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.     

Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment

The spindle assembly checkpoint (SAC) is a conserved signaling pathway that monitors faithful chromosome segregation during mitosis. As a core component of SAC, the evolutionarily conserved kinase monopolar spindle 1 (Mps1) has been implicated in regulating chromosome alignment, but the underlying molecular mechanism remains unclear. Our molecular delineation of Mps1 activity in SAC led to discovery of a previously unidentified structural determinant underlying Mps1 function at the kinetochores. Here, we show that Mps1 contains an internal region for kinetochore localization (IRK) adjacent to the tetratricopeptide repeat domain. Importantly, the IRK region determines the kinetochore localization of inactive Mps1, and an accumulation of inactive Mps1 perturbs accurate chromosome alignment and mitotic progression. Mechanistically, the IRK region binds to the nuclear division cycle 80 complex (Ndc80C), and accumulation of inactive Mps1 at the kinetochores prevents a dynamic interaction between Ndc80C and spindle microtubules (MTs), resulting in an aberrant kinetochore attachment. Thus, our results present a previously undefined mechanism by which Mps1 functions in chromosome alignment by orchestrating Ndc80C–MT interactions and highlight the importance of the precise spatiotemporal regulation of Mps1 kinase activity and kinetochore localization in accurate mitotic progression.Faithful distribution of the duplicated genome into two daughter cells during mitosis depends on proper kinetochore–microtubule (MT) attachments. Defects in kinetochore–MT attachments result in chromosome missegregation, causing aneuploidy, a hallmark of cancer (1, 2). To ensure accurate chromosome segregation, cells use the spindle assembly checkpoint (SAC) to monitor kinetochore biorientation and to control the metaphase-to-anaphase transition. Cells enter anaphase only after the SAC is satisfied, requiring that all kinetochores be attached to MTs and be properly bioriented (3, 4). The core components of SAC signaling include mitotic arrest deficient-like 1 (Mad1), Mad2, Mad3/BubR1 (budding uninhibited by benzimidazole-related 1), Bub1, Bub3, monopolar spindle 1 (Mps1), and aurora B. The full SAC function requires the correct centromere/kinetochore localization of all SAC proteins (5).Among the SAC components, Mps1 was identified originally in budding yeast as a gene required for duplication of the spindle pole body (6). Subsequently, Mps1 orthologs were found in various species, from fungi to mammals. The stringent requirement of Mps1 for SAC activity is conserved in evolution (6–13). Human Mps1 kinase (also known as “TTK”) is expressed in a cell-cycle–dependent manner and has highest expression levels and activity during mitosis. Its localization is also dynamic (8, 14). Although the molecular mechanism remains unclear, Mps1 is required to recruit Mad1 and Mad2 to unattached kinetochores, supporting its essential role in SAC activity (15–18). It also is clear that aurora B kinase activity and the outer-layer kinetochore protein nuclear division cycle 80 (Ndc80)/Hec1 are required for Mps1 localization to kinetochores, as evidenced by recent work, including ours (17, 19–24). How Mps1 activates the SAC is now becoming clear. Mps1 recruits Bub1/Bub3 and BubR1/Bub3 to kinetochores through phosphorylation of KNL1, the kinetochore receptor protein of Bub1 and BubR1 (25–30).Despite much progress in understanding Mps1 functions, it remains unclear how Mps1 is involved in regulating chromosome alignment. In budding yeast mitosis, Mps1 regulates mitotic chromosome alignment by promoting kinetochore biorientation independently of Ipl1 (aurora B in humans) (31), but in budding yeast meiosis Mps1 must collaborate with Ipl1 to mediate meiotic kinetochore biorientation (32). In humans, Mps1 regulates chromosomal alignment by modulating aurora B kinase activity (33), but recent chemical biology studies show that Mps1 kinase activity is important for proper chromosome alignment and segregation, independently of aurora B (22, 34–36). Therefore whether Mps1 regulates chromosome alignment through modulation of aurora B kinase activity is still under debate (37).In this study, we reexamined the function of human Mps1 in chromosome alignment. We found that chromosomal alignment is largely achieved in Mps1 knockdown cells, provided that cells are arrested in metaphase in the presence of MG132, a proteasome inhibitor. However, disrupting Mps1 activity via small molecule inhibitors perturbs chromosomal alignment, even in the presence of MG132. This chromosome misalignment is caused by the abnormal accumulation of inactive Mps1 in the kinetochore and the subsequent failure of correct kinetochore–MT attachments. Further, we demonstrate that inactive Mps1 does not depend on the previously reported tetratricopeptide repeat (TPR) domain for localizing to kinetochores, and we identify a previously unidentified region adjacent to the C terminus of the TPR domain that is responsible for localizing inactive Mps1 to kinetochores. Thus, our work highlights that Mps1 kinase activity is necessary in regulating chromosome alignment and that it must be tightly regulated in space and time to ensure proper localization of Mps1 at kinetochores.     

Longitudinal spread of mechanical excitation through tectorial membrane traveling waves

The mammalian inner ear separates sounds by their frequency content, and this separation underlies important properties of human hearing, including our ability to understand speech in noisy environments. Studies of genetic disorders of hearing have demonstrated a link between frequency selectivity and wave properties of the tectorial membrane (TM). To understand these wave properties better, we developed chemical manipulations that systematically and reversibly alter TM stiffness and viscosity. Using microfabricated shear probes, we show that (i) reducing pH reduces TM stiffness with little change in TM viscosity and (ii) adding PEG increases TM viscosity with little change in TM stiffness. By applying these manipulations in measurements of TM waves, we show that TM wave speed is determined primarily by stiffness at low frequencies and by viscosity at high frequencies. Both TM viscosity and stiffness affect the longitudinal spread of mechanical excitation through the TM over a broad range of frequencies. Increasing TM viscosity or decreasing stiffness reduces longitudinal spread of mechanical excitation, thereby coupling a smaller range of best frequencies and sharpening tuning. In contrast, increasing viscous loss or decreasing stiffness would tend to broaden tuning in resonance-based TM models. Thus, TM wave and resonance mechanisms are fundamentally different in the way they control frequency selectivity.The sharp frequency selectivity of auditory nerve fiber responses to sound is a hallmark of mammalian cochlear function. This remarkable signal processing originates in the mechanical stage of the cochlear signal processing chain (1–7), as evidenced by measured motions and mechanical properties of the basilar membrane (BM) (2–9) and tectorial membrane (TM) (10–24). Although the hydromechanical mechanisms underlying BM motions have been characterized based on experimental and theoretical studies, the mechanisms underlying TM motions remain unclear.The TM is an acellular matrix that overlies the hair bundles of sensory receptor cells. Based on its strategic position above the organ of Corti, conventional cochlear models (25–29) have implicated local mechanical properties (i.e., mass, stiffness) of the TM in stimulating the sensory hair bundles of hair cells and in cochlear tuning. Recent dynamic measurements of the TM, in vitro (17, 30–33) and in vivo (34), suggest that the TM supports longitudinal coupling, with large spatial extents across a broad range of frequencies. This longitudinal coupling manifests in the form of propagating traveling waves that are thought to contribute to hearing mechanisms (17, 21, 30, 35–40). Genetic modification studies provide further support that the spatial extent of TM waves may play a significant role in cochlear tuning (30, 32). Although these measurements, models, and genetic modification studies have confirmed the importance of TM mechanical properties in hearing, they have not isolated the distinct roles of TM stiffness and viscosity in generating longitudinally propagating traveling waves of the TM.To understand the contributions of TM material properties on traveling waves better, we developed chemical manipulations to alter the stiffness and viscosity of the TM selectively and reversibly. Because the TM is poroelastic (32, 41), we expect that changes in bath composition can have a direct effect on the mechanical properties of the TM mechanical matrix and its interstitial fluid, which makes up 97% of TM wet weight (42). The addition of PEG has previously been shown to generate an osmotic response that could be accounted for by the permeability of these molecules through the matrix rather than by direct changes to the matrix itself (41). In contrast, changing bath pH has little effect on the osmotic pressure or viscosity of the bath but has been shown to have a direct effect on the macromolecular matrix (43). In this paper, we apply these physicochemical manipulations to alter TM material properties reversibly, and thereby probe their role in controlling longitudinal spread of excitation through the TM.     

Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer

The surface of a living cell provides a platform for receptor signaling, protein sorting, transport, and endocytosis, whose regulation requires the local control of membrane organization. Previous work has revealed a role for dynamic actomyosin in membrane protein and lipid organization, suggesting that the cell surface behaves as an active composite composed of a fluid bilayer and a thin film of active actomyosin. We reconstitute an analogous system in vitro that consists of a fluid lipid bilayer coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors. Upon complete consumption of ATP, this system settles into distinct phases of actin organization, namely bundled filaments, linked apolar asters, and a lattice of polar asters. These depend on actin concentration, filament length, and actin/myosin ratio. During formation of the polar aster phase, advection of the self-organizing actomyosin network drives transient clustering of actin-associated membrane components. Regeneration of ATP supports a constitutively remodeling actomyosin state, which in turn drives active fluctuations of coupled membrane components, resembling those observed at the cell surface. In a multicomponent membrane bilayer, this remodeling actomyosin layer contributes to changes in the extent and dynamics of phase-segregating domains. These results show how local membrane composition can be driven by active processes arising from actomyosin, highlighting the fundamental basis of the active composite model of the cell surface, and indicate its relevance to the study of membrane organization.The cell surface mediates interactions between the cell and the outside world by serving as the site for signal transduction. It also facilitates the uptake and release of cargo and supports adhesion to substrates. These diverse roles require that the cell surface components involved in each function are spatially and temporally organized into domains spanning a few nanometers (nanoclusters) to several micrometers (microdomains). The cell surface itself may be considered as a fluid–lipid bilayer wherein proteins are embedded (1). In the living cell, this multicomponent system is supported by an actin cortex, composed of a branched network of actin and a collection of filaments (2–4).Current models of membrane organization fall into three categories: those invoking lipid–lipid and lipid–protein interactions in the plasma membrane [e.g., the fluid mosaic model (1, 5) and the lipid raft hypothesis (6)], or those that appeal to the membrane-associated actin cortex (e.g., the picket fence model) (7), or a combination of these (8, 9). Although these models based on thermodynamic equilibrium principles have successfully explained the organization and dynamics of a range of membrane components and molecules, there is a growing class of phenomena that appears inconsistent with chemical and thermal equilibrium, which might warrant a different explanation. These include aspects of the organization and dynamics of outer leaflet glycosyl-phosphatidylinositol-anchored proteins (GPI-anchored proteins) (10–13), inner leaflet Ras proteins (14), and actin-binding transmembrane proteins (13, 15, 16).Recent experimental and theoretical work has shown that these features can be explained by taking into account that many cortical and membrane proteins are driven by ATP-consuming processes that drive the system out of equilibrium (13, 15, 17). The membrane models mentioned above have by-and-large neglected this active nature of the actin cortex where actin filaments are being continuously polymerized and depolymerized (18–21), in addition to being persistently acted upon by a variety of myosin motors (22–24) that consume ATP and exert contractile stresses on cortical actin filaments, continually remodeling the architecture of the cortex (4, 21, 25). These active processes in turn can generate tangential stresses and currents on the cell surface, which could drive the dynamics and local composition of membrane components at different scales (22, 26–29).Actin polymerization is proposed to be driven at the membrane by two nucleators, the Arp2/3 complex, which creates a densely branched network, as well as formins that nucleate filaments (18, 21, 30). A number of myosin motors are also associated with the juxtamembranous actin cortex, of which nonmuscle myosin II is the major component in remodeling the cortex and creating actin flows (4, 23, 25, 26, 31, 32). Based on our observations that the clustering of cell surface components that couple directly or indirectly to cortical actin [e.g., GPI-anchored proteins, proteins of the Ezrin, Radaxin, or Moesin (ERM) family (13, 15)] depends on myosin activity, we proposed that this clustering arises from the coupling to contractile actomyosin platforms (called “actin asters”) produced at the cortex (15, 33).A coarse-grained theory describing this idea has been put forward and corroborated by the verification of its key predictions in live cells (15, 33), but a systematic identification of the underlying microscopic processes is lacking. Given the complexity of numerous processes acting at the membrane of a living cell, we use an in vitro approach to study the effect of an energy-consuming actomyosin network on the dynamics of membrane molecules that directly interact with filamentous actin.A series of in vitro studies have explored the organization of confined, dynamic filaments (both actin and microtubules) (34–39) or the role of actin architecture on membrane organization (40–46). Indeed, these studies have yielded insights into the nontrivial emergent configurations that mixtures of polar filaments and motors can adopt when fueled by ATP (34–37), in particular constitutively remodeling steady states that display characteristics of active mechanics (38, 39, 47). However, the effect of linking these mechanics to the confining lipid bilayer and its organization has not been studied.The consequences of actin polymerization on membrane organization, in particular on giant unilamellar vesicles (GUVs), have been addressed in a number of studies on the propulsion of GUVs by an actin comet tail (40, 45, 46). In those experiments, the apparent advection of membrane bound ActA or WASP toward the site of actin polymerization is mainly due to the change in binding affinity of WASP to actin through Arp2/3 (44) and the spherical geometry resulting in the drag of actin to one pole of the vesicle after symmetry break of the actin shell. That this dynamic process changes the bulk properties of the bilayer, namely the critical temperature of a phase-separating lipid bilayer, was shown by Liu and Fletcher (40) when the actin nucleator N-WASP was connected to a lipid species (PIP₂) that was capable of partitioning into one of the two phases.Besides these pioneering studies on the effects of active processes on membrane organization, little was done to directly test the effect of active lateral stresses as well as actomyosin remodeling at the membrane, particularly on the dynamics and organization of membrane-associated components.To this end, we build an active composite in vitro by stepwise addition of components: a supported lipid bilayer with an actin-binding component, actin filaments, and myosin motors. By systematically varying the concentrations of actin and myosin as well as the average actin filament length, we find distinct states of actomyosin organization at the membrane surface upon complete ATP consumption. More importantly, we find that the ATP-fueled contractile actomyosin currents induce the transient accumulation of actin-binding membrane components. As predicted, the active mechanics of actin and myosin at physiologically relevant ATP concentrations drives the system into a nonequilibrium steady state with anomalous density fluctuations and the transient clustering of actin-binding components of the lipid bilayer (15, 33). Finally, connection of this active layer of actomyosin to a phase-segregating bilayer, influences its phase behavior and coarsening dynamics.     

α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation

Physiologically, α-synuclein chaperones soluble NSF attachment protein receptor (SNARE) complex assembly and may also perform other functions; pathologically, in contrast, α-synuclein misfolds into neurotoxic aggregates that mediate neurodegeneration and propagate between neurons. In neurons, α-synuclein exists in an equilibrium between cytosolic and membrane-bound states. Cytosolic α-synuclein appears to be natively unfolded, whereas membrane-bound α-synuclein adopts an α-helical conformation. Although the majority of studies showed that cytosolic α-synuclein is monomeric, it is unknown whether membrane-bound α-synuclein is also monomeric, and whether chaperoning of SNARE complex assembly by α-synuclein involves its cytosolic or membrane-bound state. Here, we show using chemical cross-linking and fluorescence resonance energy transfer (FRET) that α-synuclein multimerizes into large homomeric complexes upon membrane binding. The FRET experiments indicated that the multimers of membrane-bound α-synuclein exhibit defined intermolecular contacts, suggesting an ordered array. Moreover, we demonstrate that α-synuclein promotes SNARE complex assembly at the presynaptic plasma membrane in its multimeric membrane-bound state, but not in its monomeric cytosolic state. Our data delineate a folding pathway for α-synuclein that ranges from a monomeric, natively unfolded form in cytosol to a physiologically functional, multimeric form upon membrane binding, and show that only the latter but not the former acts as a SNARE complex chaperone at the presynaptic terminal, and may protect against neurodegeneration.α-Synuclein is an abundant presynaptic protein that physiologically acts to promote soluble NSF attachment protein receptor (SNARE) complex assembly in vitro and in vivo (1–3). Point mutations in α-synuclein (A30P, E46K, H50Q, G51D, and A53T) as well as α-synuclein gene duplications and triplications produce early-onset Parkinson''s disease (PD) (4–10). Moreover, α-synuclein is a major component of intracellular protein aggregates called Lewy bodies, which are pathological hallmarks of neurodegenerative disorders such as PD, Lewy body dementia, and multiple system atrophy (11–14). Strikingly, neurotoxic α-synuclein aggregates propagate between neurons during neurodegeneration, suggesting that such α-synuclein aggregates are not only intrinsically neurotoxic but also nucleate additional fibrillization (15–18).α-Synuclein is highly concentrated in presynaptic terminals where α-synuclein exists in an equilibrium between a soluble and a membrane-bound state, and is associated with synaptic vesicles (19–22). The labile association of α-synuclein with membranes (23, 24) suggests that binding of α-synuclein to synaptic vesicles, and its dissociation from these vesicles, may regulate its physiological function. Membrane-bound α-synuclein assumes an α-helical conformation (25–32), whereas cytosolic α-synuclein is natively unfolded and monomeric (refs. 25, 26, 31, and 32; however, see refs. 33 and 34 and Discussion for a divergent view). Membrane binding by α-synuclein is likely physiologically important because in in vitro experiments, α-synuclein remodels membranes (35, 36), influences lipid packing (37, 38), and induces vesicle clustering (39). Moreover, membranes were found to be important for the neuropathological effects of α-synuclein (40–44).However, the relation of membrane binding to the in vivo function of α-synuclein remains unexplored, and it is unknown whether α-synuclein binds to membranes as a monomer or oligomer. Thus, in the present study we have investigated the nature of the membrane-bound state of α-synuclein and its relation to its physiological function in SNARE complex assembly. We found that soluble monomeric α-synuclein assembles into higher-order multimers upon membrane binding and that membrane binding of α-synuclein is required for its physiological activity in promoting SNARE complex assembly at the synapse.     

Modulation of a voltage-gated Na+ channel by sevoflurane involves multiple sites and distinct mechanisms

Halogenated inhaled general anesthetic agents modulate voltage-gated ion channels, but the underlying molecular mechanisms are not understood. Many general anesthetic agents regulate voltage-gated Na⁺ (Na_V) channels, including the commonly used drug sevoflurane. Here, we investigated the putative binding sites and molecular mechanisms of sevoflurane action on the bacterial Na_V channel NaChBac by using a combination of molecular dynamics simulation, electrophysiology, and kinetic analysis. Structural modeling revealed multiple sevoflurane interaction sites possibly associated with NaChBac modulation. Electrophysiologically, sevoflurane favors activation and inactivation at low concentrations (0.2 mM), and additionally accelerates current decay at high concentrations (2 mM). Explaining these observations, kinetic modeling suggests concurrent destabilization of closed states and low-affinity open channel block. We propose that the multiple effects of sevoflurane on NaChBac result from simultaneous interactions at multiple sites with distinct affinities. This multiple-site, multiple-mode hypothesis offers a framework to study the structural basis of general anesthetic action.General anesthetic agents have been in use for more than 160 y. However, we still understand relatively little about their mechanisms of action, which greatly limits our ability to design safer and more effective general anesthetic agents. Ion channels of the central nervous system are known to be key targets of general anesthetic agents, as their modulation can account for the endpoints and side effects of general anesthesia (1–4). Many families of ion channels are modulated by general anesthetic agents, including ligand-gated, voltage-gated, and nongated ion channels (2, 5–7). Mammalian voltage-gated Na⁺ (Na_V) channels, which mediate the upstroke of the action potential, are regulated by numerous inhaled general anesthetic agents (8–14), which generally cause inhibition. Previous work showed that inhaled general anesthetic agents, including sevoflurane, isoflurane, desflurane, and halothane, mediate inhibition by increasing the rate of Na⁺ channel inactivation, hyperpolarizing steady-state inactivation, and slowing recovery from inactivation (11, 15–18). Inhibition of presynaptic Na_V channels in the spinal cord is proposed to lead to inhibition of neurotransmitter release, facilitating immobilization—one of the endpoints of general anesthesia (14, 19, 20). Despite the importance of Na_V channels as general anesthetic targets, little is known about interaction sites or the mechanisms of action.What is known about anesthetic sites in Na_V channels comes primarily from the local anesthetic field. Local anesthetic agent binding to Na_V channels is well characterized. These amphiphilic drugs enter the channel pore from the intracellular side, causing open-channel block (21). Investigating molecular mechanisms of mammalian Na_V channel modulation by general anesthetic agents has been complicated by the lack of high-resolution structures of these channels as a result of their large size and pseudotetrameric organization. However, the recent discovery of the smaller, tetrameric bacterial Na⁺ channel family has provided an invaluable tool to characterize the structural features of Na_V channels and investigate their interactions with general anesthetic agents at the molecular level (22, 23). Several bacterial Na⁺ channels have been crystallized (24–27). These channels have a classical domain structure in which helices S1–S4 form the voltage sensor domain (VSD), S5 and S6 form the pore, and the S4–S5 linker connects the voltage sensor to the pore domain. One notable structural feature is the presence of “fenestrations” or hydrophobic tunnels through the pore domain (24).Although crystal structures are not yet reported, the bacterial Na⁺ channel NaChBac has been extensively characterized by electrophysiology (22, 28–36). Additionally NaChBac exhibits conserved slow open channel block in response to local and general anesthetic agents (15, 37). These anesthetic agents reduce peak current and accelerate current decay, making it conceivable that local and general anesthetic agents could share a site of action in NaChBac. The local anesthetic binding site identified in the central cavity of the mammalian Na_V1.2 channel, which mediates open channel block, is partially conserved in NaChBac (37, 38). A recent molecular dynamics (MD) modeling study found that isoflurane, which inhibits NaChBac (15), interacts with multiple regions of this channel, including the pore, the selectivity filter, and the S4–S5 linker/S6 interface (39). Although the importance of these interactions on the modulation of mammalian Na_V channels remains to be determined, the available data indicate that NaChBac is currently one of the best starting points to investigate the mechanisms of action of sevoflurane.Here, we investigated NaChBac to gain structural insight into the mechanisms of inhaled anesthetic modulation of Na_V channels. The focus of this work is sevoflurane because this anesthetic is commonly used in clinical settings and is a known inhibitor of several mammalian Na_V channels (Na_V 1.4, 1.7, and 1.8) (11, 13). A three-pronged approach incorporating MD simulation, whole-cell patch-clamp electrophysiology, and kinetic modeling suggests that sevoflurane acts on multiple sites to alter gating and permeation. Whereas the effect on gating results from modulating activation and inactivation gating at low concentrations (0.2 mM), the permeation effect is apparent at high concentrations (2 mM) and results from open channel block (2 mM). Although the net inhibitory effect of these multisite interactions is consistent with anesthetic-induced reduction of neuronal firing, general anesthesia does not simply result from a global reduction in firing. General anesthesia depends on complex mechanisms throughout the brain, which include increases and decreases in firing (3). Thus, precisely how Na⁺ channel activation by sevoflurane fits into the global effects of anesthesia remains to be seen. The present work helps elucidate the molecular mechanism of sevoflurane action on Na_V channels.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Residue-level resolution of alphavirus envelope protein interactions in pH-dependent fusion

Alphavirus envelope proteins, organized as trimers of E2–E1 heterodimers on the surface of the pathogenic alphavirus, mediate the low pH-triggered fusion of viral and endosomal membranes in human cells. The lack of specific treatment for alphaviral infections motivates our exploration of potential antiviral approaches by inhibiting one or more fusion steps in the common endocytic viral entry pathway. In this work, we performed constant pH molecular dynamics based on an atomic model of the alphavirus envelope with icosahedral symmetry. We have identified pH-sensitive residues that cause the largest shifts in thermodynamic driving forces under neutral and acidic pH conditions for various fusion steps. A series of conserved interdomain His residues is identified to be responsible for the pH-dependent conformational changes in the fusion process, and ligand binding sites in their vicinity are anticipated to be potential drug targets aimed at inhibiting viral infections.Alphaviruses, mosquito-borne human pathogens causing severe inflammations and fatal fevers, have infected many millions of people in recent outbreaks worldwide since 2005 (1–3). The lack of a vaccine or specific treatment prompts investigations of the fundamental mechanisms of the alphaviral lifecycle to facilitate the development of effective antiviral therapies (4). Alphaviruses have been reported to enter the cell through receptor-mediated endocytosis. Here, alphaviruses are ferried toward the perinuclear space of the host cell inside vesicles towed by molecular motors and delivered to specific locations for productive replication (5–11). Even when direct entry into the cytoplasm is possible (11–15), the endocytic entry pathway facilitates the transportation of viruses across the crowded cytoplasmic space and delays detection by the immune system without leaving empty capsid or envelope as obvious evidence of the viral infection exposed outside the host cell (10, 11). Before the delivery of its viral genome into the cytoplasm of a host cell, the alphavirus must undergo a critical step of low pH-triggered membrane fusion, which is a common mechanism in the endocytic viral entry pathway among many different viruses. Understanding the mechanism of the low pH-triggered alphaviral membrane fusion is essential for the development of therapies against alphavirus as well as other viruses using similar endocytic entry mechanisms.Recent studies of the lifecycle of alphavirus reveal that a precursor, p62, is first synthesized as a chaperon forming a heterodimer with E1, which is essential for viral budding (16); p62 protects the E1 protein in the low-pH environment of the secretory pathway before being cleaved by cellular furin to produce mature E2-E1 and a smaller fragment, E3 (17–21). After the virus buds from the cytoplasmic membrane, E3 is released from the virus particle under neutral pH conditions outside the host cell (13, 22–24).On the surface of a mature alphavirus, 80 (E2–E1)₃ viral spikes, organized in T = 4 icosahedral symmetry on the viral lipid membrane, enclose the viral capsid and genome (25–43). On internalization of the mature virus in the endosome of the host cell in a new round of infection cycle, the increasingly acidified endosomal environment triggers a series of conformational changes in the alphaviral spike (E2–E1)₃ (38), including the dissociation of E2 (42, 44, 45), release of a fusion loop on E1 (46, 47), and trimerization of E1 (48). The fusion loop, roughly residues 83–100 on the cd loop of each E1 protein (13, 49, 50), in the newly formed E1 homotrimer (HT), inserts into the endosomal membrane. Then, the E1 proteins fold back, pulling the viral and endosomal membranes together and thus, promoting membrane fusion (13, 24).Recently solved high-resolution structures of the alphavirus envelope proteins E2–E1 fitted into cryo-EM data representing the intact virus under both acidic and neutral pH conditions (43, 51, 52) provide excellent atomic models for studies of the low pH-triggered fusion process. The structure of Chikungunya virus (CHIKV) obtained at pH 8.0 represents the initial mature state (M state) of the (E2–E1)₃ viral spike before the fusion process (51). Under pH 5.6, domain B (DB) of E2, which protects the E1 fusion loop, is observed to be disordered in Sindbis virus (52). The rest of the domains of the (E2–E1)₃ spike show moderate conformational differences with an rmsd = 4.0 Å among C_α atoms compared with the structures obtained at pH 8.0 for CHIKV (43, 51). The structure of the envelope proteins in acidic conditions most likely depicts a fusion intermediate (FI) state (52) before E2 dissociation during the low pH-triggered fusion process. In addition, the crystal structure of the folded-back E1 HT (53) is a good model to describe the postfusion state.Based on these atomic models of the E2 and E1 envelope proteins and our previously developed constant pH molecular dynamics (CPHMD) method (54–58), we simulated the envelope proteins with icosahedral symmetry under various pH conditions covering pH 2.0–9.0. We used pH replica exchange in CPHMD and calculated pK_a values using pH titration fitting, which has been shown as a reliable and accurate approach to capture pK_a values of protein residues in various systems (59–64). Through the CPHMD modeling, we calculated the pK_a of the possible pH-sensitive residues (Asp, Glu, and His) in the M, FI, dissociated E2 (Dis), and HT states. We, therefore, derive the shifts in the thermodynamic stabilities originating from each titrating residue for the steps from the M to the FI state (M→FI) of (E2–E1)₃, from the FI to the Dis state (FI→Dis) of E2 proteins, and from the FI to the HT state (FI→HT) of E1 proteins as shown in Fig. 1D. For these processes, we assume that the virus is in the endosomal environment, and we do not consider possible receptor-induced conformational changes. Our residue-level resolution simulations and analyses allow us to identify the critical functional residues with significant pK_a shifts and changes in thermodynamic stability in the low pH-triggered fusion activation. Our results suggest that the most pH-sensitive residues are highly conserved among different alphaviral species and that these critical residues control the pH threshold of fusion activities, provide guidance to further mutagenesis experiments, and lead to more fundamental understanding of low pH-triggered alphaviral membrane fusion.Open in a separate windowFig. 1.Structure and organization of alphaviral envelope proteins. (A) The alphaviral envelope modeled in our simulations. (B) The alphaviral envelope proteins in an MAU. (C) The heterodimer of E2 (DA–DB–DC) and E1 (DI–DII–DIII). (D) Structures of a viral spike in different conformational states simulated for shifts in pK_a values and thermodynamic stabilities. E1 proteins are shown in blue, cyan, and light blue. E2 proteins are shown in red, magenta, and pink.     

Enhanced receptor–clathrin interactions induced by N-glycan–mediated membrane micropatterning

Glycan–protein interactions are emerging as important modulators of membrane protein organization and dynamics, regulating multiple cellular functions. In particular, it has been postulated that glycan-mediated interactions regulate surface residence time of glycoproteins and endocytosis. How this precisely occurs is poorly understood. Here we applied single-molecule-based approaches to directly visualize the impact of glycan-based interactions on the spatiotemporal organization and interaction with clathrin of the glycosylated pathogen recognition receptor dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). We find that cell surface glycan-mediated interactions do not influence the nanoscale lateral organization of DC-SIGN but restrict the mobility of the receptor to distinct micrometer-size membrane regions. Remarkably, these regions are enriched in clathrin, thereby increasing the probability of DC-SIGN–clathrin interactions beyond random encountering. N-glycan removal or neutralization leads to larger membrane exploration and reduced interaction with clathrin, compromising clathrin-dependent internalization of virus-like particles by DC-SIGN. Therefore, our data reveal that cell surface glycan-mediated interactions add another organization layer to the cell membrane at the microscale and establish a novel mechanism of extracellular membrane organization based on the compartments of the membrane that a receptor is able to explore. Our work underscores the important and complex role of surface glycans regulating cell membrane organization and interaction with downstream partners.Glycans are fundamental cellular components ubiquitously present in the extracellular matrix and cell membrane as glycoproteins or glycolipids. Glycan-binding proteins such as galectins, siglecs, and selectins are mostly multivalent and thus thought to cross-link glycoproteins into higher-order aggregates, creating a cell surface glycan-based connectivity also called glycan lattice or network (1–3). By concentrating specific glycoproteins or glycolipids while excluding other cell surface molecules, surface glycan-based connectivity can organize the plasma membrane into specialized domains that perform unique functions (1, 3–6). Nevertheless, direct observation of glycan-mediated ligand cross-linking in living cells remains challenging (7). Notwithstanding, there is no doubt that surface glycan-based connectivity is essential in the control of multiple biological processes including immune cell activation and homeostasis, cell proliferation and differentiation, and receptor turnover and endocytosis (1, 5, 6, 8).Clathrin-mediated endocytosis (CME) constitutes the primary pathway of cargo internalization in mammalian cells regulating the surface expression of receptors (9). Formation of clathrin-coated pits (CCPs) starts by nucleation of coat assembly at distributed positions in the inner surface of the plasma membrane, where it continues to grow or dissolve rapidly unless coat stabilization occurs (10, 11). One event that clearly correlates with successful CCP stabilization is cargo loading (11). Recent studies show that cargo molecules diffuse randomly on the cell membrane until they meet growing CCPs, with the extent of cargo interactions regulating CCP maturation (12). As such, factors that affect cargo mobility within/at the cell surface will inevitably impact on CCP maturation and successful internalization. In the context of surface glycan–protein interactions, it has been shown that glycoproteins with an intact glycan-based connectivity exhibit reduced lateral mobility and this correlates with compromised endocytosis (3, 13–17). How this precisely occurs is poorly defined, although fluorescence recovery after photobleaching on the EGF receptor (EGFR) suggested that cell surface glycan-based interactions restrict EGFR dynamics and localization into membrane regions away from endocytic platforms (14, 17). Whether this is a general mechanism for glycosylated proteins or specific to EGFR is not known. Moreover, visualization of receptor interactions with the endocytic machinery under the influence of the glycan network has not yet been attained.In this work we applied superresolution nanoscopy and developed a dedicated dual-color single-molecule spatio-dynamic exploration approach to visualize the impact of glycan-based interactions on the spatiotemporal organization and clathrin interaction of a glycosylated membrane receptor involved in pathogen recognition and uptake. We focused on the transmembrane glycoprotein dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) given its importance in supporting primary immune responses such as pathogen recognition and uptake on immature dendritic cells (imDCs), signaling, and cell adhesion (6, 18–20). Moreover, DC-SIGN contains a single N-glycosylation site, organizes in nanoclusters at the cell membrane (19, 21–23), and internalizes bound antigens via CPPs for subsequent processing and presentation to T cells (20, 24–26). Our work provides insights on how surface glycan-mediated interactions tune spatiotemporal micropatterning of receptors on the cell membrane, potentially regulating interactions with the endocytic machinery and underscoring the importance and complex role of surface glycans on cell membrane organization and function.     

High-resolution structures of the M2 channel from influenza A virus reveal dynamic pathways for proton stabilization and transduction

The matrix 2 (M2) protein from influenza A virus is a proton channel that uses His37 as a selectivity filter. Here we report high-resolution (1.10 Å) cryogenic crystallographic structures of the transmembrane domain of M2 at low and high pH. These structures reveal that waters within the pore form hydrogen-bonded networks or “water wires” spanning 17 Å from the channel entrance to His37. Pore-lining carbonyl groups are well situated to stabilize hydronium via second-shell interactions involving bridging water molecules. In addition, room temperature crystallographic structures indicate that water becomes increasingly fluid with increasing temperature and decreasing pH, despite the higher electrostatic field. Complementary molecular dynamics simulations reveal a collective switch of hydrogen bond orientations that can contribute to the directionality of proton flux as His37 is dynamically protonated and deprotonated in the conduction cycle.Proton transport and conduction is essential to life. Proteins conduct protons over long distances through membranes to facilitate proton-coupled electron transfer and the formation and utilization of proton gradients. The M2 proton channel from the influenza A virus (1) is not only a medically important protein but also a simple, well-defined system for studying proton transport through confined spaces (2–4). This channel is the target of the anti-flu drug amantadine. M2 is activated at low pH by protonation of His37, which also participates in proton conduction by shuttling protons into the interior of the virus (5–7). His37 lies near the center of the bilayer, where it is connected to the viral exterior by a water-filled pore through which protons must pass to gain access to the viral interior (8–13).Visualizing the flow of protons within protein channels such as M2 is one of the long-standing challenges in molecular biophysics. Based on computational studies (9, 14–19) it has been suggested that protons reach His37 through “water wires” via the Grotthuss mechanism, but there is little high-resolution information concerning the path by which protons are conducted. A previously solved 1.65-Å crystal structure (9) showed six ordered waters immediately above the His37 tetrad, but ordered waters spanning the entire aqueous pore of M2 have not been observed until now. Previous MD simulations suggested a pore with mobile waters (12, 15), whereas the results of NMR and IR experiments are more consistent with an environment that is more similar to bulk water at low pH (13, 19, 20). However, it is difficult to deconvolute the changes in the water structure and dynamics when the protonation of His37 is raised from those induced indirectly via the conformation of the protein’s main chain.The M2 channel is known to have at least two conformational states that are populated to differing extents at low versus high pH (1, 10, 12). One, seen primarily at high pH, has been characterized extensively by solution NMR (21, 22), solid-state NMR (SSNMR) (10, 12), and X-ray crystallography (9). A second form is observed in dynamic equilibrium at lower pH (21–23), as evidenced by a broadening of magnetic resonances that thus far has made it impractical to determine a high-resolution structure of the protein in this state by SSNMR or solution NMR. X-ray crystallographic studies, however, have provided structures of both states (8, 9), which differ primarily in the conformation of the C terminus where protons exit the channel. Here we have obtained crystals that diffract to high resolution (1.10 Å) at both low and high pH, allowing visualization of water wires leading to His37 as a function of pH. The conformations of the backbone at the two pH values are essentially identical, permitting us to isolate changes in the organization of the water without any confounding factors.     

Predictive Value of Brain Natriuretic Peptides in Patients on Peritoneal Dialysis: Results from the ADEMEX Trial

Background and objectives: Natriuretic peptides have been suggested to be of value in risk stratification in dialysis patients. Data in patients on peritoneal dialysis remain limited.Design, setting, participants, & measurements: Patients of the ADEMEX trial (ADEquacy of peritoneal dialysis in MEXico) were randomized to a control group [standard 4 × 2L continuous ambulatory peritoneal dialysis (CAPD); n = 484] and an intervention group (CAPD with a target creatinine clearance ≥60L/wk/1.73 m²; n = 481). Natriuretic peptides were measured at baseline and correlated with other parameters as well as evaluated for effects on patient outcomes.Results: Control group and intervention group were comparable at baseline with respect to all measured parameters. Baseline values of natriuretic peptides were elevated and correlated significantly with levels of residual renal function but not with body size or diabetes. Baseline values of N-terminal fragment of B-type natriuretic peptide (NT-proBNP) but not proANP(1–30), proANP(31–67), or proANP(1–98) were independently highly predictive of overall survival and cardiovascular mortality. Volume removal was also significantly correlated with patient survival.Conclusions. NT-proBNP have a significant predictive value for survival of CAPD patients and may be of value in guiding risk stratification and potentially targeted therapeutic interventions.Plasma levels of cardiac natriuretic peptides are elevated in patients with chronic kidney disease, owing to impairment of renal function, hypertension, hypervolemia, and/or concomitant heart disease (1–7). Atrial natriuretic peptide (ANP) and particularly brain natriuretic peptide (BNP) levels are linked independently to left ventricular mass (3–5,8–16) and function (3,6–17) and predict total and cardiovascular mortality (1,3,8,10,12,18) as well as cardiac events (12,19). ANP and BNP decrease significantly during hemodialysis treatment but increase again during the interdialytic interval (1,2,4,6,7,14,17,20–23). Levels in patients on peritoneal dialysis (PD) have been found to be lower than in patients on hemodialysis (11,24–26), but the correlations with left ventricular function and structure are maintained in both types of dialysis modalities (11,15,27,28).The high mortality of patients on peritoneal dialysis and the failure of dialytic interventions to alter this mortality (29,30) necessitate renewed attention into novel methods of stratification and identification of patients at highest risk to be targeted for specific interventions. Cardiac natriuretic peptides are increasingly considered to fulfill this role in nonrenal patients. Evaluations of cardiac natriuretic peptides in patients on PD have been limited by small numbers (3,9,11,12,15,24–26) and only one study examined correlations between natriuretic peptide levels and outcomes (12). The PD population enrolled in the ADEMEX trial offered us the opportunity to evaluate cardiac natriuretic peptides and their value in predicting outcomes in the largest clinical trial ever performed on PD (29,30). It is hoped that such an evaluation would identify patients at risk even in the absence of overt clinical disease and hence facilitate or encourage interventions with salutary outcomes.     

Dynamic PIP2 interactions with voltage sensor elements contribute to KCNQ2 channel gating

The S4 segment and the S4–S5 linker of voltage-gated potassium (Kv) channels are crucial for voltage sensing. Previous studies on the Shaker and Kv1.2 channels have shown that phosphatidylinositol-4,5-bisphosphate (PIP₂) exerts opposing effects on Kv channels, up-regulating the current amplitude, while decreasing the voltage sensitivity. Interactions between PIP₂ and the S4 segment or the S4–S5 linker in the closed state have been highlighted to explain the effects of PIP₂ on voltage sensitivity. Here, we show that PIP₂ preferentially interacts with the S4–S5 linker in the open-state KCNQ2 (Kv7.2) channel, whereas it contacts the S2–S3 loop in the closed state. These interactions are different from the PIP₂–Shaker and PIP₂–Kv1.2 interactions. Consistently, PIP₂ exerts different effects on KCNQ2 relative to the Shaker and Kv1.2 channels; PIP₂ up-regulates both the current amplitude and voltage sensitivity of the KCNQ2 channel. Disruption of the interaction of PIP₂ with the S4–S5 linker by a single mutation decreases the voltage sensitivity and current amplitude, whereas disruption of the interaction with the S2–S3 loop does not alter voltage sensitivity. These results provide insight into the mechanism of PIP₂ action on KCNQ channels. In the closed state, PIP₂ is anchored at the S2–S3 loop; upon channel activation, PIP₂ interacts with the S4–S5 linker and is involved in channel gating.A series of ion channels, such as inward rectifier K⁺ (Kir) channels, transient receptor potential channels, and voltage-gated channels, are sensitive to the presence of phosphatidylinositol-4,5-bisphosphate (PIP₂) in membranes (1–4). Structural studies on Kir channels (1, 2, 5) demonstrated that PIP₂ directly interacts with the channels. Subsequent studies supported that PIP₂ also interacts directly with voltage-gated potassium (Kv) channels (6–19). Several positive residues that may be critical for PIP₂ activity have been identified (7, 11, 18, 20–24). Previous studies on Kv1.2 and Shaker channels showed that PIP₂ exerts opposing effects on Kv channels, up-regulating the current amplitude, while leading to a decrease in voltage sensitivity (7, 18). The S4 segment and the S4–S5 linker of Kv channels are crucial for voltage sensing. The interactions of PIP₂ with the S4 segments and the S4–S5 linkers of the closed-state Shaker and Kv1.2 channels underlie the loss-of-function effect of PIP₂ on voltage sensitivity (7, 18).The KCNQ (Kv7) family of slowly activated outwardly rectifying potassium channels is one of the Kv channel families that are sensitive to the presence of PIP₂ in the membrane. KCNQ channels have been widely studied because of their important biological and pharmacological functions. Retigabine, a first-in-class K⁺ channel opener used for the treatment of epilepsy, adopts a unique mechanism to enhance the activity of KCNQ channels (25). PIP₂ is important for the functions of KCNQ channels. Reduction of PIP₂ affinity caused by congenic mutations of KCNQ channels is associated with long QT syndrome, suggesting critical physiological implications of PIP₂ on KCNQ channels (23, 26). We reported that PIP₂ also alters the pharmacological selectivity of KCNQ potassium channels (6). Zaydman et al. (27) showed that the coupling of voltage sensing and pore opening in the KCNQ1 channel requires PIP₂ and suggested there is a PIP₂ interaction site at the interface between the voltage-sensing domain (VSD) and the central pore domain (PD). However, the effects and interactions of PIP₂ on KCNQ channels are not well understood.Here, by combining molecular dynamics (MD) simulations, mutagenesis, and electrophysiological determinations, we observed that the effects and interactions of PIP₂ on KCNQ2 are different relative to the Shaker and Kv1.2 channels. PIP₂ up-regulates both the current amplitude and voltage sensitivity of the KCNQ2 channel. PIP₂ preferentially interacts with the S4–S5 linker of the open-state KCNQ2 channel and does not interact with the S4 segment or S4-S5 linker of the closed state. In the closed state, PIP₂ only interacts with the S2–S3 loop. Furthermore, our electrophysiological experiments suggest that disruption of the interaction of PIP₂ with the S4–S5 linker may decrease the voltage sensitivity and current amplitude, whereas disruption of the interaction with the S2–S3 loop only alters the current amplitude of the channel. These results provide insights into the mechanism of PIP₂ action on Kv channels.     

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.     

An early secretory pathway mediated by GNOM-LIKE 1 and GNOM is essential for basal polarity establishment in Arabidopsis thaliana

Spatial regulation of the plant hormone indole-3-acetic acid (IAA, or auxin) is essential for plant development. Auxin gradient establishment is mediated by polarly localized auxin transporters, including PIN-FORMED (PIN) proteins. Their localization and abundance at the plasma membrane are tightly regulated by endomembrane machinery, especially the endocytic and recycling pathways mediated by the ADP ribosylation factor guanine nucleotide exchange factor (ARF-GEF) GNOM. We assessed the role of the early secretory pathway in establishing PIN1 polarity in Arabidopsis thaliana by pharmacological and genetic approaches. We identified the compound endosidin 8 (ES8), which selectively interferes with PIN1 basal polarity without altering the polarity of apical proteins. ES8 alters the auxin distribution pattern in the root and induces a strong developmental phenotype, including reduced root length. The ARF-GEF–defective mutants gnom-like 1 (gnl1-1) and gnom (van7) are significantly resistant to ES8. The compound does not affect recycling or vacuolar trafficking of PIN1 but leads to its intracellular accumulation, resulting in loss of PIN1 basal polarity at the plasma membrane. Our data confirm a role for GNOM in endoplasmic reticulum (ER)–Golgi trafficking and reveal that a GNL1/GNOM-mediated early secretory pathway selectively regulates PIN1 basal polarity establishment in a manner essential for normal plant development.Due to their sessile lifestyle, the development of plants is characterized by continuous growth, generating the capacity to adapt to environmental conditions. Such flexibility has been made possible by a set of morphological adjustments that are accomplished through altered growth regulation of different plant organs, such as leaves or roots. Most aspects of plant development are regulated by the differential distribution of the plant hormone indole-3-acetic acid (IAA, or auxin) between cells or tissues (reviewed by ref. 1). The formation of auxin maxima is generated concomitantly by local auxin biosynthesis, metabolism, and directional transport (2–8).Polar auxin transport occurs in a cell-to-cell manner and is dependent on plasma membrane-localized auxin influx and efflux carriers (reviewed by ref. 9). Among them, the PIN-FORMED (PIN) auxin efflux carriers are essential for plant development, and single or multiple pin mutants display phenotypes typical for auxin transport defects, such as tropism, embryo development, organogenesis, and root meristem patterning defects (6, 7, 10–14). A polar subcellular localization has been shown for most of the plasma membrane-localized auxin transporters, in particular for the PIN proteins (PIN1-4 and PIN7) and, to some extent, also for the ATP-BINDING CASSETTE SUBFAMILY B proteins (ABCBs) and AUXIN RESISTANT 1 (AUX1) (11–13, 15–20). The PIN proteins are known to be essential for targeting and redirecting auxin flux, which modulates the spatial pattern of expression of auxin response markers (21). PINs can be targeted toward the apical (shootward), basal (rootward), or lateral plasma membrane depending upon the PIN protein identity, the cell type, and the developmental context (reviewed by ref. 22). In the root, PIN1 is localized basally toward the root tip in stele provascular cells (12). PIN2 is also localized basally in young cortex cells close to the root meristem but is localized apically in mature cortex cells, epidermal cells, and the lateral root cap (16, 22, 23).Until now, it has been unclear whether newly synthesized PIN proteins are initially secreted to the plasma membrane in a polar or apolar manner. In Arabidopsis thaliana, the current model for PIN polar localization establishment and maintenance at the plasma membrane is based on endocytosis, polar recycling, and restriction of lateral diffusion (reviewed by ref. 24). PIN proteins are internalized via clathrin-mediated endocytosis (25, 26) and can cycle back to plasma membrane domains via distinct trafficking routes. Recycling and endocytosis of PIN1 depend on the endosome-localized fraction of the ADP ribosylation factor guanine nucleotide exchange factor (ARF-GEF) GNOM (27, 28), which is sensitive to the fungal toxin brefeldin A (BFA) (29). ARF-GEFs are essential regulators of vesicle formation and, among the eight ARF-GEFs in Arabidopsis, GNOM is the only one reported as being essential specifically for basal PIN recycling, whereas apical PIN and AUX1 localization and dynamics are not affected in gnom mutants (30, 31). Additionally, although apical targeting of AUX1 is resistant to BFA, subcellular AUX1 trafficking is BFA-sensitive, suggesting that trafficking of apical proteins may require both BFA-sensitive and -insensitive, GNOM-independent, ARF-GEF–mediated pathways (30, 32).In addition to GNOM, other Arabidopsis ARF-GEFs have been characterized, including GNOM-LIKE 1 (GNL1), which localizes to Golgi stacks and is BFA-resistant (33, 34). GNL1 acts in the early secretory pathway where it regulates COPI-mediated recycling of endoplasmic reticulum (ER)–resident proteins from the Golgi back to the ER (33, 34). Moreover, GNOM has recently been shown to predominantly localize to Golgi stacks (35) where it plays a minor but redundant function to GNL1 in ER-Golgi trafficking (33). The other Arabidopsis ARF-GEFs include GNL2, which is expressed specifically in pollen (36), and the five BIG ARF-GEFs, BIG1 to -5. BIG5, which is BFA-sensitive, has been described under the name BFA-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE 1 (BEN1) as mediating early endosomal trafficking (37). BIG1 to -4, of which BIG3 is BFA-resistant whereas BIG1, -2, and -4 are BFA-sensitive, have recently been described as acting redundantly in the late secretory pathway from the trans Golgi network (TGN) to the plasma membrane, as well as in late vacuolar trafficking (38).Endosomal PIN homeostasis is tightly controlled by the retromer complex through the regulation of PIN protein trafficking to the vacuole, thus controlling polar PIN abundance within the cell (39–43). Additionally, a large amount of data has demonstrated that not only trafficking routes per se are essential to determine the polar localization of PIN proteins but also internal protein signals such as posttranslational phosphorylation via the protein kinase PINOID (PID) and the protein phosphatase 2A (PP2A) (44–46). Despite recent progress, our understanding of the mechanisms establishing basal polarity remains limited. In the present work, we aimed to unravel the details of PIN basal polarity establishment by identifying selective inhibitors of this process.A number of genetic screens have been successfully used to discover new components of the endomembrane system (for examples, see refs. 34, 37, and 47–51). However, most of the molecular actors regulating endomembrane trafficking are either essential to plant survival or belong to large protein families, leading to lethality of knock-out mutants or lack of a phenotype due to redundancy. The use of fast-acting molecules suitable for the highly dynamic nature of the endomembrane system circumvents these problems and has deepened our understanding of interconnected networks of trafficking routes (52–58). While BFA has expanded our knowledge of the GNOM-dependent recycling pathway (27), other small compounds can be used to dissect different trafficking routes. In recent studies, automated screening of small molecules based on inhibition of tobacco pollen tube growth led to the isolation of a set of compounds interfering with the endomembrane system (52). Through the screening of 46,418 diverse molecules, 360 were identified as inhibitors of pollen germination (53). To dissect the trafficking routes of plasma membrane proteins specifically, a secondary screen was established based on confocal laser-scanning microscopy, leading to the identification of 123 compounds named plasma membrane recycling compound set A (PMRA), which induce mislocalization of plasma membrane markers in the Arabidopsis root meristem (53).In the present study, we reasoned that using the PMRA endomembrane trafficking modulators in combination with BFA could unravel trafficking routes regulating basal plasma membrane targeting. We designed a chemical screen to identify PMRA molecules that modulated the accumulation of PIN1 in BFA-induced agglomerations. We subsequently identified the endosidin 8 (ES8) compounds, including the original compound ES8.0 and its more potent analog ES8.1, which selectively modify PIN1 basal plasma membrane targeting in Arabidopsis with minimal effects on apical plasma membrane proteins. Using this pharmacological approach, we herein confirm that GNOM plays a role in ER-Golgi trafficking independently of its role in recycling and reveal that a GNL1/GNOM-dependent early secretory pathway is essential for targeting PIN1 toward the basal plasma membrane. Furthermore, we demonstrate that this pathway is specific for basal polarity establishment, revealing an essential and previously unknown regulatory mechanism for establishing cell polarity and regulating auxin transport and plant development.     

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.     

Enhancing the antitumor efficacy of a cell-surface death ligand by covalent membrane display

TNF superfamily death ligands are expressed on the surface of immune cells and can trigger apoptosis in susceptible cancer cells by engaging cognate death receptors. A recombinant soluble protein comprising the ectodomain of Apo2 ligand/TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) has shown remarkable preclinical anticancer activity but lacked broad efficacy in patients, possibly owing to insufficient exposure or potency. We observed that antibody cross-linking substantially enhanced cytotoxicity of soluble Apo2L/TRAIL against diverse cancer cell lines. Presentation of the ligand on glass-supported lipid bilayers enhanced its ability to drive receptor microclustering and apoptotic signaling. Furthermore, covalent surface attachment of Apo2L/TRAIL onto liposomes—synthetic lipid-bilayer nanospheres—similarly augmented activity. In vivo, liposome-displayed Apo2L/TRAIL achieved markedly better exposure and antitumor activity. Thus, covalent synthetic-membrane attachment of a cell-surface ligand enhances efficacy, increasing therapeutic potential. These findings have translational implications for liposomal approaches as well as for Apo2L/TRAIL and other clinically relevant TNF ligands.Apoptosis is a mode of regulated cell death that is critical for the elimination of unwanted cells in metazoans (1). Extensive efforts have been aimed at developing therapeutic strategies to reactivate apoptosis in cancer cells (2–4). Two distinct signaling pathways converge on “executioner” caspases -3 and -7 to trigger apoptosis: the intrinsic pathway, controlled by Bcl-2 (B-cell lymphoma 2) family members (5), and the extrinsic pathway, controlled by extracellular death ligands (6, 7). Certain immune-effector cells express surface death ligands such as TNFα, FasL, and Apo2 ligand/TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) to engage cognate death receptors on susceptible target cells. Ligation promotes death receptor microclustering, which drives recruitment and activation of caspase-8 to initiate apoptosis (7–9). Although significant clinical progress has been made with small-molecule Bcl-2 antagonists to reactivate the intrinsic pathway in tumors (2), efforts to engage the extrinsic pathway with protein-based proapoptotic receptor agonists (PARAs) have been disappointing to date (10–14). A leading strategy is to target death receptor 4 (DR4) and/or DR5, which are overexpressed on various tumors. A recombinant soluble version of the cognate ligand, human Apo2L/TRAIL (dulanermin), and several agonistic anti-DR4 or anti-DR5 antibodies have been studied in preclinical models and in patients (10–16). Despite remarkable preclinical efficacy, only rare clinical responses have been observed. Potential reasons for the lack of clinical activity include insufficient tumor exposure (16, 17) and/or weak engagement of the extrinsic pathway (10, 13). Such PARAs have been relatively well tolerated in clinical trials, suggesting there might be a therapeutic window for increasing tumor exposure and/or potency of these agents (18). Because recombinant soluble Apo2L/TRAIL more faithfully resembles the endogenous ligand, we used it as a starting point for augmenting efficacy.     

The ATM–p53 pathway suppresses aneuploidy-induced tumorigenesis

The spindle assembly checkpoint (SAC) is essential for proper sister chromatid segregation. Defects in this checkpoint can lead to chromosome missegregation and aneuploidy. An increasing body of evidence suggests that aneuploidy can play a causal role in tumorigenesis. However, mutant mice that are prone to aneuploidy have only mild tumor phenotypes, suggesting that there are limiting factors in the aneuploidy-induced tumorigenesis. Here we provide evidence that p53 is such a limiting factor. We show that aneuploidy activates p53 and that loss of p53 drastically accelerates tumor development in two independent aneuploidy models. The p53 activation depends on the ataxia-telangiectasia mutated (ATM) gene product and increased levels of reactive oxygen species. Thus, the ATM-p53 pathway safeguards not only DNA damage but also aneuploidy.Faithful transmission of genetic materials is of fundamental importance to the survival of all organisms. In eukaryotes, replicated chromosomes are held together as sister chromatids by the cohesin complexes established during the replication and are segregated to daughter cells in mitosis. The timing of the sister chromatid separation is controlled by the spindle assembly checkpoint (SAC), which monitors the status of microtubule attachment at kinetochores. The SAC is activated when kinetochores are not attached (i.e., occupied) by microtubules and/or when there is a lack of tension at sister kinetochores (1, 2), under both of which situations separation of sister chromatids needs to be actively prevented or missegregation of chromosomes would ensue. The activation of SAC leads to the inhibition of the anaphase-promoting complex or cyclosome (APC/C), a multisubunit E3 ubiquitin ligase that targets securin and cyclin B1 for destruction (3–6). Both securin and cyclin B1 are recognized and brought to APC/C by the adaptor protein Cdc20. Not surprisingly, APC^Cdc20 is inhibited by the SAC. The inhibition is carried out by two proteins, Mad2 and BubR1 (for more detailed and recent reviews, see refs. 7 and 8). Genetic analyses in budding yeasts unequivocally demonstrated that the spindle assembly checkpoint was essential in maintaining chromosomal stability (9, 10). Studies of engineered mouse strains carrying mutations in SAC components also indicated the importance of the checkpoint in maintaining chromosome stability (11–19), and BUBR1 was found mutated in a rare human disorder, mosaic variegated aneuploidy (20).A hallmark of human cancers is genomic instability including chromosomal instability (CIN). CIN can be numerical changes in whole chromosomes (aneuploidy) or structural alterations such as translocations. Aneuploidy is found in nearly all of the major human tumor types (21), and it was the abnormal chromosome numbers in cancerous cells that prompted Boveri to propose nearly a century ago that cancer was caused by aneuploidy (22). Nearly all SAC-compromised mouse strains develop spontaneous tumors, although the rates vary substantially (for a summary see ref. 23). Together with the finding of BUBR1 mutation in mosaic variegated aneuploidy, a condition that predisposes patients to childhood cancers (20), the tumor results in SAC mutants strongly argue that aneuploidy can induce tumorigenesis. However, the spontaneous tumor development in SAC mutant mice is usually late onset and at relatively low rates, indicating that aneuploidy does not present a serious risk of tumor development. The low risk of aneuploidy-induced tumorigenesis suggests that there are limiting factors. One such factor could be the general unfitness of aneuploid cells when compared with euploid cells, which is true from budding yeasts to mammals (24, 25). This unfitness likely stems from the imbalances in gene dosage that lead to changes in a score of physiological parameters including energy metabolism (24, 25). Mouse embryonic fibroblasts (MEFs) trisomy for chromosome 1, 13, 16, or 19 grew less robustly than the diploid MEFs and showed resistance to transformation (24). These findings support the notion that aneuploidy can be tumor suppressing under certain circumstances (15, 26). However, the fact that most human cancer cells are aneuploid (21) indicates that there must be ways to overcome the unfitness barrier and that once this barrier is overcome, aneuploidy is beneficial to the tumor development.Here we report that p53 is another limiting factor in aneuploidy-induced tumorigenesis. We provide evidence that p53 is activated by aneuploidy and the activation depends on ataxia-telangiectasia mutated (ATM). We further show that aneuploidy resulted in heightened energy metabolism and increased levels of intracellular reactive oxygen species, which caused oxidative DNA damage and ATM activation.     

Application of desorption electrospray ionization mass spectrometry imaging in breast cancer margin analysis

Distinguishing tumor from normal glandular breast tissue is an important step in breast-conserving surgery. Because this distinction can be challenging in the operative setting, up to 40% of patients require an additional operation when traditional approaches are used. Here, we present a proof-of-concept study to determine the feasibility of using desorption electrospray ionization mass spectrometry imaging (DESI-MSI) for identifying and differentiating tumor from normal breast tissue. We show that tumor margins can be identified using the spatial distributions and varying intensities of different lipids. Several fatty acids, including oleic acid, were more abundant in the cancerous tissue than in normal tissues. The cancer margins delineated by the molecular images from DESI-MSI were consistent with those margins obtained from histological staining. Our findings prove the feasibility of classifying cancerous and normal breast tissues using ambient ionization MSI. The results suggest that an MS-based method could be developed for the rapid intraoperative detection of residual cancer tissue during breast-conserving surgery.Breast cancer is the most commonly diagnosed carcinoma in women in the United States and Western countries. Breast conservation surgery (BCS) has become the preferred treatment option for many women with early-stage breast cancer (1). BCS entails resection of the tumor, with a clean margin of normal tissue around it. Surgery is usually followed by radiation therapy. Results from seven large randomized prospective studies, with the largest two having over 20 y of follow-up, have shown equal survival when comparing BCS coupled with whole-breast radiation and mastectomy (2, 3).Normally, breast surgeons aim to remove a patient’s tumor, along with a rim of normal tissue that is free of cancer. Preoperative mammography, ultrasonography, or MRI may be used by the surgeon to guide adequate resection (4–6). Despite numerous improvements in imaging and surgical technique, the need for reexcision to achieve complete tumor resection in the United States typically ranges from 20–40% (7–15), and has been reported as being as high as 60% (16). The importance of reexcision is underscored by numerous studies, which have shown that incomplete resection of tumor and positive margins are associated with increased locoregional recurrence compared with negative margins (12, 17–20). Furthermore, the landmark meta-analysis performed by the Early Breast Cancer Trialists’ Collaborative Group (18, 21) directly linked local recurrence to survival, placing great emphasis on the surgeon’s role in minimizing local recurrence by obtaining adequate margins.Breast tumor reexcisions are accompanied by a number of undesirable problems: The completion of therapy is delayed, infection rates are increased, cost is increased, there can be a negative psychological impact on the patient, and there can be diminished aesthetic outcomes (22–24). The development of an intraoperative technique that allows the fast and accurate identification of residual tumor at surgical resection margins could decrease the reexcision rate, and therefore improve the care delivered to patients with cancer who are receiving BCS.To this end, multiple intraoperative methods have been explored, with various benefits as well as limitations. These methods include touch frozen section analysis (25), touch preparation cytology (26), specimen radiography (27, 28), rf spectroscopy (29, 30), Raman spectroscopy (31), radioguided occult lesion localization (32), near-IR fluorescence (33, 34), and high-frequency ultrasound (35–37). The intraoperative application of MRI, which has been successfully applied in brain surgery (38–42), is limited in its application in BCS. These limitations include MRI interpretation in the presence of acute surgical changes; lack of real-time imaging, requiring the interruption of surgery; and accurate localization of tumor based on images requiring development of fiducials (43–46).Mass spectrometry imaging (MSI) has been applied to investigate the molecular distribution of proteins, lipids, and metabolites without the use of labels (47, 48). In particular, the newly developed ambient ionization technique of desorption electrospray ionization (DESI) allows direct tissue analysis with little to no sample preparation (49, 50). Therefore, with the advantage of easy use, DESI-MSI has great potential in the application of intraoperative tumor assessment. The development of DESI-MSI enables the correlation of lipid distribution in two or three dimensions with tissue morphology (47, 51) and the distinction of cancerous from noncancerous tissues based on lipidomic information (52–54). Distinctive lipid profiles associated with different human cancers have been investigated by DESI-MSI (55–58). Moreover, the grades and subtypes of human brain tumors have been discriminated using this technique. Additionally, tumor margins have been delineated using DESI-MSI, and the results have been correlated with histopathological examination (59, 60).It has been reported that breast cancer demonstrates metabolic profiles that are distinct from those metabolic profiles found in normal breast tissue. This finding suggests a potential for using metabolite information for breast cancer diagnosis and tumor margin identification (61, 62). Here, we demonstrate an MS-based methodology for using lipidomic information to distinguish cancerous from noncancerous tissue and to delineate tumor boundaries.     

From the Cover: Why the proportion of transmission during early-stage HIV infection does not predict the long-term impact of treatment on HIV incidence

Antiretroviral therapy (ART) reduces the infectiousness of HIV-infected persons, but only after testing, linkage to care, and successful viral suppression. Thus, a large proportion of HIV transmission during a period of high infectiousness in the first few months after infection (“early transmission”) is perceived as a threat to the impact of HIV “treatment-as-prevention” strategies. We created a mathematical model of a heterosexual HIV epidemic to investigate how the proportion of early transmission affects the impact of ART on reducing HIV incidence. The model includes stages of HIV infection, flexible sexual mixing, and changes in risk behavior over the epidemic. The model was calibrated to HIV prevalence data from South Africa using a Bayesian framework. Immediately after ART was introduced, more early transmission was associated with a smaller reduction in HIV incidence rate—consistent with the concern that a large amount of early transmission reduces the impact of treatment on incidence. However, the proportion of early transmission was not strongly related to the long-term reduction in incidence. This was because more early transmission resulted in a shorter generation time, in which case lower values for the basic reproductive number (R₀) are consistent with observed epidemic growth, and R₀ was negatively correlated with long-term intervention impact. The fraction of early transmission depends on biological factors, behavioral patterns, and epidemic stage and alone does not predict long-term intervention impacts. However, early transmission may be an important determinant in the outcome of short-term trials and evaluation of programs.Recent studies have confirmed that effective antiretroviral therapy (ART) reduces the transmission of HIV among stable heterosexual couples (1–3). This finding has generated interest in understanding the population-level impact of HIV treatment on reducing the rate of new HIV infections in generalized epidemic settings (4). Research, including mathematical modeling (5–10), implementation research (11), and major randomized controlled trials (12–14), are focused on how ART provision might be expanded strategically to maximize its public health benefits (15, 16).One concern is that if a large fraction of HIV transmission occurs shortly after a person becomes infected, before the person can be diagnosed and initiated on ART, this will limit the potential impact of HIV treatment on reducing HIV incidence (9, 17, 18). Data suggest that persons are more infectious during a short period of “early infection” after becoming infected with HIV (19–22), although there is debate about the extent, duration, and determinants of elevated infectiousness (18, 23). The amount of transmission that occurs also will depend on patterns of sexual behavior and sexual networks (17, 24–27). There have been estimates for the contribution of early infection to transmission from mathematical models (7, 17, 21, 24–26) and phylogenetic analyses (28–31), but these vary widely, from 5% to above 50% (23).In this study, we use a mathematical model to quantify how the proportion of transmission that comes from persons who have been infected recently affects the impact of treatment scale-up on HIV incidence. The model is calibrated to longitudinal HIV prevalence data from South Africa using a Bayesian framework. Thus, the model accounts for not only the early epidemic growth rate highlighted in previous research (5, 9, 18), but also the heterogeneity and sexual behavior change to explain the peak and decline in HIV incidence observed in sub-Saharan African HIV epidemics (32, 33).The model calibration allows uncertainty about factors that determine the amount of early transmission, including the relative infectiousness during early infection, heterogeneity in propensity for sexual risk behavior, assortativity in sexual partner selection, reduction in risk propensity over the life course, and population-wide reductions in risk behavior in response to the epidemic (32, 33). This results in multiple combinations of parameter values that are consistent with the observed epidemic and variation in the amount of early transmission. We simulated the impact of a treatment intervention and report how the proportion of early transmission correlates with the reduction in HIV incidence from the intervention over the short- and long-term.