue path length of 5 mm and a maximal path length of 11 mm, acts through “crossfire” throughout tumor masses, providing a strategy for killing tumor cells at a distance of several cell diameters, including Reed–Sternberg cells that lack CD25 expression provided that T cells in their vicinity express the target antigen (16, 23, 24). In the current phase II trial we treated 46 patients with recurrent or refractory HL with ⁹⁰Y-daclizumab every 6–10 wk for up to seven doses, depending on hematological recovery. The activity of ⁹⁰Y used in the present trial was determined on the basis of three previous phase I/II dose-escalation trials of ⁹⁰Y–anti-CD25 performed in patients with lymphoproliferative disorders (16).     

The Two-pore channel (TPC) interactome unmasks isoform-specific roles for TPCs in endolysosomal morphology and cell pigmentation

The two-pore channels (TPC1 and TPC2) belong to an ancient family of intracellular ion channels expressed in the endolysosomal system. Little is known about how regulatory inputs converge to modulate TPC activity, and proposed activation mechanisms are controversial. Here, we compiled a proteomic characterization of the human TPC interactome, which revealed that TPCs complex with many proteins involved in Ca²⁺ homeostasis, trafficking, and membrane organization. Among these interactors, TPCs were resolved to scaffold Rab GTPases and regulate endomembrane dynamics in an isoform-specific manner. TPC2, but not TPC1, caused a proliferation of endolysosomal structures, dysregulating intracellular trafficking, and cellular pigmentation. These outcomes required both TPC2 and Rab activity, as well as their interactivity, because TPC2 mutants that were inactive, or rerouted away from their endogenous expression locale, or deficient in Rab binding, failed to replicate these outcomes. Nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca²⁺ release was also impaired using either a Rab binding-defective TPC2 mutant or a Rab inhibitor. These data suggest a fundamental role for the ancient TPC complex in trafficking that holds relevance for lysosomal proliferative scenarios observed in disease.Two-pore channels (TPCs) are an ancient family of intracellular ion channels and a likely ancestral stepping stone in the evolution of voltage-gated Ca²⁺ and Na⁺ channels (1). Architecturally, TPCs resemble a halved voltage-gated Ca²⁺/Na⁺ channel with cytosolic NH₂ and COOH termini, comprising two repeats of six transmembrane spanning helices with a putative pore-forming domain between the fifth and sixth membrane-spanning regions. Since their discovery in vertebrate systems, many studies have investigated the properties of these channels (2–7) that may support such a lengthy evolutionary pedigree.In this context, demonstration that (i) the two human TPC isoforms (TPC1 and TPC2) are uniquely distributed within the endolysosomal system (2, 3) and that (ii) TPC channel activity is activated by the Ca²⁺ mobilizing molecule nicotinic acid adenine dinucleotide phosphate (NAADP) (4–6) generated considerable excitement that TPCs function as effectors of this mercurial second messenger long known to trigger Ca²⁺ release from “acidic stores.” The spectrum of physiological activities that have been linked to NAADP signaling over the last 25 years (8, 9) may therefore be realized through regulation of TPC activity. However, recent studies have questioned the idea that TPCs are NAADP targets (10, 11), demonstrating instead that TPCs act as Na⁺ channels regulated by the endolysosomal phosphoinositide PI(3,5)P₂. Such controversy (12, 13) underscores how little we know about TPC regulatory inputs and the dynamic composition of TPC complexes within cells.Here, to generate unbiased insight into the cell biology of the TPC complex, we report a proteomic analysis of human TPCs. The TPC interactome establishes a useful community resource as a “rosetta stone” for interrogating the cell biology of TPCs and their regulation. The dataset reveals a predomination of links between TPCs and effectors controlling membrane organization and trafficking, relevant for disease states involving lysosomal proliferation where TPC functionality may be altered (14).     

Ion-binding properties of a K+ channel selectivity filter in different conformations

K⁺ channels are membrane proteins that selectively conduct K⁺ ions across lipid bilayers. Many voltage-gated K⁺ (K_V) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K⁺ channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to K_V channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na⁺ or low [K⁺]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K⁺ ion binding to channels with an inactivated or conductive selectivity filter is different from K⁺ ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.K⁺ channels are found in all three domains of life, where they selectively conduct K⁺ ions across cell membranes. Specific stimuli trigger the activation of K⁺ channels, which results in a hinged movement of the inner helix bundle (1–7). This opening on the intracellular side of the membrane initiates ion conduction across the membrane by allowing ions to enter into the channel. After a period, many channels spontaneously inactivate to attenuate the response (8–17). The inactivation process is a timer that terminates the flow of ions in the presence of an activator to help shape the response of the system. Two dominant types of inactivation have been characterized in voltage-dependent channels: N-type and C-type (18). N-type inactivation is fast and involves an N-terminal positively charged “ball” physically plugging the pore of the channel when the membrane is depolarized. C-type inactivation, on the other hand, is a slower process involving a conformational change in the selectivity filter that is initiated by a functional link between the intracellular gate and the selectivity filter (10, 19).Several experimental observations indicate a role for the selectivity filter in C-type inactivation. First, mutations in and around the selectivity filter can alter the kinetics of inactivation (20–23). Second, increasing concentrations of extracellular K⁺ ions decrease the rate of inactivation, as if the ions are stabilizing the conductive conformation of the channel to prevent a conformational change in the selectivity filter (14, 16, 17, 22). Finally, a loss of selectivity of K⁺ over Na⁺ has been observed during the inactivation process in Shaker channels, suggesting a role for the selectivity filter (24, 25). Together, these data indicate that channels in their inactivated and conductive conformations interact with K⁺ ions differently, and suggest that C-type inactivation involves a conformational change in the selectivity filter. Although several structures of K⁺ channels in their conductive state have been solved using X-ray crystallography, there is at present no universally accepted model for the C-type inactivated channel (1, 3–5, 9, 19, 26–28) (Fig. 1B).Open in a separate windowFig. 1.Macroscopic recordings and structural models of KcsA K⁺ channel. (A) Macroscopic currents of WT KcsA obtained by a pH jump from pH 8 to pH 4 reveal channel inactivation. Two models representing the conformation of the channel are shown below. (B) Conductive [Left, Protein Data Bank (PDB) ID code 1K4C] and constricted (Right, PDB ID code 1K4D) conformations of the selectivity filter are shown as sticks, and the ion-binding sites are indicated with green spheres. The thermodynamic properties of the conductive, constricted, and inactivated (Middle) conformations are the subject of this study.Inactivation in the K⁺ channel from Streptomyces lividians (KcsA) has many of the same functional properties of C-type inactivation, which has made it a model to understand its structural features (20). KcsA channels transition from their closed to open gate upon changing the intracellular pH from high to low (Fig. 1A). The rapid flux of ions through the channel is then attenuated by channel inactivation, where most open WT channels are not conducting, suggesting that crystal structures of open KcsA channels would reveal the inactivated channel. In some crystal structures of truncated WT KcsA solved with an open gate, the selectivity filter appears in the constricted conformation, similar to the conformation observed in structures of the KcsA channel determined in the presence of only Na⁺ ions or low concentrations of K⁺ ions (3, 10, 29, 30) (Fig. 1B). Solid-state and solution NMR also indicate that the selectivity filter of the KcsA channel is in the constricted conformation when the cytoplasmic gate is open (31–33).However, a recently published study shows that even when the constricted conformation of KcsA’s selectivity filter is prevented by a nonnatural amino acid substitution, the channel inactivates like WT channels, suggesting the constricted filter does not correspond to the functionally observed inactivation in KcsA (28). In this study, we use isothermal titration calorimetry (ITC) to quantify the ion-binding properties of WT and mutant KcsA K⁺ channels with their selectivity filters in different conformations and EPR spectroscopy to determine the conformation of the channels’ intracellular gates. A comparison of these ion-binding properties leads us to conclude that the conductive and inactivated filters are energetically more similar to each other than the constricted and inactivated filters.     

Reducing the genetic code induces massive rearrangement of the proteome

Expanding the genetic code is an important aim of synthetic biology, but some organisms developed naturally expanded genetic codes long ago over the course of evolution. Less than 1% of all sequenced genomes encode an operon that reassigns the stop codon UAG to pyrrolysine (Pyl), a genetic code variant that results from the biosynthesis of Pyl-tRNA^Pyl. To understand the selective advantage of genetically encoding more than 20 amino acids, we constructed a markerless tRNA^Pyl deletion strain of Methanosarcina acetivorans (ΔpylT) that cannot decode UAG as Pyl or grow on trimethylamine. Phenotypic defects in the ΔpylT strain were evident in minimal medium containing methanol. Proteomic analyses of wild type (WT) M. acetivorans and ΔpylT cells identified 841 proteins from >7,000 significant peptides detected by MS/MS. Protein production from UAG-containing mRNAs was verified for 19 proteins. Translation of UAG codons was verified by MS/MS for eight proteins, including identification of a Pyl residue in PylB, which catalyzes the first step of Pyl biosynthesis. Deletion of tRNA^Pyl globally altered the proteome, leading to >300 differentially abundant proteins. Reduction of the genetic code from 21 to 20 amino acids led to significant down-regulation in translation initiation factors, amino acid metabolism, and methanogenesis from methanol, which was offset by a compensatory (100-fold) up-regulation in dimethyl sulfide metabolic enzymes. The data show how a natural proteome adapts to genetic code reduction and indicate that the selective value of an expanded genetic code is related to carbon source range and metabolic efficiency.Synthesizing whole genomes (1) and eliminating codons (2) are novel methods for rewriting the genetic code that may dramatically alter the repertoire of genetically encoded amino acids. Expansion of the genetic code has led to exciting technologies, including site-directed protein labeling and production of proteins with hardwired posttranslational modifications (3). The current approaches to cotranslationally insert noncanonical amino acids (ncAAs) into proteins rely on the reassigning of one of three stop codons (4).Although these approaches were highly successful in incorporating over 100 ncAAs into proteins (3), they limit the expansion of the code to no more than 2 additional amino acids at a time and significantly challenge the cellular production host by unnaturally extending proteins and reducing growth rate (5). Alternate methods focus on quadruplet codons (6, 7) and recoding (8) or reassigning sense codons (9–13). Attempts to reassign a sense codon in Mycoplasma capricolum were defied by tRNA misacylation by endogenous aminoacyl-tRNA synthetases (9). This result indicates that, although extensively rewriting the genetic code may be possible, it comes with unexpected challenges related to cellular fitness and translation fidelity. These considerations will impact efforts to engineer cells to synthesize proteins with multiple ncAAs or create biologically contained strains that require an expanded code for survival (14).Opening codons by reducing the genetic code is highly promising, but it is unknown how removing 1 amino acid from the genetic code might impact the proteome or cellular viability. Many genetic code variations are found in nature (15), including stop or sense codon reassignments, codon recoding, and natural code expansion (16). Pyrrolysine (Pyl) is a rare example of natural genetic code expansion. Evidence for genetically encoded Pyl is found in <1% of all sequenced genomes (17). In these organisms, Pyl is encoded by the UAG codon, which requires tRNA^Pyl, pyrrolysyl-tRNA synthetase (PylRS), and the products of three genes (pylBCD) that synthesize Pyl from two molecules of lysine (18). The PylRS enzyme was engineered to genetically encode >100 ncAAs (19). The Pyl encoding system has already been used to expand the genetic codes of Escherichia coli (20–22), mammalian cells, and animals (23).Despite the use of Pyl in synthetic biology, little is known about the role of Pyl in its native environment or the evolutionary pressures that sustain expanded genetic codes in nature. The Pyl-decoding trait is found in methanogenic archaea of the orders Methanosarcinales and Methanomassiliicoccales (24) and certain anaerobic bacteria (17). In addition to producing 74% of global methane emissions, methanogens are remarkable for their ability to survive with only the most basic carbon and energy sources (25). Methanosarcina shows the greatest substrate range among methanogens and survives on acetate, carbon monoxide, methylamines, methanol, or dimethyl sulfide (DMS). Their broad substrate range depends, in part, on the presence of Pyl in the active site of several methylamine methyltransferases (26). Hundreds of Methanosracina genes contain in-frame TAG codons (27), but natural Pyl incorporation was only shown in methylamine methyltransferases (17, 28) and tRNA^His guanylyltransferase (Thg1) (29).Methanosarcina acetivorans provides an ideal model system to identify Pyl-containing proteins and study the impact of genetic code reduction on the proteome and physiology of the cell. We constructed a markerless tRNA^Pyl deletion (ΔpylT) strain of M. acetivorans C2A and used three independent mass spectrometry (MS) approaches to characterize soluble proteomes from M. acetivorans grown on minimal medium containing trimethylamine (TMA) or methanol and ΔpylT cells grown on methanol. The data reveal previously unidentified biochemical roles for Pyl and Pyl-containing proteins and indicate that the expanded genetic code of Methanosarcina is intricately linked with cellular metabolism and the composition of the proteome.     

Arginine oscillation explains Na+ independence in the substrate/product antiporter CaiT

Most secondary-active transporters transport their substrates using an electrochemical ion gradient. In contrast, the carnitine transporter (CaiT) is an ion-independent, l-carnitine/γ-butyrobetaine antiporter belonging to the betaine/carnitine/choline transporter family of secondary transporters. Recently determined crystal structures of CaiT from Escherichia coli and Proteus mirabilis revealed an inverted five-transmembrane-helix repeat similar to that in the amino acid/Na⁺ symporter LeuT. The ion independence of CaiT makes it unique in this family. Here we show that mutations of arginine 262 (R262) make CaiT Na⁺-dependent. The transport activity of R262 mutants increased by 30–40% in the presence of a membrane potential, indicating substrate/Na⁺ cotransport. Structural and biochemical characterization revealed that R262 plays a crucial role in substrate binding by stabilizing the partly unwound TM1′ helix. Modeling CaiT from P. mirabilis in the outward-open and closed states on the corresponding structures of the related symporter BetP reveals alternating orientations of the buried R262 sidechain, which mimic sodium binding and unbinding in the Na⁺-coupled substrate symporters. We propose that a similar mechanism is operative in other Na⁺/H⁺-independent transporters, in which a positively charged amino acid replaces the cotransported cation. The oscillation of the R262 sidechain in CaiT indicates how a positive charge triggers the change between outward-open and inward-open conformations as a unifying critical step in LeuT-type transporters.The carnitine/γ-butyrobetaine antiporter CaiT belongs to the betaine/carnitine/choline transporter family of secondary transporters that transfer substrates containing a quaternary ammonium group (1, 2) in and out of the cell. In Escherichia coli and other enterobacteria, such as Proteus mirabilis, carnitine is taken up by CaiT and converted to γ-butyrobetaine via the reaction intermediate crotonobetaine (3–5), which serves as an external electron acceptor under anaerobic growth conditions (4). Biochemical studies of E. coli CaiT (EcCaiT) have shown that it is a constitutively active, Na⁺/H⁺-independent antiporter (6).Crystal structures of CaiT from P. mirabilis (PmCaiT) and E. coli (EcCaiT) were recently determined with and without bound substrate (7, 8). These structures revealed a trimeric assembly of CaiT, as previously found (9). The protein was in an inward-facing conformation with two substrate molecules bound per EcCaiT monomer: one in the central transport site and another in an external binding site (7). Fluorescent binding assays with the protein reconstituted into liposomes indicated that substrate binding was cooperative. This suggested a regulatory role for the external binding site, which was proposed to increase substrate affinity and initiate substrate transport (7). Strikingly, the crystal structures revealed that CaiT adopts a fold similar to that of the LeuT-type transporters (7, 10, 11). This places it in the amino acid–polyamine-organocation (APC) superfamily (12), which shares a conserved architecture of two inverted repeats of five transmembrane (TM) helices each, implying common mechanistic principles. Among the APC transporters, the leucine transporter LeuT from the neurotransmitter/sodium symporter family (10), the betaine transporter BetP from the betaine/choline/carnitine transporter family (13), the benzyl-hydantoin transporter Mhp1 of the nucleobase/cation symport 1 family, and the Na⁺/galactose symporter vSGLT of the solute/sodium symporter family are substrate/sodium symporters (14, 15). Although the substrate to sodium stoichiometry varies for each Na⁺-dependent LeuT-type transporter, most of them possess a conserved sodium-binding site (Na2 site) at which the binding and dissociation of a sodium ion is proposed to facilitate structural changes that lead to substrate transport (16–18). Although an additional sodium-binding site (Na1 site) exists in transporters such as LeuT and BetP, the position or even the presence of this site is not strictly conserved among the Na⁺-dependent LeuT-type transporters (14, 15, 19–21).A small number of LeuT-type transporters, namely, AdiC (arginine/agmatine antiporter), ApcT (broad-specificity amino acid transporter), and CaiT, are Na⁺-independent (6, 22–25). The crystal structure of ApcT revealed that a lysine residue (K158) occupies a position equivalent to the Na2 site in LeuT. This lysine is proposed to undergo a protonation/deprotonation event that leads to conformational changes facilitating substrate transport (25). In CaiT, a methionine residue (M331) occupies a position equivalent to Na1 in LeuT, whereas a positively charged arginine residue (R262) occupies the Na2 site (7). Previously, we have shown that mutating M331 reduces transport activity but does not induce Na⁺ dependence in CaiT (7). Here we report that point mutations of R262 render CaiT inactive. Strikingly, the transport activity was partially restored by the addition of sodium, thus making these mutants Na⁺-dependent. Unlike wild-type, the transport activity of R262 mutants increased by 30–40% when a membrane potential was applied, suggesting that Na⁺ and substrate were cotransported. To find out whether and how the mutation affects the Na2 site, we determined the crystal structure of CaiT R262E. Although we did not find any major changes in the mutant protein, comparison with the substrate-bound EcCaiT wild-type protein revealed that the γ-butyrobetaine substrate adopts a different orientation at the central binding site, in which it directly interacts with the unwound part of TM1′. (To make the nomenclature consistent, we adopt the same helix numbering as in LeuT. Because CaiT has two extra helices at the N terminus in comparison with LeuT, TM3 in CaiT corresponds to TM1 in LeuT and is denoted as TM1′, and the remainder of the CaiT helices follow suit.) Because R262 is known to play a role in stabilizing the unwound part of TM1′ (7), we propose that R262 is crucial for substrate binding, similar to Na⁺ in the Na2 site of LeuT and BetP (17, 19). Indeed, our fluorescent binding assays using R262 mutants showed markedly decreased substrate affinity. Modeling CaiT in various conformations with BetP as a template revealed that R262 undergoes an oscillatory movement, contributing to different hydrogen bond networks in each conformation. We suggest that this movement of the positively charged R262 sidechain in CaiT mimics Na⁺ binding/unbinding in Na⁺-dependent LeuT-type transporters and plays a central role in the transport mechanism.     

Network compensation of cyclic GMP-dependent protein kinase II knockout in the hippocampus by Ca2+-permeable AMPA receptors

Gene knockout (KO) does not always result in phenotypic changes, possibly due to mechanisms of functional compensation. We have studied mice lacking cGMP-dependent kinase II (cGKII), which phosphorylates GluA1, a subunit of AMPA receptors (AMPARs), and promotes hippocampal long-term potentiation (LTP) through AMPAR trafficking. Acute cGKII inhibition significantly reduces LTP, whereas cGKII KO mice show no LTP impairment. Significantly, the closely related kinase, cGKI, does not compensate for cGKII KO. Here, we describe a previously unidentified pathway in the KO hippocampus that provides functional compensation for the LTP impairment observed when cGKII is acutely inhibited. We found that in cultured cGKII KO hippocampal neurons, cGKII-dependent phosphorylation of inositol 1,4,5-trisphosphate receptors was decreased, reducing cytoplasmic Ca²⁺ signals. This led to a reduction of calcineurin activity, thereby stabilizing GluA1 phosphorylation and promoting synaptic expression of Ca²⁺-permeable AMPARs, which in turn induced a previously unidentified form of LTP as a compensatory response in the KO hippocampus. Calcineurin-dependent Ca²⁺-permeable AMPAR expression observed here is also used during activity-dependent homeostatic synaptic plasticity. Thus, a homeostatic mechanism used during activity reduction provides functional compensation for gene KO in the cGKII KO hippocampus.Some gene deletions yield no phenotypic changes because of functional compensation by closely related or duplicate genes (1). However, such duplicate gene activity may not be the main compensatory mechanism in mouse (2), although this possibility is still controversial (3). A second mechanism of compensation is provided by alternative metabolic pathways or regulatory networks (4). Although such compensatory mechanisms have been extensively studied, especially in yeast and nematode (1), the roles of metabolic and network compensatory pathways are not well understood in mouse.Long-term potentiation (LTP) and long-term depression (LTD) are long-lasting forms of synaptic plasticity that are thought to be the cellular basis for learning and memory and proper formation of neural circuits during development (5). NMDA receptor (NMDAR)-mediated synaptic plasticity is a generally agreed postsynaptic mechanism in the hippocampus (5). In particular, synaptic Ca²⁺ influx through NMDARs is critical for LTP and LTD through control of various protein kinases and phosphatases (6). LTP is in part dependent upon the activation of protein kinases, which phosphorylate target proteins (6). Several kinases are activated during the induction of LTP, including cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinases (cGKs) (6). In contrast, LTD results from activation of phosphatases that dephosphorylate target proteins (6), and calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase, is important for LTD expression (7). AMPA receptors (AMPARs) are postsynaptic glutamate receptors that mediate rapid excitatory transmission in the central nervous system (8). During LTP, activated kinases phosphorylate AMPARs, leading to synaptic trafficking of the receptors to increase synapse activity (5). For LTD, activation of postsynaptic phosphatases induces internalization of AMPARs from the synaptic membrane, thereby reducing synaptic strength (5). Therefore, both protein kinases and phosphatases control synaptic trafficking of AMPARs, underlying LTP and LTD.AMPARs are tetrameric ligand-gated ion channels that consist of a combinatorial assembly of four subunits (GluA1–4) (9). Studies of GluA1 knockout (KO) mice show that GluA1 is critical for LTP in the CA1 region of the hippocampus (10). GluA1 homomers, like all GluA2-lacking/GluA1-containing receptors, are sensitive to polyamine block and are Ca²⁺-permeable, whereas GluA2-containing AMPARs are Ca²⁺-impermeable (9). Moreover, GluA1 is the major subunit that is trafficked from recycling endosomes to the synaptic membrane in response to neuronal activity (11). Phosphorylation of GluA1 within its intracellular carboxyl-terminal domain (CTD) can regulate AMPAR membrane trafficking (12). Several CTD phosphorylations regulate trafficking (6). In particular, PKA and cGKII both phosphorylate serine 845 of GluA1, increasing the level of extrasynaptic receptors (13, 14). Therefore, activation of PKA and cGKII during LTP induction increases GluA1 phosphorylation, which enhances AMPAR activity at synapses. On the other hand, calcineurin dephosphorylates serine 845 of GluA1, which enables GluA1-containing AMPARs to be endocytosed from the plasma membrane during LTD (15, 16). This removes synaptic AMPARs, leading to reduction of receptor function during LTD. Taken together, the activity-dependent trafficking of synaptic GluA1 is regulated by the status of phosphorylation in the CTD, which provides a critical mechanism underlying LTP and LTD.Several studies have shown that acute inhibition of cGKII impairs hippocampal LTP (13, 17, 18). However, cGKII KO animals show apparently normal LTP in the hippocampus (19), suggesting that a form of functional compensation takes place in the KO hippocampus. Here, we show that cGKII KO reduces Ca²⁺ signals by decreasing cGKII-dependent phosphorylation of inositol 1,4,5-trisphosphate receptors (IP3Rs), which in turn lowers calcineurin activity in hippocampal neurons, which stabilizes phosphorylation of GluA1 in homomeric, Ca²⁺-permeable AMPARs (CPARs). This elevates CPARs at the synapse as a previously unidentified compensatory mechanism for hippocampal LTP in cGKII-deficient animals that is alternative to the form of LTP expressed in WT.     

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head–bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.Kinesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1–6). Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7–10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4, 11, 12), consistent with the motor walking in a hand-over-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7–10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head–bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads–bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4, 11, 14–20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7, 19, 21, 22) and single-molecule tracking studies (4, 8–11) have failed to detect this 1HB state during stepping. Single-molecule fluorescence approaches have detected a 1HB intermediate at limiting ATP concentrations (11, 12, 14, 15), but apart from one study that used autocorrelation analysis to detect a 3-ms intermediate (17), the 1HB state has been undetectable at physiological ATP concentrations.Single-molecule microscopy is a powerful tool for studying the kinetics of structural changes in macromolecules (23). Tracking steps and potential substeps for kinesin-1 at saturating ATP has until now been hampered by the high stepping rates of the motor (up to 100 s⁻¹), which necessitates high frame rates, and the small step size (8.2 nm), which necessitates high spatial precision (7). Here, we apply interferometric scattering microscopy (iSCAT), a recently established single-molecule tool with high spatiotemporal resolution (24–27) to directly visualize the structural changes underlying kinesin stepping. By labeling one motor domain in a dimeric motor, we detect a 1HB intermediate state in which the tethered head resides over the bound head for half the duration of the stepping cycle at saturating ATP. We further show that at physiological stepping rates, ATP binding is required to enter this 1HB state and that ATP hydrolysis is required to exit it. This work leads to a significant revision of the sequence and kinetics of mechanochemical transitions that make up the kinesin-1 stepping cycle and provides a framework for understanding functional diversity across the kinesin superfamily.     

Medial septal GABAergic projection neurons promote object exploration behavior and type 2 theta rhythm

Exploratory drive is one of the most fundamental emotions, of all organisms, that are evoked by novelty stimulation. Exploratory behavior plays a fundamental role in motivation, learning, and well-being of organisms. Diverse exploratory behaviors have been described, although their heterogeneity is not certain because of the lack of solid experimental evidence for their distinction. Here we present results demonstrating that different neural mechanisms underlie different exploratory behaviors. Localized Ca_v3.1 knockdown in the medial septum (MS) selectively enhanced object exploration, whereas the null mutant (KO) mice showed enhanced-object exploration as well as open-field exploration. In MS knockdown mice, only type 2 hippocampal theta rhythm was enhanced, whereas both type 1 and type 2 theta rhythm were enhanced in KO mice. This selective effect was accompanied by markedly increased excitability of septo-hippocampal GABAergic projection neurons in the MS lacking T-type Ca²⁺ channels. Furthermore, optogenetic activation of the septo-hippocampal GABAergic pathway in WT mice also selectively enhanced object exploration behavior and type 2 theta rhythm, whereas inhibition of the same pathway decreased the behavior and the rhythm. These findings define object exploration distinguished from open-field exploration and reveal a critical role of T-type Ca²⁺ channels in the medial septal GABAergic projection neurons in this behavior.When confronted with an unfamiliar environment, or physical or social objects, animals often exhibit behavior patterns that can broadly be termed exploration, such as moving around the environment, touching or sniffing novel objects, and interacting with social stimuli (1). Social exploration involves complex processes that differ from those involved in the nonsocial exploration (2). Several distinctions were proposed to categorize the different forms of nonsocial exploratory behaviors from a motivational perspective (3). Behaviorally, two types of nonsocial exploration are observed in rodents and humans (3–5): object exploration and spatial or environmental exploration in the absence of objects. Object exploration is the behavior to explore discrete novel objects. This activity is elicited and sustained by the physical presence of an object. Several types of preference or “novelty” tests have been developed to investigate object exploration in rodents (3, 5–7). Environmental or spatial exploration in the absence of objects refers to the inquisitive activity of an animal in a new space, where the eliciting and sustaining stimulus is the “place” itself. Various forms of open-field tests have been used to investigate environmental or spatial exploration in rodents (3, 5, 8). Experimentally, however, the distinction can be less obvious because both can occur together (4, 7–9). Spatial exploration is suggested to be hippocampal-dependent (10)—although that is controversial (11)—whereas object exploration is suggested to be hippocampal-independent (12). Thus, it is still a matter of debate whether animal exploration belongs to a unitary category or not (9). To resolve this issue, neural definitions of these two previously proposed exploratory behaviors are needed.Interestingly, the medial septum (MS), where Ca_v3.1 T-type Ca²⁺ channels are highly expressed (13), is suggested to be critical for exploratory behaviors (5, 14–16). Moreover, the MS is also the nodal point for ascending afferent systems involved in the generation of hippocampal theta rhythms, the largest synchronous oscillatory signals in the mammalian brain, which are implicated in diverse brain functions (17, 18). Although the heterogeneity of hippocampal theta rhythms has long been under debate (19), recent studies based on genetic mutations in mice and optogenetics provide strong support for theta rhythm heterogeneity (20–22). However, their exact behavioral correlates are still debated. Ca_v3.1 Ca²⁺ channels play an important role in diverse behaviors, as well as the generation of physiologic and pathophysiologic brain rhythms (23). Notably, T-type, low-threshold Ca²⁺ currents are assumed to be a candidate ionic mechanism of theta rhythm genesis (24), analogous to the role of T-type channels in the generation of oscillations in the reticular nucleus of the thalamus (25). Nevertheless the involvement of T-type Ca²⁺ channels in hippocampal theta rhythms or exploratory behavior has not been examined. Here, we analyzed global KO mice and mice with MS-specific inactivation of the Ca_v3.1 gene encoding T-type Ca²⁺ channels, focusing on finding the neural mechanism that control the exploratory behaviors. Using a combination of tools, we provide evidence that object and open field exploratory behaviors are processed differently in the brain. Furthermore, Ca_v3.1 T-type Ca²⁺ channels in the septo-hippocampal GABAergic projection neurons are critically involved in controlling object exploration through modulating hippocampal type 2 theta rhythm.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

Effects of lymphocyte profile on development of EBV-induced lymphoma subtypes in humanized mice

Epstein-Barr virus (EBV) infection causes both Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL). The present study reveals that EBV-induced HL and NHL are intriguingly associated with a repopulated immune cell profile in humanized mice. Newborn immunodeficient NSG mice were engrafted with human cord blood CD34⁺ hematopoietic stem cells (HSCs) for a 8- or 15-wk reconstitution period (denoted ^8whN and ^15whN, respectively), resulting in human B-cell and T-cell predominance in peripheral blood cells, respectively. Further, novel humanized mice were established via engraftment of hCD34⁺ HSCs together with nonautologous fetal liver-derived mesenchymal stem cells (MSCs) or MSCs expressing an active notch ligand DLK1, resulting in mice skewed with human B or T cells, respectively. After EBV infection, whereas NHL developed more frequently in B-cell–predominant humanized mice, HL was seen in T-cell–predominant mice (P = 0.0013). Whereas human splenocytes from NHL-bearing mice were positive for EBV-associated NHL markers (hBCL2⁺, hCD20⁺, hKi67⁺, hCD20⁺/EBNA1⁺, and EBER⁺) but negative for HL markers (LMP1⁻, EBNA2⁻, and hCD30⁻), most HL-like tumors were characterized by the presence of malignant Hodgkin’s Reed–Sternberg (HRS)-like cells, lacunar RS (hCD30⁺, hCD15⁺, IgJ⁻, EBER⁺/hCD30⁺, EBNA1⁺/hCD30⁺, LMP⁺/EBNA2⁻, hCD68⁺, hBCL2⁻, hCD20^-/weak, Phospho STAT6⁺), and mummified RS cells. This study reveals that immune cell composition plays an important role in the development of EBV-induced B-cell lymphoma.Epstein Barr virus (EBV) infects human B lymphocytes and epithelial cells in >90% of the human population (1, 2). EBV infection is widely associated with the development of diverse human disorders that include Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphomas (NHL), including diffused large B-cell lymphoma (DLBCL), follicular B-cell lymphoma (FBCL), endemic Burkitt’s lymphoma (BL), and hemophagocytic lymphohistiocytosis (HLH) (3).HL is a malignant lymphoid neoplasm most prevalent in adolescents and young adults (4–6). Hodgkin/Reed–Sternberg (HRS) cells are the sole malignant cells of HL. HRS cells are characterized by CD30⁺/CD15⁺/BCL6⁻/CD20^+/− markers and appear large and multinucleated owing to multiple nuclear divisions without cytokinesis. Although HRS cells are malignant in the body, surrounding inflammatory cells greatly outnumber them. These reactive nonmalignant inflammatory cells, including lymphocytes, histiocytes, eosinophils, fibroblasts, neutrophils, and plasma cells, compose the vast majority of the tumor mass. The presence of HRS cells in the context of this inflammatory cellular background is a critical hallmark of the HL diagnosis (4). Approximately 50% of HL cases are EBV-associated (EBVaHL) (7–11). EBV-positive HRS cells express EBV latent membrane protein (LMP) 1 (LMP1), LMP2A, LMP2B, and EBV nuclear antigen (EBNA) 1 (EBNA1), but lack EBNA2 (latency II marker) (12). LMP1 is consistently expressed in all EBV-associated cases of classical HL (13, 14). LMP1 mimics activated CD40 receptors, induces NF-κB, and allows cells to become malignant while escaping apoptosis (15).The etiologic role of EBV in numerous disorders has been studied in humanized mouse models in diverse experimental conditions. Humanized mouse models recapitulate key characteristics of EBV infection-associated disease pathogenesis (16–24). Different settings have given rise to quite distinct phenotypes, including B-cell type NHL (DLBCL, FBCL, and unspecified B-cell lymphomas), natural killer/T cell lymphoma (NKTCL), nonmalignant lymphoproliferative disorder (LPD), extremely rare HL, HLH, and arthritis (16–24). Despite considerable efforts (16–24), EBVaHL has not been properly produced in the humanized mouse setting model, owing to inappropriate animal models and a lack of in-depth analyses. After an initial report of infected humanized mice, HRS-like cells appeared to be extremely rare in the spleens of infected humanized mice; however, the findings were inconclusive (18). Here we report direct evidence of EBVaHL or HL-like neoplasms in multiple humanized mice in which T cells were predominant over B cells. Our study demonstrates that EBV-infected humanized mice display additional EBV-associated pathogenesis, including DLBCL and hemophagocytic lymphohistiocytosis (16, 17).     

Overexpression of junctophilin-2 does not enhance baseline function but attenuates heart failure development after cardiac stress

Heart failure is accompanied by a loss of the orderly disposition of transverse (T)-tubules and a decrease of their associations with the junctional sarcoplasmic reticulum (jSR). Junctophilin-2 (JP2) is a structural protein responsible for jSR/T-tubule docking. Animal models of cardiac stresses demonstrate that down-regulation of JP2 contributes to T-tubule disorganization, loss of excitation-contraction coupling, and heart failure development. Our objective was to determine whether JP2 overexpression attenuates stress-induced T-tubule disorganization and protects against heart failure progression. We therefore generated transgenic mice with cardiac-specific JP2 overexpression (JP2-OE). Baseline cardiac function and Ca²⁺ handling properties were similar between JP2-OE and control mice. However, JP2-OE mice displayed a significant increase in the junctional coupling area between T-tubules and the SR and an elevated expression of the Na⁺/Ca²⁺ exchanger, although other excitation-contraction coupling protein levels were not significantly changed. Despite similar cardiac function at baseline, overexpression of JP2 provided significantly protective benefits after pressure overload. This was accompanied by a decreased percentage of surviving mice that developed heart failure, as well as preservation of T-tubule network integrity in both the left and right ventricles. Taken together, these data suggest that strategies to maintain JP2 levels can prevent the progression from hypertrophy to heart failure.In working ventricular myocytes, normal excitation-contraction (E-C) coupling requires precise communication between voltage-gated L-type Ca²⁺ channels (Cav1.2) located in clusters within transverse (T)-tubules and, less frequently, on the plasmalemma, and Ca²⁺ release channels/ryanodine receptor channels (RyRs) that are also clustered on the junctional sarcoplasmic reticulum (jSR) membrane (1–4). In normal hearts, flat jSR cisternae containing a continuous row of polymerized calsequestrin (CsQ2) either wrap around a T-tubule segment or abut against the plasmalemma (5, 6) and are coupled to the surface membranes via apposed clusters of RyR2 and Cav1.2 (7). These junctional sites are called dyads. However, although the jSR cisternae constitute a single continuous compartment, the clusters of RyR2 do not occupy the whole jSR surface but are in smaller groups (8, 9). Hence, each dyad is composed of several smaller RyR2/Cav1.2 complexes, also called couplons. Functional interaction between Cav1.2 and RyR2 at these sites ensure synchronous SR Ca²⁺ release and coordinated contraction (1, 10, 11). There is evidence that impaired cardiac E-C coupling/Ca²⁺ handling is a key mediator of heart failure (12, 13). One underlying mechanism for the defective Ca²⁺ release is the progressive loss of T-tubule network organization and of the relationship between RyR2 and Cav1.2 (14–16). Therefore, preventing loss of jSR/T-tubule junctions and of T-tubule organization may represent a new strategy for therapeutic intervention in heart failure.In normal cardiomyocytes, the formation of dyads requires junctophilin 2 (JP2), a structural protein that provides a physical connection between the T-tubule and SR membranes (17). JP2’s eight N-terminal “membrane occupation and recognition nexus” domains bind to the plasmalemma (T-tubules), and its C-terminal transmembrane domain tethers the opposite end to the SR membrane (17). Decreased JP2 levels have been observed in human heart failure patients and in failing hearts from animal models of cardiac disease (16, 18–22). Knockdown of JP2 results in acute heart failure that is associated with the loss of junctional membrane complex, disrupted T-tubule organization, and Ca²⁺ handling dysfunction (23). In addition, embryonic myocytes with JP2 deficiency have defective cardiac dyads, including more SR segments with no T-tubule couplings as well as reduced intracellular Ca²⁺ transients (17). These data collectively suggest that loss of JP2 contributes to the functional defects in heart failure. Therefore, interesting questions are: Is the JP2 deficiency effect linked to the resultant disruption of jSR/T-tubule junctions and of T-tubule network integrity, as suggested by previous findings (16–18, 23)? Conversely, could exogenous overexpression of JP2 in cardiomyocytes improve Ca²⁺ handling and protect against the development of heart failure?To answer this question, we generated transgenic mice with cardiac-specific overexpression of JP2. Moderate overexpression of JP2 led to a significant increase in the junctional coupling area between T-tubule and SR membrane, but surprisingly, it did not enhance cardiac function or increase SR Ca²⁺ release at baseline. However, interestingly, JP2-overexpressing mice were resistant to left ventricular pressure overload-induced heart failure, demonstrating that JP2 overexpression is protective. These data suggest that preventing the loss of JP2 could be a potential therapeutic strategy for heart failure treatment.     

Quantum mechanical calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl,oxygen-rebound mechanism

Lytic polysaccharide monooxygenases (LPMOs) exhibit a mononuclear copper-containing active site and use dioxygen and a reducing agent to oxidatively cleave glycosidic linkages in polysaccharides. LPMOs represent a unique paradigm in carbohydrate turnover and exhibit synergy with hydrolytic enzymes in biomass depolymerization. To date, several features of copper binding to LPMOs have been elucidated, but the identity of the reactive oxygen species and the key steps in the oxidative mechanism have not been elucidated. Here, density functional theory calculations are used with an enzyme active site model to identify the reactive oxygen species and compare two hypothesized reaction pathways in LPMOs for hydrogen abstraction and polysaccharide hydroxylation; namely, a mechanism that employs a η¹-superoxo intermediate, which abstracts a substrate hydrogen and a hydroperoxo species is responsible for substrate hydroxylation, and a mechanism wherein a copper-oxyl radical abstracts a hydrogen and subsequently hydroxylates the substrate via an oxygen-rebound mechanism. The results predict that oxygen binds end-on (η¹) to copper, and that a copper-oxyl–mediated, oxygen-rebound mechanism is energetically preferred. The N-terminal histidine methylation is also examined, which is thought to modify the structure and reactivity of the enzyme. Density functional theory calculations suggest that this posttranslational modification has only a minor effect on the LPMO active site structure or reactivity for the examined steps. Overall, this study suggests the steps in the LPMO mechanism for oxidative cleavage of glycosidic bonds.Carbohydrates are the most diverse set of biomolecules, and thus, many enzyme classes have evolved to assemble, modify, and depolymerize carbohydrates, including glycosyltransferases, glycoside hydrolases, carbohydrate esterases, and polysaccharide lyases (1). Recently, a new enzymatic paradigm was discovered that employs copper-dependent oxidation to cleave glycosidic bonds in polysaccharides (2–13). These newly classified enzymes, termed lytic polysaccharide monooxygenases (LPMOs), broadly resemble other copper monooxygenases and some hydroxylation catalysts (14–21).The discovery that LPMOs use an oxidative mechanism has attracted interest both because it is a unique paradigm for carbohydrate modification that employs a powerful C–H activation mechanism, and also because LPMOs synergize with hydrolytic enzymes in biomass conversion to sugars because they act directly on the crystalline polysaccharide surface without the requirement for depolymerization (4, 22, 23), making them of interest in biofuels production. LPMOs were originally characterized as Family 61 glycoside hydrolases (GH61s, reclassified as auxiliary activity 9, AA9) or Family 33 carbohydrate-binding modules (CBM33s, reclassified as AA10), which are structurally similar enzymes found in fungi and nonfungal organisms (22), respectively. In 2005, Vaaje-Kolstad et al. described the synergism (24) of a chitin-active CBM33 (chitin-binding protein, CBP21) with hydrolases, but the mechanism was not apparent. Harris et al. demonstrated that a GH61 boosts hydrolytic enzyme activity on lignocellulosic biomass (2). Vaaje-Kolstad et al. subsequently showed that CBP21 employs an oxidative mechanism to cleave glycosidic linkages in chitin (4).Following these initial discoveries, multiple features of LPMOs have been elucidated. LPMOs use copper (5–7) and produce either aldonic acids or 4-keto sugars at oxidized chain ends, believed to result from hydroxylation at the C1 or C4 carbon, respectively. Hydroxylation at the C1 carbon is proposed to spontaneously undergo elimination to a lactone followed by hydrolytic ring opening to an aldonic acid, whereas hydroxylation and elimination at C4 yields a 4-keto sugar at the nonreducing end (5–12). The active site is a mononuclear type(II) copper center ligated by a “histidine brace” (5, 12), comprising a bidentate N-terminal histidine ligand via the amino terminus and an imidazole ring nitrogen atom and another histidine residue also via a ring nitrogen atom. Hemsworth et al. reported a bacterial LPMO structure wherein the active site copper ion was photoreduced to Cu(I) (12), and Aachmann et al. demonstrated that Cu(I) binds with higher affinity than Cu(II) in CBP21 (13). A structural study of a fungal LPMO revealed an N-terminal methylation on a nitrogen atom in the imidazole ring of unknown function (5), but some LPMOs are active without this modification (6, 11). LPMOs require reducing agents for activity such as ascorbate (2–8, 10–12), and cellobiose dehydrogenase (CDH), a common fungal secretome component, can potentiate LPMO activity in lieu of a small-molecule reducing agent (7, 8).Overall, many structural and mechanistic insights have been reported since the discoveries that LPMOs are oxidative enzymes (4–10). However, many questions remain regarding LPMO function (22, 25). Here, we examine the LPMO catalytic mechanism with density functional theory (DFT) calculations on an active site model (ASM) of a fungal LPMO. We seek to (i) understand the identity of the reactive oxygen species (ROS), (ii) compare two hypothesized catalytic mechanisms, and (iii) examine the role of N-terminal methylation in catalysis.     

From the Cover: The point of no return in vetoing self-initiated movements

In humans, spontaneous movements are often preceded by early brain signals. One such signal is the readiness potential (RP) that gradually arises within the last second preceding a movement. An important question is whether people are able to cancel movements after the elicitation of such RPs, and if so until which point in time. Here, subjects played a game where they tried to press a button to earn points in a challenge with a brain–computer interface (BCI) that had been trained to detect their RPs in real time and to emit stop signals. Our data suggest that subjects can still veto a movement even after the onset of the RP. Cancellation of movements was possible if stop signals occurred earlier than 200 ms before movement onset, thus constituting a point of no return.It has been repeatedly shown that spontaneous movements are preceded by early brain signals (1–8). As early as a second before a simple voluntary movement, a so-called readiness potential (RP) is observed over motor-related brain regions (1–3, 5). The RP was found to precede the self-reported time of the “‘decision’ to act” (ref. 3, p. 623). Similar preparatory signals have been observed using invasive electrophysiology (8, 9) and functional MRI (7, 10), and have been demonstrated also for choices between multiple-response options (6, 7, 10), for abstract decisions (10), for perceptual choices (11), and for value-based decisions (12). To date, the exact nature and causal role of such early signals in decision making is debated (12–20).One important question is whether a person can still exert a veto by inhibiting the movement after onset of the RP (13, 18, 21, 22). One possibility is that the onset of the RP triggers a causal chain of events that unfolds in time and cannot be cancelled. The onset of the RP in this case would be akin to tipping the first stone in a row of dominoes. If there is no chance of intervening, the dominoes will gradually fall one-by-one until the last one is reached. This has been coined a ballistic stage of processing (23, 24). A different possibility is that participants can still terminate the process, akin to taking out a domino at some later stage in the chain and thus preventing the process from completing. Here, we directly tested this in a real-time experiment that required subjects to terminate their decision to move once a RP had been detected by a brain–computer interface (BCI) (25–31).