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Significance Polycomb repressive complex 2 (PRC2) is a major epigenetic machinery that maintains transcriptionally silent heterochromatin in the nucleus and plays critical roles in embryonic development and oncogenesis. It is generally thought that PRC2 propagates repressive histone marks by modifying neighboring nucleosomes in strictly linear progression. However, the behavior of PRC2 on native-like chromatin substrates remains incompletely characterized, making the precise mechanism of PRC2-mediated heterochromatin maintenance elusive. Here we use single-molecule force spectroscopy and computational modeling to dissect the interactions between PRC2 and polynucleosome arrays. Our results provide direct evidence that PRC2 can simultaneously engage nonadjacent nucleosome pairs. The demonstration of PRC2's ability to bridge noncontiguous chromosomal segments furthers our understanding of how Polycomb complexes spread epigenetic modifications and compact chromatin., Polycomb repressive complex 2 (PRC2) installs and spreads repressive histone methylation marks on eukaryotic chromosomes. Because of the key roles that PRC2 plays in development and disease, how this epigenetic machinery interacts with DNA and nucleosomes is of major interest. Nonetheless, the mechanism by which PRC2 engages with native-like chromatin remains incompletely understood. In this work, we employ single-molecule force spectroscopy and molecular dynamics simulations to dissect the behavior of PRC2 on polynucleosome arrays. Our results reveal an unexpectedly diverse repertoire of PRC2 binding configurations on chromatin. Besides reproducing known binding modes in which PRC2 interacts with bare DNA, mononucleosomes, and adjacent nucleosome pairs, our data also provide direct evidence that PRC2 can bridge pairs of distal nucleosomes. In particular, the “1–3” bridging mode, in which PRC2 engages two nucleosomes separated by one spacer nucleosome, is a preferred low-energy configuration. Moreover, we show that the distribution and stability of different PRC2–chromatin interaction modes are modulated by accessory subunits, oncogenic histone mutations, and the methylation state of chromatin. Overall, these findings have implications for the mechanism by which PRC2 spreads histone modifications and compacts chromatin. The experimental and computational platforms developed here provide a framework for understanding the molecular basis of epigenetic maintenance mediated by Polycomb-group proteins.

Significance Muscle myosins polymerize into thick filaments that drive muscle contraction by interaction with actin thin filaments. The details of muscle myosin polymerization into thick filaments are not known. Current hypotheses are that elongated myosin monomers form antiparallel dimers that polymerize into filaments. However, we find polymerization in vitro of skeletal, cardiac, and smooth muscle myosins involves formation of monomers with folded tails, tail-folded dimers, and tail-folded tetramers. These observations should stimulate studies of the pathway of formation of muscle myosins in vitro and in vivo including the possible roles of the multiple myosin-associated proteins on in vivo polymerization., Muscle contraction depends on the cyclical interaction of myosin and actin filaments. Therefore, it is important to understand the mechanisms of polymerization and depolymerization of muscle myosins. Muscle myosin 2 monomers exist in two states: one with a folded tail that interacts with the heads (10S) and one with an unfolded tail (6S). It has been thought that only unfolded monomers assemble into bipolar and side-polar (smooth muscle myosin) filaments. We now show by electron microscopy that, after 4 s of polymerization in vitro in both the presence (smooth muscle myosin) and absence of ATP, skeletal, cardiac, and smooth muscle myosins form tail-folded monomers without tail–head interaction, tail-folded antiparallel dimers, tail-folded antiparallel tetramers, unfolded bipolar tetramers, and small filaments. After 4 h, the myosins form thick bipolar and, for smooth muscle myosin, side-polar filaments. Nonphosphorylated smooth muscle myosin polymerizes in the presence of ATP but with a higher critical concentration than in the absence of ATP and forms only bipolar filaments with bare zones. Partial depolymerization in vitro of nonphosphorylated smooth muscle myosin filaments by the addition of MgATP is the reverse of polymerization.

Significance Cardiovascular disease continues to be the leading cause of death worldwide, and is frequently associated with heart failure. Efforts to develop better therapeutics for heart failure have been held back by limited understanding of the normal control of contraction on the timescale of the heartbeat. We used synchrotron X-ray diffraction to determine the dynamic structural changes in the myosin motors that drive contraction in the heart muscle, and show that myosin filament-based control mechanisms determine the time course and strength of contraction, allowing those mechanisms to be targeted for developing new therapies for heart disease., Myosin-based mechanisms are increasingly recognized as supplementing their better-known actin-based counterparts to control the strength and time course of contraction in both skeletal and heart muscle. Here we use synchrotron small-angle X-ray diffraction to determine the structural dynamics of local domains of the myosin filament during contraction of heart muscle. We show that, although myosin motors throughout the filament contribute to force development, only about 10% of the motors in each filament bear the peak force, and these are confined to the filament domain containing myosin binding protein-C, the “C-zone.” Myosin motors in domains further from the filament midpoint are likely to be activated and inactivated first in each contraction. Inactivated myosin motors are folded against the filament core, and a subset of folded motors lie on the helical tracks described previously. These helically ordered motors are also likely to be confined to the C-zone, and the associated motor conformation reforms only slowly during relaxation. Myosin filament stress-sensing determines the strength and time course of contraction in conjunction with actin-based regulation. These results establish the fundamental roles of myosin filament domains and the associated motor conformations in controlling the strength and dynamics of contraction in heart muscle, enabling those structures to be targeted to develop new therapies for heart disease.

Significance We identified a group of DNA repair genes directly induced by EZH2 and repressed by EZH2 inhibitors. Expression of these genes predicts the response of wild-type EZH2-harboring solid tumors to EZH2 inhibitors. Most importantly, our findings lay the foundation for the development of a combination therapy that combines EZH2 inhibitors and DNA damaging agents or drugs that block DNA repair for the treatment of castration-resistant prostate cancer and other solid tumors., Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring EZH2 gain-of-function mutations that enhance its polycomb repressive function. We have previously reported that EZH2 can act as a transcriptional activator in castration-resistant prostate cancer (CRPC). Now we show that EZH2 inhibitors can also block the transactivation activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomics profiling of cells treated with EZH2 inhibitors demonstrated that in addition to derepressing gene expression, these compounds also robustly down-regulate a set of DNA damage repair (DDR) genes, especially those involved in the base excision repair (BER) pathway. Methylation of the pioneer factor FOXA1 by EZH2 contributes to the activation of these genes, and interaction with the transcriptional coactivator P300 via the transactivation domain on EZH2 directly turns on the transcription. In addition, CRISPR-Cas9–mediated knockout screens in the presence of EZH2 inhibitors identified these BER genes as the determinants that underlie the growth-inhibitory effect of EZH2 inhibitors. Interrogation of public data from diverse types of solid tumors expressing wild-type EZH2 demonstrated that expression of DDR genes is significantly correlated with EZH2 dependency and cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhances their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential combination cancer therapies.

The precise assembly and disassembly of actin filaments is required for several cellular processes, and their regulation has been scrutinized for decades. Twenty years ago, a handful of studies marked the advent of a new type of experiment to study actin dynamics: using optical microscopy to look at individual events, taking place on individual filaments in real time. Here, we summarize the main characteristics of this approach and how it has changed our ability to understand actin assembly dynamics. We also highlight some of its caveats and reflect on what we have learned over the past 20 years, leading us to propose a set of guidelines, which we hope will contribute to a better exploitation of this powerful tool.

Significance Cellular cryo-electron tomography suffers from severely compromised Z resolution due to the missing wedges of information not collected during the acquisition of tilt series. This paper shows that application of entropy-regularized deconvolution to transmission electron tomography substantially fills in this missing information, allowing for improved Z resolution and better interpretation of cellular structures., Cryo-electron tomography (cryo-ET) allows for the high-resolution visualization of biological macromolecules. However, the technique is limited by a low signal-to-noise ratio (SNR) and variance in contrast at different frequencies, as well as reduced Z resolution. Here, we applied entropy-regularized deconvolution (ER-DC) to cryo-ET data generated from transmission electron microscopy (TEM) and reconstructed using weighted back projection (WBP). We applied deconvolution to several in situ cryo-ET datasets and assessed the results by Fourier analysis and subtomogram analysis (STA).

Type IV pili (T4P) are dynamic surface appendages that promote virulence, biofilm formation, horizontal gene transfer, and motility in diverse bacterial species. Pilus dynamic activity is best characterized in T4P that use distinct ATPase motors for pilus extension and retraction. Many T4P systems, however, lack a dedicated retraction motor, and the mechanism underlying this motor-independent retraction remains a mystery. Using the Vibrio cholerae competence pilus as a model system, we identify mutations in the major pilin gene that enhance motor-independent retraction. These mutants likely diminish pilin-pilin interactions within the filament to produce less-stable pili. One mutation adds a bulky residue to α1C, a universally conserved feature of T4P. We found that inserting a bulky residue into α1C of the retraction motor-dependent Acinetobacter baylyi competence T4P enhances motor-independent retraction. Conversely, removing bulky residues from α1C of the retraction motor-independent, V. cholerae toxin-coregulated T4P stabilizes the filament and diminishes pilus retraction. Furthermore, alignment of pilins from the broader type IV filament (T4F) family indicated that retraction motor-independent T4P, gram-positive Com pili, and type II secretion systems generally encode larger residues within α1C oriented toward the pilus core compared to retraction motor-dependent T4P. Together, our data demonstrate that motor-independent retraction relies, in part, on the inherent instability of the pilus filament, which may be a conserved feature of diverse T4Fs. This provides evidence for a long-standing yet previously untested model in which pili retract in the absence of a motor by spontaneous depolymerization.

Significance Alzheimeŕs disease is one of the major global health challenges. Neuronal cell dysfunction and death are connected to the self-assembly of the amyloid β peptide (Aβ42) into oligomeric and fibrillar aggregates. The fibril surface can catalyze the formation of toxic oligomers via secondary nucleation. Access to a high-resolution structure of Aβ42 fibrils would provide a valuable basis for design of inhibitors of oligomer generation and toxicity in the form of fibril-binders and thus significantly contribute to the development of therapeutics against Alzheimer’s disease. A combination of methods may be most fruitful toward this aim. We show that small-angle X-ray scattering data, in combination with a solid-state NMR structure of the filament core, can reveal a detailed fibril model., Amyloid fibrils are associated with a number of neurodegenerative diseases, including fibrils of amyloid β42 peptide (Aβ42) in Alzheimer’s disease. These fibrils are a source of toxicity to neuronal cells through surface-catalyzed generation of toxic oligomers. Detailed knowledge of the fibril structure may thus facilitate therapeutic development. We use small-angle scattering to provide information on the fibril cross-section dimension and shape for Aβ42 fibrils prepared in aqueous phosphate buffer at pH = 7.4 and pH 8.0 under quiescent conditions at 37 °C from pure recombinant Aβ42 peptide. Fitting the data using a continuum model reveals an elliptical cross-section and a peptide mass-per-unit length compatible with two filaments of two monomers, four monomers per plane. To provide a more detailed atomistic model, the data were fitted using as a starting state a high-resolution structure of the two-monomer arrangement in filaments from solid-state NMR (Protein Data Bank ID 5kk3). First, a twofold symmetric model including residues 11 to 42 of two monomers in the filament was optimized in terms of twist angle and local packing using Rosetta. A two-filament model was then built and optimized through fitting to the scattering data allowing the two N-termini in each filament to take different conformations, with the same conformation in each of the two filaments. This provides an atomistic model of the fibril with twofold rotation symmetry around the fibril axis. Intriguingly, no polydispersity as regards the number of filaments was observed in our system over separate samples, suggesting that the two-filament arrangement represents a free energy minimum for the Aβ42 fibril.

External enforcement policies aimed to reduce violations differ on two key components: the probability of inspection and the severity of the punishment. Different lines of research offer different insights regarding the relative importance of each component. In four studies, students and Prolific crowdsourcing participants (N(total) = 816) repeatedly faced temptations to commit violations under two enforcement policies. Controlling for expected value, we found that a policy combining a high probability of inspection with a low severity of fines (HILS) was more effective than an economically equivalent policy that combined a low probability of inspection with a high severity of fines (LIHS). The advantage of prioritizing inspection frequency over punishment severity (HILS over LIHS) was greater for participants who, in the absence of enforcement, started out with a higher violation rate. Consistent with studies of decisions from experience, frequent enforcement with small fines was more effective than rare severe fines even when we announced the severity of the fine in advance to boost deterrence. In addition, in line with the phenomenon of underweighting of rare events, the effect was stronger when the probability of inspection was rarer (as in most real-life inspection probabilities) and was eliminated under moderate inspection probabilities. We thus recommend that policymakers looking to effectively reduce recurring violations among noncriminal populations should consider increasing inspection rates rather than punishment severity.

Significance Platelet activation induces reorganization of the cell and the formation of cellular protrusions, pseudopodia. These processes are accompanied by remodeling of the actin cytoskeleton and the activation of platelet integrins, which mediate strong adhesion to the extracellular matrix. In this work, we analyzed the actin polarity and integrin architecture in pseudopodia. A nonuniform polarity of actin filaments in pseudopodia indicates that these protrusions may be involved in contractile acto-myosin forces. Heterogeneity in integrin conformation was found, while solely a bent integrin structure was resolved, ∼50 to 70 nm above the support., During activation the platelet cytoskeleton is reorganized, inducing adhesion to the extracellular matrix and cell spreading. These processes are critical for wound healing and clot formation. Initially, this task relies on the formation of strong cellular–extracellular matrix interactions, exposed in subendothelial lesions. Despite the medical relevance of these processes, there is a lack of high-resolution structural information on the platelet cytoskeleton controlling cell spreading and adhesion. Here, we present in situ structural analysis of membrane receptors and the underlying cytoskeleton in platelet protrusions by applying cryoelectron tomography to intact platelets. We utilized three-dimensional averaging procedures to study receptors at the plasma membrane. Analysis of substrate interaction-free receptors yielded one main structural class resolved to 26 Å, resembling the αIIbβ3 integrin folded conformation. Furthermore, structural analysis of the actin network in pseudopodia indicates a nonuniform polarity of filaments. This organization would allow generation of the contractile forces required for integrin-mediated cell adhesion.

Dynamin oligomerizes into helical filaments on tubular membrane templates and, through constriction, cleaves them in a GTPase-driven way. Structural observations of GTP-dependent cross-bridges between neighboring filament turns have led to the suggestion that dynamin operates as a molecular ratchet motor. However, the proof of such mechanism remains absent. Particularly, it is not known whether a powerful enough stroke is produced and how the motor modules would cooperate in the constriction process. Here, we characterized the dynamin motor modules by single-molecule Förster resonance energy transfer (smFRET) and found strong nucleotide-dependent conformational preferences. Integrating smFRET with molecular dynamics simulations allowed us to estimate the forces generated in a power stroke. Subsequently, the quantitative force data and the measured kinetics of the GTPase cycle were incorporated into a model including both a dynamin filament, with explicit motor cross-bridges, and a realistic deformable membrane template. In our simulations, collective constriction of the membrane by dynamin motor modules, based on the ratchet mechanism, is directly reproduced and analyzed. Functional parallels between the dynamin system and actomyosin in the muscle are seen. Through concerted action of the motors, tight membrane constriction to the hemifission radius can be reached. Our experimental and computational study provides an example of how collective motor action in megadalton molecular assemblies can be approached and explicitly resolved.

Significance Houseplant enthusiasts know the power of plant regeneration—small cuttings can regrow into whole plants. Yet normally, plant tissues and cells maintain distinct and stable identities. A biochemical modification to histones (H3K27me3) is thought to lock in cell identity by preventing access to specific regions of DNA. Here, we investigated how H3K27me3 was used to maintain the identity of stomatal guard cells. We identified changes in H3K27me3 distribution and gene expression in normal guard cells and after we induced “reprogramming” into alternative cell identities. This linked the loss of H3K27me3 at stomatal precursor genes to a return to an earlier fate, but increased H3K27me3 at a wound-induced reprogramming gene indicated how plant cells may sense and resist inappropriate loss of identity., Plant cells maintain remarkable developmental plasticity, allowing them to clonally reproduce and to repair tissues following wounding; yet plant cells normally stably maintain consistent identities. Although this capacity was recognized long ago, our mechanistic understanding of the establishment, maintenance, and erasure of cellular identities in plants remains limited. Here, we develop a cell-type–specific reprogramming system that can be probed at the genome-wide scale for alterations in gene expression and histone modifications. We show that relationships among H3K27me3, H3K4me3, and gene expression in single cell types mirror trends from complex tissue, and that H3K27me3 dynamics regulate guard cell identity. Further, upon initiation of reprogramming, guard cells induce H3K27me3-mediated repression of a regulator of wound-induced callus formation, suggesting that cells in intact tissues may have mechanisms to sense and resist inappropriate dedifferentiation. The matched ChIP-sequencing (seq) and RNA-seq datasets created for this analysis also serve as a resource enabling inquiries into the dynamic and global-scale distribution of histone modifications in single cell types in plants.

Significance The structural dynamics of the dendritic synapse, arising from the remodeling of actin cytoskeletons, has been widely associated with memory and cognition. The remodeling is regulated by intracellular Ca2+ levels. Under low Ca2+ concentration, actin filaments are bundled by a calcium signaling protein, CaMKII. When the Ca2+ concentration is raised, CaMKII dissociates from actin and opens the window for actin remodeling. At present, the molecular details of the actin bundling and regulation are elusive. Herein we use experimental tools along with molecular simulations to construct a model of how CaMKII bundles actin and how the CaMKII–actin architecture is regulated by Ca2+ signals. In this way, our results explain how Ca2+ signals ultimately change the structure of the dendritic synapse., Calcium/calmodulin-dependent kinase II (CaMKII) plays a key role in the plasticity of dendritic spines. Calcium signals cause calcium−calmodulin to activate CaMKII, which leads to remodeling of the actin filament (F-actin) network in the spine. We elucidate the mechanism of the remodeling by combining computer simulations with protein array experiments and electron microscopic imaging, to arrive at a structural model for the dodecameric complex of CaMKII with F-actin. The binding interface involves multiple domains of CaMKII. This structure explains the architecture of the micrometer-scale CaMKII/F-actin bundles arising from the multivalence of CaMKII. We also show that the regulatory domain of CaMKII may bind either calmodulin or F-actin, but not both. This frustration, along with the multipartite nature of the binding interface, allows calmodulin transiently to strip CaMKII from actin assemblies so that they can reorganize. This observation therefore provides a simple mechanism by which the structural dynamics of CaMKII establishes the link between calcium signaling and the morphological plasticity of dendritic spines.

Significance The advent of high-speed atomic force microscopy (HS-AFM) changed the field of biology considerably. HS-AFM is the only method where in situ dynamics of biological samples and imaging can be coupled with a spatial resolution of 1 to 5 nm in the horizontal direction. Unlike electron or cryo-electron microscopy, HS-AFM does not require fixation or freezing of the samples, and has the ability to derive kinetic parameters by recording the live movements of single-molecule dynamics. In this paper, we used HS-AFM to investigate directly the mechanisms of cardiac muscle activation. We visualized the muscle regulatory tropomyosin–troponin complex movements during activation by calcium or myosin (motor that drives contraction), and the structural transitions that happen during these events., High-speed atomic force microscopy (HS-AFM) can be used to study dynamic processes with real-time imaging of molecules within 1- to 5-nm spatial resolution. In the current study, we evaluated the 3-state model of activation of cardiac thin filaments (cTFs) isolated as a complex and deposited on a mica-supported lipid bilayer. We studied this complex for dynamic conformational changes 1) at low and high [Ca2+] (pCa 9.0 and 4.5), and 2) upon myosin binding to the cTF in the nucleotide-free state or in the presence of ATP. HS-AFM was used to directly visualize the tropomyosin–troponin complex and Ca2+-induced tropomyosin movements accompanied by structural transitions of actin monomers within cTFs. Our data show that cTFs at relaxing or activating conditions are not ultimately in a blocked or activated state, respectively, but rather the combination of states with a prevalence that is dependent on the [Ca2+] and the presence of weakly or strongly bound myosin. The weakly and strongly bound myosin induce similar changes in the structure of cTFs as confirmed by the local dynamical displacement of individual tropomyosin strands in the center of a regulatory unit of cTF at the relaxed and activation conditions. The displacement of tropomyosin at the relaxed conditions had never been visualized directly and explains the ability of myosin binding to TF at the relaxed conditions. Based on the ratios of nonactivated and activated segments within cTFs, we proposed a mechanism of tropomyosin switching from different states that includes both weakly and strongly bound myosin.

Significance Vascular diseases remain a major health burden, and AAAs lack effective medical therapy. We demonstrate a seminal role for APLN in AAA pathogenesis based on loss-of-function and gain-of-function approaches and included human vascular SMCs and AA tissue obtained from patients. We identified NEP as a dominant inactivating enzyme for native APLN-17. This allowed us to design and synthesize a stable and bioactive APLN analog resistant to NEP degradation that showed profound therapeutic effects against AAA. Our study clearly defines the APLN pathway as a central node in the pathogenesis of AAA and elucidate a therapeutic strategy of enhancing the APLN pathway by using a stable APLN analog to treat AAA., Abdominal aortic aneurysm (AAA) remains the second most frequent vascular disease with high mortality but has no approved medical therapy. We investigated the direct role of apelin (APLN) in AAA and identified a unique approach to enhance APLN action as a therapeutic intervention for this disease. Loss of APLN potentiated angiotensin II (Ang II)-induced AAA formation, aortic rupture, and reduced survival. Formation of AAA was driven by increased smooth muscle cell (SMC) apoptosis and oxidative stress in Apln−/y aorta and in APLN-deficient cultured murine and human aortic SMCs. Ang II-induced myogenic response and hypertension were greater in Apln−/y mice, however, an equivalent hypertension induced by phenylephrine, an α-adrenergic agonist, did not cause AAA or rupture in Apln−/y mice. We further identified Ang converting enzyme 2 (ACE2), the major negative regulator of the renin-Ang system (RAS), as an important target of APLN action in the vasculature. Using a combination of genetic, pharmacological, and modeling approaches, we identified neutral endopeptidase (NEP) that is up-regulated in human AAA tissue as a major enzyme that metabolizes and inactivates APLN-17 peptide. We designed and synthesized a potent APLN-17 analog, APLN-NMeLeu9-A2, that is resistant to NEP cleavage. This stable APLN analog ameliorated Ang II-mediated adverse aortic remodeling and AAA formation in an established model of AAA, high-fat diet (HFD) in Ldlr−/− mice. Our findings define a critical role of APLN in AAA formation through induction of ACE2 and protection of vascular SMCs, whereas stable APLN analogs provide an effective therapy for vascular diseases.

Significance Condensin and cohesin are heteropentameric complexes containing two structural maintenance of chromosomes (SMC) subunits and three non-SMC subunits. SMC dimers form head and hinge domains connected by long coiled coils. Suppressor screening for head-associated non-SMC, and SMC hinge mutants of fission yeast, reveals that condensin is regulated by SUMOylation, ubiquitination, and phosphorylation, while cohesin is regulated by RNA elimination and chromatin remodeling and releasing factors. So, they are regulated by distinct pathways. However, hinge interface mutations are commonly suppressed by mutations in the kleisin N terminus. The results support a “hold and release” model, in which the head and hinge interact to form arched coiled coils that hold and release chromosomal DNAs. The head-kleisin and hinge may cooperate to regulate arched coiled coils’ orientation, which affects their interaction with DNAs., Cohesin and condensin play fundamental roles in sister chromatid cohesion and chromosome segregation, respectively. Both consist of heterodimeric structural maintenance of chromosomes (SMC) subunits, which possess a head (containing ATPase) and a hinge, intervened by long coiled coils. Non-SMC subunits (Cnd1, Cnd2, and Cnd3 for condensin; Rad21, Psc3, and Mis4 for cohesin) bind to the SMC heads. Here, we report a large number of spontaneous extragenic suppressors for fission yeast condensin and cohesin mutants, and their sites were determined by whole-genome sequencing. Mutants of condensin’s non-SMC subunits were rescued by impairing the SUMOylation pathway. Indeed, SUMOylation of Cnd2, Cnd3, and Cut3 occurs in midmitosis, and Cnd3 K870 SUMOylation functionally opposes Cnd subunits. In contrast, cohesin mutants rad21 and psc3 were rescued by loss of the RNA elimination pathway (Erh1, Mmi1, and Red1), and loader mutant mis4 was rescued by loss of Hrp1-mediated chromatin remodeling. In addition, distinct regulations were discovered for condensin and cohesin hinge mutants. Mutations in the N-terminal helix bundle [containing a helix–turn–helix (HTH) motif] of kleisin subunits (Cnd2 and Rad21) rescue virtually identical hinge interface mutations in cohesin and condensin, respectively. These mutations may regulate kleisin’s interaction with the coiled coil at the SMC head, thereby revealing a common, but previously unknown, suppression mechanism between the hinge and the kleisin N domain, which is required for successful chromosome segregation. We propose that in both condensin and cohesin, the head (or kleisin) and hinge may interact and collaboratively regulate the resulting coiled coils to hold and release chromosomal DNAs.

Significance Physiological regulation and behavior depend less on neurons than on neuronal circuits. Neurosignal integration is the basis of neurocircuit function. The modalities of neuroinformation integration are evolutionarily conserved in animals and humans. Here, we identified two modalities of neurosignal integration in two different circuits by which serotonergic ADFs regulate pharyngeal pumping in Caenorhabditis elegans: disinhibition in a feedforward circuit consisting of ADF, RIC, and SIA neurons and disexcitation, a modality of neurosignal integration suggested by this study, in a feedback circuit consisting of ADF, RIC, AWB, and ADF neurons., Feeding is vital for animal survival and is tightly regulated by the endocrine and nervous systems. To study the mechanisms of humoral regulation of feeding behavior, we investigated serotonin (5-HT) and octopamine (OA) signaling in Caenorhabditis elegans, which uses pharyngeal pumping to ingest bacteria into the gut. We reveal that a cross-modulation mechanism between 5-HT and OA, which convey feeding and fasting signals, respectively, mainly functions in regulating the pumping and secretion of both neuromodulators via ADF/RIC/SIA feedforward neurocircuit (consisting of ADF, RIC, and SIA neurons) and ADF/RIC/AWB/ADF feedback neurocircuit (consisting of ADF, RIC, AWB, and ADF neurons) under conditions of food supply and food deprivation, respectively. Food supply stimulates food-sensing ADFs to release more 5-HT, which augments pumping via inhibiting OA secretion by RIC interneurons and, thus, alleviates pumping suppression by OA-activated SIA interneurons/motoneurons. In contrast, nutrient deprivation stimulates RICs to secrete OA, which suppresses pumping via activating SIAs and maintains basal pumping and 5-HT production activity through excitation of ADFs relayed by AWB sensory neurons. Notably, the feedforward and feedback circuits employ distinct modalities of neurosignal integration, namely, disinhibition and disexcitation, respectively.

Significance Budding yeast harbors a simple point centromere, which is originally believed to be sequence dependent without much epigenetic regulation and is transcription incompatible, as inserting a strong promoter upstream inactivates the centromere completely. Here, we demonstrate that an optimal level centromeric noncoding RNA is required for budding yeast centromere activity. Centromeric transcription is induced in S phase, coinciding with the assembly of new centromeric proteins. Too much or too little centromeric noncoding RNA leads to centromere malfunction. Overexpression of centromeric noncoding RNA reduces the protein levels and chromatin localization of inner centromere and kinetochore proteins, such as CENP-A, CENP-C, and the chromosome passenger complex. This work shows that point centromere is epigenetically regulated by noncoding RNA., In budding yeast, which possesses simple point centromeres, we discovered that all of its centromeres express long noncoding RNAs (cenRNAs), especially in S phase. Induction of cenRNAs coincides with CENP-ACse4 loading time and is dependent on DNA replication. Centromeric transcription is repressed by centromere-binding factor Cbf1 and histone H2A variant H2A.ZHtz1. Deletion of CBF1 and H2A.ZHTZ1 results in an up-regulation of cenRNAs; an increased loss of a minichromosome; elevated aneuploidy; a down-regulation of the protein levels of centromeric proteins CENP-ACse4, CENP-A chaperone HJURPScm3, CENP-CMif2, SurvivinBir1, and INCENPSli15; and a reduced chromatin localization of CENP-ACse4, CENP-CMif2, and Aurora BIpl1. When the RNA interference system was introduced to knock down all cenRNAs from the endogenous chromosomes, but not the cenRNA from the circular minichromosome, an increase in minichromosome loss was still observed, suggesting that cenRNA functions in trans to regulate centromere activity. CenRNA knockdown partially alleviates minichromosome loss in cbf1Δ, htz1Δ, and cbf1Δ htz1Δ in a dose-dependent manner, demonstrating that cenRNA level is tightly regulated to epigenetically control point centromere function.

Significance Diverse demands on cardiac muscle require the fine-tuning of contraction. Cardiac myosin binding protein-C (cMyBP-C) is involved in this regulation; however, its precise molecular mechanism of action remains uncertain. By imaging the interactions of single myosin and cMyBP-C molecules interacting with suspended thin filaments in vitro we observe cMyBP-C N-terminal fragments assist activation and modulate contraction velocity by affecting myosin binding to the thin filament. Fluorescent imaging of Cy3-labeled cMyBP-C revealed that it diffusively scans the thin filament and then strongly binds to displace tropomyosin and activate at low calcium. At high calcium, cMyBP-C decorates the filament more extensively, reducing myosin binding through competing with binding sites. Understanding the mechanism of MyBP-C action has important implications for heart disease., Cardiac muscle contraction is triggered by calcium binding to troponin. The consequent movement of tropomyosin permits myosin binding to actin, generating force. Cardiac myosin-binding protein C (cMyBP-C) plays a modulatory role in this activation process. One potential mechanism for the N-terminal domains of cMyBP-C to achieve this is by binding directly to the actin-thin filament at low calcium levels to enhance the movement of tropomyosin. To determine the molecular mechanisms by which cMyBP-C enhances myosin recruitment to the actin-thin filament, we directly visualized fluorescently labeled cMyBP-C N-terminal fragments and GFP-labeled myosin molecules binding to suspended actin-thin filaments in a fluorescence-based single-molecule microscopy assay. Binding of the C0C3 N-terminal cMyBP-C fragment to the thin filament enhanced myosin association at low calcium levels. However, at high calcium levels, C0C3 bound in clusters, blocking myosin binding. Dynamic imaging of thin filament-bound Cy3-C0C3 molecules demonstrated that these fragments diffuse along the thin filament before statically binding, suggesting a mechanism that involves a weak-binding mode to search for access to the thin filament and a tight-binding mode to sensitize the thin filament to calcium, thus enhancing myosin binding. Although shorter N-terminal fragments (Cy3-C0C1 and Cy3-C0C1f) bound to the thin filaments and displayed modes of motion on the thin filament similar to that of the Cy3-C0C3 fragment, the shorter fragments were unable to sensitize the thin filament. Therefore, the longer N-terminal fragment (C0C3) must possess the requisite domains needed to bind specifically to the thin filament in order for the cMyBP-C N terminus to modulate cardiac contractility.

Significance This study answers two long-standing questions about FtsZ dynamics and its relationship to septal peptidoglycan (PG) synthesis in Streptococcus pneumoniae. In previous models, FtsZ concertedly moves from midcell septa to MapZ rings that have reached the equators of daughter cells. Instead, the results presented here show that FtsZ, FtsA, and EzrA filaments/bundles move continuously out from early septa as part of MapZ rings. In addition, this study establishes that the movement of bPBP2x:FtsW complexes in septal PG synthesis depends on and likely mirrors new PG synthesis and is not correlated with the treadmilling of FtsZ filaments/bundles. These findings are consistent with a mechanism where septal FtsZ rings organize directional movement of bPBP2x:FtsW complexes dependent upon PG substrate availability., Bacterial cell division and peptidoglycan (PG) synthesis are orchestrated by the coordinated dynamic movement of essential protein complexes. Recent studies show that bidirectional treadmilling of FtsZ filaments/bundles is tightly coupled to and limiting for both septal PG synthesis and septum closure in some bacteria, but not in others. Here we report the dynamics of FtsZ movement leading to septal and equatorial ring formation in the ovoid-shaped pathogen, Streptococcus pneumoniae. Conventional and single-molecule total internal reflection fluorescence microscopy (TIRFm) showed that nascent rings of FtsZ and its anchoring and stabilizing proteins FtsA and EzrA move out from mature septal rings coincident with MapZ rings early in cell division. This mode of continuous nascent ring movement contrasts with a failsafe streaming mechanism of FtsZ/FtsA/EzrA observed in a ΔmapZ mutant and another Streptococcus species. This analysis also provides several parameters of FtsZ treadmilling in nascent and mature rings, including treadmilling velocity in wild-type cells and ftsZ(GTPase) mutants, lifetimes of FtsZ subunits in filaments and of entire FtsZ filaments/bundles, and the processivity length of treadmilling of FtsZ filament/bundles. In addition, we delineated the motion of the septal PBP2x transpeptidase and its FtsW glycosyl transferase-binding partner relative to FtsZ treadmilling in S. pneumoniae cells. Five lines of evidence support the conclusion that movement of the bPBP2x:FtsW complex in septa depends on PG synthesis and not on FtsZ treadmilling. Together, these results support a model in which FtsZ dynamics and associations organize and distribute septal PG synthesis, but do not control its rate in S. pneumoniae.

Significance Through conducting quantitative proteomics upon Salmonella infection, we identified a SIK2 signaling network, implementing the kinase into a so far concealed biological function. Our data exposed SIK2 as a central orchestrator of an actin regulatory network, coordinating the stability of Salmonella-containing vacuole (SCV) and cellular actin assembly, in order to limit the acute phase of the infection. Most strikingly, SIK2 is not exclusively acting locally on actin assembly associated with the SCV but impacts the actin cytoskeleton architecture in its entirety upon Salmonella infection. Our work provides a mechanistic framework for how the actin cytoskeleton is regulated and how it helps to control an acute Salmonella infection., Salmonella is an intracellular pathogen of a substantial global health concern. In order to identify key players involved in Salmonella infection, we performed a global host phosphoproteome analysis subsequent to bacterial infection. Thereby, we identified the kinase SIK2 as a central component of the host defense machinery upon Salmonella infection. SIK2 depletion favors the escape of bacteria from the Salmonella-containing vacuole (SCV) and impairs Xenophagy, resulting in a hyperproliferative phenotype. Mechanistically, SIK2 associates with actin filaments under basal conditions; however, during bacterial infection, SIK2 is recruited to the SCV together with the elements of the actin polymerization machinery (Arp2/3 complex and Formins). Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. We propose that SIK2 controls the formation of a protective SCV actin shield shortly after invasion and orchestrates the actin cytoskeleton architecture in its entirety to control an acute Salmonella infection after bacterial invasion.

Intracellular procollagen folding begins at the protein's C-terminal propeptide (C-Pro) domain, which initiates triple-helix assembly and defines the composition and chain register of fibrillar collagen trimers. The C-Pro domain is later proteolytically cleaved and excreted from the body, while the mature triple helix is incorporated into the extracellular matrix. The procollagen C-Pro domain possesses a single N-glycosylation site that is widely conserved in all the fibrillar procollagens across humans and diverse other species. Given that the C-Pro domain is removed once procollagen folding is complete, the N-glycan might be presumed to be important for folding. Surprisingly, however, there is no difference in the folding and secretion of N-glycosylated versus non-N-glycosylated collagen type-I, leaving the function of the N-glycan unclear. We hypothesized that the collagen N-glycan might have a context-dependent function, specifically, that it could be required to promote procollagen folding only when proteostasis is challenged. We show that removal of the N-glycan from misfolding-prone C-Pro domain variants does indeed cause serious procollagen and ER proteostasis defects. The N-glycan promotes folding and secretion of destabilized C-Pro variants by providing access to the ER's lectin-based chaperone machinery. Finally, we show that the C-Pro N-glycan is actually critical for the folding and secretion of even wild-type procollagen under ER stress conditions. Such stress is commonly incurred during development, wound healing, and other processes in which collagen production plays a key role. Collectively, these results establish an essential, context-dependent function for procollagen's previously enigmatic N-glycan, wherein the carbohydrate moiety buffers procollagen folding against proteostatic challenge.

Fusion-associated small transmembrane (FAST) proteins are a diverse family of non-structural viral proteins that, once expressed on the plasma membrane of infected cells, drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the inter-membrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP-mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tails of p22 and p14 can be exchanging to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we replace p22’s branched actin nucleator, N-WASP, with the parallel filament nucleator, formin, its ability to drive fusion is maintained, indicating that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.

Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), which minimally requires two other subunits, EED and SUZ12, for enzymatic activity. EZH2 has been traditionally known to mediate histone H3K27 trimethylation, a hallmark of silent chromatin. Emerging evidence indicates that EZH2 also activates gene expression in cancer cells in a context distinct from canonical PRC2. The molecular mechanism underlying the functional conversion of EZH2 from a gene repressor to an activator is unclear. Here, we show that EZH2 harbors a hidden, partially disordered transactivation domain (TAD) capable of interacting with components of active transcription machinery, mimicking archetypal acidic activators. The EZH2 TAD comprises the SRM (Stimulation-Responsive Motif) and SANT1 (SWI3, ADA2, N-CoR, and TFIIIB 1) regions that are normally involved in H3K27 methylation. The crystal structure of an EZH2-EED binary complex indicates that the EZH2 TAD mediates protein oligomerization in a noncanonical PRC2 context and is entirely sequestered. The EZH2 TAD can be unlocked by cancer-specific EZH2 phosphorylation events to undergo structural transitions that may enable subsequent transcriptional coactivator binding. The EZH2 TAD directly interacts with the transcriptional coactivator and histone acetyltransferase p300 and activates gene expression in a p300-dependent manner in cells. The corresponding TAD may also account for the gene activation function of EZH1, the paralog of EZH2. Distinct kinase signaling pathways that are known to abnormally convert EZH2 into a gene activator in cancer cells can now be understood in a common structural context of the EZH2 TAD.

Significance Some substances are known to exist in several different structurally disordered solid states and transform between them similarly to structural phase transitions between crystalline polymorphs. This interesting and yet not fully understood phenomenon is called polyamorphism, and notable examples include water, SiO 2 , Si, etc. Here we present an example of such behavior in a simple molecular substance, SO 2 . By using experimental high-pressure techniques, we observe a reversible transition between different amorphous states around 26 GPa. Experimental results are well supported by ab initio simulations, which identify the high-pressure amorphous form as a nonmolecular, polymeric one consisting of intertwined chains. Our findings contribute to the fundamental understanding of structure of disordered matter as well as high-pressure behavior of simple archetypal molecules.

Microtubules (MTs) are essential components of the eukaryotic cytoskeleton that serve as “highways” for intracellular trafficking. In addition to the well-known active transport of cargo by motor proteins, many MT-binding proteins seem to adopt diffusional motility as a transportation mechanism. However, because of the limited spatial resolution of current experimental techniques, the detailed mechanism of protein diffusion has not been elucidated. In particular, the precise role of tubulin tails and tail modifications in the diffusion process is unclear. Here, using coarse-grained molecular dynamics simulations validated against atomistic simulations, we explore the molecular mechanism of protein diffusion along MTs. We found that electrostatic interactions play a central role in protein diffusion; the disordered tubulin tails enhance affinity but slow down diffusion, and diffusion occurs in discrete steps. While diffusion along wild-type MT is performed in steps of dimeric tubulin, the removal of the tails results in a step of monomeric tubulin. We found that the energy barrier for diffusion is larger when diffusion on MTs is mediated primarily by the MT tails rather than the MT body. In addition, globular proteins (EB1 and PRC1) diffuse more slowly than an intrinsically disordered protein (Tau) on MTs. Finally, we found that polyglutamylation and polyglycylation of tubulin tails lead to slower protein diffusion along MTs, although polyglycylation leads to faster diffusion across MT protofilaments. Taken together, our results explain experimentally observed data and shed light on the roles played by disordered tubulin tails and tail modifications in the molecular mechanism of protein diffusion along MTs.

The yeast prion protein Sup35, which contains intrinsically disordered regions, forms amyloid fibrils responsible for a prion phenotype [ PSI + ]. Using high-speed atomic force microscopy (HS-AFM), we directly visualized the prion determinant domain (Sup35NM) and the formation of its oligomers and fibrils at subsecond and submolecular resolutions. Monomers with freely moving tail-like regions initially appeared in the images, and subsequently oligomers with distinct sizes of ∼1.7 and 3 to 4 nm progressively accumulated. Nevertheless, these oligomers did not form fibrils, even after an incubation for 2 h in the presence of monomers. Fibrils appeared after much longer monomer incubation. The fibril elongation occurred smoothly without discrete steps, suggesting gradual conversions of the incorporated monomers into cross-β structures. The individual oligomers were separated from each other and also from the fibrils by respective, identical lengths on the mica surface, probably due to repulsion caused by the freely moving disordered regions. Based on these HS-AFM observations, we propose that the freely moving tails of the monomers are incorporated into the fibril ends, and then the structural conversions to cross-β structures gradually occur.

Cytoskeletons are self-organized networks based on polymerized proteins: actin, tubulin, and driven by motor proteins, such as myosin, kinesin, and dynein. Their positive Darwinian evolution enables them to approach optimized functionality (self-organized criticality). Dynein has three distinct titled subunits, but how these units connect to function as a molecular motor is mysterious. Dynein binds to tubulin through two coiled coil stalks and a stalk head. The energy used to alter the head binding and propel cargo along tubulin is supplied by ATP at a ring 1,500 amino acids away. Here, we show how many details of this extremely distant interaction are explained by water waves quantified by thermodynamic scaling. Water waves have shaped all proteins throughout positive Darwinian evolution, and many aspects of long-range water–protein interactions are universal (described by self-organized criticality). Dynein water waves resembling tsunami produce nearly optimal energy transport over 1,500 amino acids along dynein’s one-dimensional peptide backbone. More specifically, this paper identifies many similarities in the function and evolution of dynein compared to other cytoskeleton proteins such as actin, myosin, and tubulin.

In Alzheimer’s disease (AD), human Tau is phosphorylated at S199 (hTau-S199-P) by the protein kinase glycogen synthase kinase 3β (GSK3β). HTau-S199-P mislocalizes to dendritic spines, which induces synaptic dysfunction at the early stage of AD. The AKT kinase, once phosphorylated, inhibits GSK3β by phosphorylating it at S9. In AD patients, the abundance of phosphorylated AKT with active GSK3β implies that phosphorylated AKT was unable to inactivate GSK3β. However, the underlying mechanism of the inability of phosphorylated AKT to phosphorylate GSK3β remains unknown. Here, we show that total AKT and phosphorylated AKT was sulfhydrated at C77 due to the induction of intracellular hydrogen sulfide (H(2)S). The increase in intracellular H(2)S levels resulted from the induction of the proinflammatory cytokine, IL-1β, which is a pathological hallmark of AD. Sulfhydrated AKT does not interact with GSK3β, and therefore does not phosphorylate GSK3β. Thus, active GSK3β phosphorylates Tau aberrantly. In a transgenic knockin mouse (AKT-KI(+/+)) that lacked sulfhydrated AKT, the interaction between AKT or phospho-AKT with GSK3β was restored, and GSK3β became phosphorylated. In AKT-KI(+/+) mice, expressing the pathogenic human Tau mutant (hTau-P301L), the hTau S199 phosphorylation was ameliorated as GSK3β phosphorylation was regained. This event leads to a decrease in dendritic spine loss by reducing dendritic localization of hTau-S199-P, which improves cognitive dysfunctions. Sulfhydration of AKT was detected in the postmortem brains from AD patients; thus, it represents a posttranslational modification of AKT, which primarily contributes to synaptic dysfunction in AD.

Polycomb group proteins are essential regulators of developmental processes across animals. Despite their importance, studies on Polycomb are often restricted to classical model systems and, as such, little is known about the evolution of these important chromatin regulators. Here we focus on Polycomb Repressive Complex 1 (PRC1) and trace the evolution of core components of canonical and non-canonical PRC1 complexes in animals. Previous work suggested that a major expansion in the number of PRC1 complexes occurred in the vertebrate lineage. Here we show that the expansion of the PCGF protein family, an essential step for the establishment of the large diversity of PRC1 complexes found in vertebrates, predates the bilaterian-cnidarian ancestor. This means that the genetic repertoire necessary to form all major vertebrate PRC1 complexes emerged early in animal evolution, over 550 million years ago. We further show thatPCGF5, a gene conserved in cnidarians and vertebrates but lost in all other studied groups, is expressed in the nervous system in the sea anemoneNematostella vectensis, similar to its mammalian counterpart. Together this work provides an evolutionary framework to understand PRC1 complex diversity and evolution and establishesNematostellaas a promising model system in which this can be further explored.

Virtually all proton-pumping terminal respiratory oxygen reductases are members of the heme-copper oxidoreductase superfamily. Most of these enzymes use reduced cytochrome c as a source of electrons, but a group of enzymes have evolved to directly oxidize membrane-bound quinols, usually menaquinol or ubiquinol. All of the quinol oxidases have an additional transmembrane helix (TM0) in subunit I that is not present in the related cytochrome c oxidases. The current work reports the 3.6-Å-resolution X-ray structure of the cytochrome aa 3 -600 menaquinol oxidase from Bacillus subtilis containing 1 equivalent of menaquinone. The structure shows that TM0 forms part of a cleft to accommodate the menaquinol-7 substrate. Crystals which have been soaked with the quinol-analog inhibitor HQNO ( N -oxo-2-heptyl-4-hydroxyquinoline) or 3-iodo-HQNO reveal a single binding site where the inhibitor forms hydrogen bonds to amino acid residues shown previously by spectroscopic methods to interact with the semiquinone state of menaquinone, a catalytic intermediate.

Significance Inherited pathogenic mutations in genes required for copper delivery to cytochrome c oxidase (CcO) perturb mitochondrial energy metabolism and result in fatal mitochondrial disease. A prior attempt to treat human patients with these mutations by direct copper supplementation was not successful, possibly because of inefficient copper delivery to the mitochondria. We performed a targeted search to identify compounds that can efficiently transport copper across biological membranes and identified elesclomol (ES), an investigational anticancer drug, as the most efficient copper delivery agent. ES rescues CcO function in yeast, zebrafish, and mammalian models of copper deficiency by increasing cellular and mitochondrial copper content. Thus, our study offers a possibility of repurposing this anticancer drug for the treatment of disorders of copper metabolism., Copper is an essential cofactor of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Inherited loss-of-function mutations in several genes encoding proteins required for copper delivery to CcO result in diminished CcO activity and severe pathologic conditions in affected infants. Copper supplementation restores CcO function in patient cells with mutations in two of these genes, COA6 and SCO2, suggesting a potential therapeutic approach. However, direct copper supplementation has not been therapeutically effective in human patients, underscoring the need to identify highly efficient copper transporting pharmacological agents. By using a candidate-based approach, we identified an investigational anticancer drug, elesclomol (ES), that rescues respiratory defects of COA6-deficient yeast cells by increasing mitochondrial copper content and restoring CcO activity. ES also rescues respiratory defects in other yeast mutants of copper metabolism, suggesting a broader applicability. Low nanomolar concentrations of ES reinstate copper-containing subunits of CcO in a zebrafish model of copper deficiency and in a series of copper-deficient mammalian cells, including those derived from a patient with SCO2 mutations. These findings reveal that ES can restore intracellular copper homeostasis by mimicking the function of missing transporters and chaperones of copper, and may have potential in treating human disorders of copper metabolism.

Significance Orthopedic implant infections require long-term antibiotic therapy and surgical debridement to successfully retain the implant; however, therapeutic failure can lead to implant removal. Here an injectable PEG-based hydrogel that adheres to exposed tissue and fracture surfaces is engineered to deliver the antimicrobial enzyme lysostaphin to infected, implant-fixed, mouse femoral fractures. Lysostaphin encapsulation within the hydrogel enhances enzyme stability while providing enhanced antibiofilm activity and serving as a controlled delivery platform. In a preclinical animal model of orthopedic-implant infection, we show that lysostaphin-delivering hydrogels outperform prophylactic antibiotic therapy and soluble lysostaphin, by eradicating infection while promoting bone repair. Importantly, lysostaphin-delivering hydrogels are effective against antibiotic-resistant infections. This lysostaphin delivery platform could be highly effective at treating and preventing implant infections., Orthopedic implant infections are a significant clinical problem, with current therapies limited to surgical debridement and systemic antibiotic regimens. Lysostaphin is a bacteriolytic enzyme with high antistaphylococcal activity. We engineered a lysostaphin-delivering injectable PEG hydrogel to treat Staphylococcus aureus infections in bone fractures. The injectable hydrogel formulation adheres to exposed tissue and fracture surfaces, ensuring efficient, local delivery of lysostaphin. Lysostaphin encapsulation within this synthetic hydrogel maintained enzyme stability and activity. Lysostaphin-delivering hydrogels exhibited enhanced antibiofilm activity compared with soluble lysostaphin. Lysostaphin-delivering hydrogels eradicated S. aureus infection and outperformed prophylactic antibiotic and soluble lysostaphin therapy in a murine model of femur fracture. Analysis of the local inflammatory response to infections treated with lysostaphin-delivering hydrogels revealed indistinguishable differences in cytokine secretion profiles compared with uninfected fractures, demonstrating clearance of bacteria and associated inflammation. Importantly, infected fractures treated with lysostaphin-delivering hydrogels fully healed by 5 wk with bone formation and mechanical properties equivalent to those of uninfected fractures, whereas fractures treated without the hydrogel carrier were equivalent to untreated infections. Finally, lysostaphin-delivering hydrogels eliminate methicillin-resistant S. aureus infections, supporting this therapy as an alternative to antibiotics. These results indicate that lysostaphin-delivering hydrogels effectively eliminate orthopedic S. aureus infections while simultaneously supporting fracture repair.

Significance Accumulation of islet amyloid polypeptide (IAPP)-containing amyloid fibrils is the main pathological finding in pancreatic islets in type 2 diabetes. The formation of these IAPP amyloid fibrils is considered toxic and may constitute a major cause for the loss of insulin-producing beta cells. The protein domain BRICHOS is present in several different proproteins and possesses antiamyloid chaperone activity. This study demonstrates expression of the BRICHOS-containing protein Bri2 in human pancreatic beta cells and its colocalization with IAPP. The Bri2 BRICHOS domain effectively prevents IAPP from forming fibrils and protects cells from the toxicity associated with IAPP fibrillation. It is concluded that the Bri2 BRICHOS domain may act as an endogenous inhibitor of IAPP amyloid formation in pancreatic beta cells., Aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils in islets of Langerhans is associated with type 2 diabetes, and formation of toxic IAPP species is believed to contribute to the loss of insulin-producing beta cells. The BRICHOS domain of integral membrane protein 2B (Bri2), a transmembrane protein expressed in several peripheral tissues and in the brain, has recently been shown to prevent fibril formation and toxicity of Aβ42, an amyloid-forming peptide in Alzheimer disease. In this study, we demonstrate expression of Bri2 in human islets and in the human beta-cell line EndoC-βH1. Bri2 colocalizes with IAPP intracellularly and is present in amyloid deposits in patients with type 2 diabetes. The BRICHOS domain of Bri2 effectively inhibits fibril formation in vitro and instead redirects IAPP into formation of amorphous aggregates. Reduction of endogenous Bri2 in EndoC-βH1 cells with siRNA increases sensitivity to metabolic stress leading to cell death while a concomitant overexpression of Bri2 BRICHOS is protective. Also, coexpression of IAPP and Bri2 BRICHOS in lateral ventral neurons of Drosophila melanogaster results in an increased cell survival. IAPP is considered to be the most amyloidogenic peptide known, and described findings identify Bri2, or in particular its BRICHOS domain, as an important potential endogenous inhibitor of IAPP aggregation and toxicity, with the potential to be a possible target for the treatment of type 2 diabetes.

Significance Kinetochores are large protein networks located on centromeres that mediate chromosome segregation during mitosis and maintain genomic stability. Mis12 complex (Mis12C) functions as a scaffold that targets Ndc80 and Knl1 complexes to the centromere by associating with CENP-C. Here, we provide insights into the molecular mechanism underlying the CENP-C–dependent kinetochore recruitment of Mis12C, which is negatively regulated by Aurora B-dependent CENP-C phosphorylation. Replacement of Schizosaccharomyces pombe Cnp3 with a phosphorylation-mimicking mutant, Cnp3T28E, results in defective chromosome segregation caused by improper kinetochore assembly. These findings indicate that Aurora B-dependent phosphorylation of CENP-C plays a role in interrupting the connection between the inner and outer kinetochore and is thus involved in the error correction/spindle assembly checkpoint pathway to prevent chromosome missegregation during mitosis., Kinetochores are superprotein complexes that orchestrate chromosome segregation via a dynamic interaction with spindle microtubules. A physical connection between CENP-C and the Mis12–Ndc80–Knl1 (KMN) protein network is an important pathway that is used to assemble kinetochores on CENP-A nucleosomes. Multiple outer kinetochore components are phosphorylated by Aurora B kinase to activate the spindle assembly checkpoint (SAC) and to ensure accurate chromosome segregation. However, it is unknown whether Aurora B can phosphorylate inner kinetochore components to facilitate proper mitotic chromosome segregation. Here, we reported the structure of the fission yeast Schizosaccharomyces pombe Mis12–Nnf1 complex and showed that N-terminal residues 26–50 in Cnp3 (the CENP-C homolog of S. pombe) are responsible for interacting with the Mis12 complex. Interestingly, Thr28 of Cnp3 is a substrate of Ark1 (the Aurora B homolog of S. pombe), and phosphorylation impairs the interaction between the Cnp3 and Mis12 complex. The expression of a phosphorylation-mimicking Cnp3 mutant results in defective chromosome segregation due to improper kinetochore assembly. These results establish a previously uncharacterized regulatory mechanism involved in CENP-C–Mis12-facilitated kinetochore attachment error correction to ensure accurate chromosome segregation during mitosis.

Significance Nitric oxide synthase-2 (NOS2) and cyclooxygenase-2 (COX2) are inflammation-associated enzymes with oncogenic function in breast cancer. We show that crosstalk between NOS2/COX2 promotes aggressive phenotypes and that elevated coexpression of NOS2/COX2 in tumors predict significantly reduced patient survival (33%) when compared with 95% survival of estrogen receptor-negative patients with low NOS2/COX2 tumor expression. In addition, we have identified a tumor subtype-specific mechanism showing involvement of TNFα and/or endoplasmic reticulum stress as key players in this autocrine loop. Importantly, the simultaneous inhibition of NOS2/COX2 significantly reduced tumor growth in a xenograft murine model, suggesting that targeted inhibition of NOS2/COX2 may be therapeutically beneficial., Proinflammatory signaling pathways are commonly up-regulated in breast cancer. In estrogen receptor-negative (ER−) and triple-negative breast cancer (TNBC), nitric oxide synthase-2 (NOS2) and cyclooxygenase-2 (COX2) have been described as independent predictors of disease outcome. We further explore these findings by investigating the impact of their coexpression on breast cancer survival. Elevated coexpression of NOS2/COX2 proteins is a strong predictor of poor survival among ER− patients (hazard ratio: 21). Furthermore, we found that the key products of NOS2 and COX2, NO and prostaglandin E2 (PGE2), respectively, promote feed-forward NOS2/COX2 crosstalk in both MDA-MB-468 (basal-like) and MDA-MB-231 (mesenchymal-like) TNBC cell lines in which NO induced COX2 and PGE2 induced NOS2 proteins. COX2 induction by NO involved TRAF2 activation that occurred in a TNFα-dependent manner in MDA-MB-468 cells. In contrast, NO-mediated TRAF2 activation in the more aggressive MDA-MB-231 cells was TNFα independent but involved the endoplasmic reticulum stress response. Inhibition of NOS2 and COX2 using amino-guanidine and aspirin/indomethacin yielded an additive reduction in the growth of MDA-MB-231 tumor xenografts. These findings support a role of NOS2/COX2 crosstalk during disease progression of aggressive cancer phenotypes and offer insight into therapeutic applications for better survival of patients with ER− and TNBC disease.

Significance In the crowded interior of a cell, diffusion alone is insufficient to master varying transport requirements for cell sustenance and growth. The dynamic actin cytoskeleton is an essential cellular component that provides transport and cytoplasmic streaming in plant cells, but little is known about its system-level organization. Here, we resolve key challenges in understanding system-level actin-based transport. We present an automated image-based, network-driven framework that accurately incorporates both actin cytoskeleton and organelle trafficking. We demonstrate that actin cytoskeleton network properties support efficient transport in both growing and elongated hypocotyl cells. We show that organelle transport can be predicted from the system-wide cellular organization of the actin cytoskeleton. Our framework can be readily applied to investigate cytoskeleton-based transport in other organisms., The actin cytoskeleton is an essential intracellular filamentous structure that underpins cellular transport and cytoplasmic streaming in plant cells. However, the system-level properties of actin-based cellular trafficking remain tenuous, largely due to the inability to quantify key features of the actin cytoskeleton. Here, we developed an automated image-based, network-driven framework to accurately segment and quantify actin cytoskeletal structures and Golgi transport. We show that the actin cytoskeleton in both growing and elongated hypocotyl cells has structural properties facilitating efficient transport. Our findings suggest that the erratic movement of Golgi is a stable cellular phenomenon that might optimize distribution efficiency of cell material. Moreover, we demonstrate that Golgi transport in hypocotyl cells can be accurately predicted from the actin network topology alone. Thus, our framework provides quantitative evidence for system-wide coordination of cellular transport in plant cells and can be readily applied to investigate cytoskeletal organization and transport in other organisms.

A lysine-to-methionine mutation at lysine 27 of histone 3 (H3K27M) has been shown to promote oncogenesis in a subset of pediatric gliomas. While there is evidence that this "oncohistone" mutation acts by inhibiting the histone methyltransferase PRC2, the details of this proposed mechanism nevertheless continue to be debated. Recent evidence suggests that PRC2 must simultaneously bind both H3K27M and H3K27me3 to experience competitive inhibition of its methyltransferase activity. In this work, we used PRC2 inhibitor treatments in a transgenic H3K27M cell line to validate this dependence in a cellular context. We further used designer chromatin inhibitors to probe the geometric constraints of PRC2 engagement of H3K27M and H3K27me3 in a biochemical setting. We found that PRC2 binds to a bivalent inhibitor unit consisting of an H3K27M and an H3K27me3 nucleosome and exhibits a distance dependence in its affinity for such an inhibitor, which favors closer proximity of the 2 nucleosomes within a chromatin array. Together, our data precisely delineate fundamental aspects of the H3K27M inhibitor and support a model wherein PRC2 becomes trapped at H3K27M-H3K27me3 boundaries.

Skeletal muscle myosin-binding protein C (MyBP-C) is a myosin thick filament-associated protein; localized through its C terminus to distinct regions (C-zones) of the sarcomere. MyBP-C modulates muscle contractility, presumably through its N terminus extending from the thick filament and interacting with either the myosin head region and/or the actin thin filament. Two isoforms of MyBP-C (fast- and slow-type) are expressed in a muscle-type specific manner. Are the expression, localization, and Ca2+-dependent modulatory capacities of these isoforms different in fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles derived from Sprague-Dawley rats? By mass spectrometry, four MyBP-C isoforms (one fast-type MyBP-C and three N-terminally spliced slow-type MyBP-C) were expressed in EDL but only the three slow-type MyBP-C isoforms in SOL. Using EDL and SOL native thick filaments in which the MyBP-C stoichiometry and localization are preserved, native thin filament sliding over these thick filaments showed that only in the C-zone, MyBP-C Ca2+-sensitizes the thin filament and slows thin filament velocity. These modulatory properties depended on MyBP-C’s N-terminus, as N-terminal proteolysis attenuated MyBP-C’s functional capacities. To determine each MyBP-C isoform’s contribution to thin filament Ca2+-sensitization and slowing in the C-zone, we used a combination ofin vitromotility assays using expressed recombinant N-terminal fragments andin silicomechanistic modeling. Our results suggest that each skeletal MyBP-C isoform’s N terminus is functionally distinct and has modulatory capacities that depend on the muscle-type in which they are expressed, providing the potential for molecular tuning of skeletal muscle performance through differential MyBP-C expression.SIGNIFICANCEMyosin-binding protein C (MyBP-C) is a critical component of the skeletal muscle sarcomere, muscle’s smallest contractile unit. MyBP-C’s importance is evident by genetic mutations leading to human myopathies, such as distal arthrogryposis (i.e. club foot). However, the molecular basis of MyBP-C’s functional impact on skeletal muscle contractility is far from certain. Complicating matters further is the expression of fast- and slow-type MyBP-C isoforms that depend on whether the muscle is fast- or slow-twitch. Using multi-scale proteomic, biophysical and mathematical modeling approaches, we define the expression, localization, and modulatory capacities of these distinct skeletal MyBP-C isoforms in rat skeletal muscles. Each MyBP-C isoform appears to modulate muscle contractility differentially; providing the capacity to fine-tune muscle’s mechanical performance as physiological demands arise.

Significance In the disease familial amyloidosis, Finnish type (FAF) the mechanism by which point mutations in gelsolin domain 2 (G2) lead to furin cleavage is not understood for the intact protein. Here, we determine that FAF mutants adopt similar conformations to the wild-type protein. However, the mutations appear to affect the dynamics of domain:domain interactions. Thus, proper domain:domain interactions are needed to protect G2 from protease cleavage. We make mutations in the following domain (G3) that functionally mimic the FAF mutations in G2. We conclude that G2 is on the limits of stability, and perturbations that affect domain:domain stabilizing interactions tip the balance toward cleavage. These data explain how multiple FAF mutations give rise to amyloid formation., In the disease familial amyloidosis, Finnish type (FAF), also known as AGel amyloidosis (AGel), the mechanism by which point mutations in the calcium-regulated actin-severing protein gelsolin lead to furin cleavage is not understood in the intact protein. Here, we provide a structural and biochemical characterization of the FAF variants. X-ray crystallography structures of the FAF mutant gelsolins demonstrate that the mutations do not significantly disrupt the calcium-free conformations of gelsolin. Small-angle X-ray–scattering (SAXS) studies indicate that the FAF calcium-binding site mutants are slower to activate, whereas G167R is as efficient as the wild type. Actin-regulating studies of the gelsolins at the furin cleavage pH (6.5) show that the mutant gelsolins are functional, suggesting that they also adopt relatively normal active conformations. Deletion of gelsolin domains leads to sensitization to furin cleavage, and nanobody-binding protects against furin cleavage. These data indicate instability in the second domain of gelsolin (G2), since loss or gain of G2-stabilizing interactions impacts the efficiency of cleavage by furin. To demonstrate this principle, we engineered non-FAF mutations in G3 that disrupt the G2-G3 interface in the calcium-activated structure. These mutants led to increased furin cleavage. We carried out molecular dynamics (MD) simulations on the FAF and non-FAF mutant G2-G3 fragments of gelsolin. All mutants showed an increase in the distance between the center of masses of the 2 domains (G2 and G3). Since G3 covers the furin cleavage site on G2 in calcium-activated gelsolin, this suggests that destabilization of this interface is a critical step in cleavage.

The mTORC1 pathway regulates cell growth and proliferation by properly coupling critical processes such as gene expression, protein translation, and metabolism to the availability of growth factors and hormones, nutrients, cellular energetics, oxygen status, and cell stress. Although multiple cytoplasmic substrates of mTORC1 have been identified, how mTORC1 signals within the nucleus remains incompletely understood. Here, we report a mechanism by which mTORC1 modulates the phosphorylation of multiple nuclear events. We observed a significant nuclear enrichment of GSK3 when mTORC1 was suppressed, which promotes phosphorylation of several proteins such as GTF2F1 and FOXK1. Importantly, nuclear localization of GSK3 is sufficient to suppress cell proliferation. Additionally, expression of a nuclear exporter of GSK3, FRAT, restricts the nuclear localization of GSK3, represses nuclear protein phosphorylation, and prevents rapamycin-induced cytostasis. Finally, we observe a correlation between rapamycin resistance and FRAT expression in multiple-cancer cell lines. Resistance to Food and Drug Administration (FDA)-approved rapamycin analogs (rapalogs) is observed in many tumor settings, but the underling mechanisms remain incompletely understood. Given that FRAT expression levels are frequently elevated in various cancers, our observations provide a potential biomarker and strategy for overcoming rapamycin resistance.

A descendant of the red algal lineage, diatoms are unicellular eukaryotic algae characterized by thylakoid membranes that lack the spatial differentiation of stroma and grana stacks found in green algae and higher plants. While the photophysiology of diatoms has been studied extensively, very little is known about the spatial organization of the multimeric photosynthetic protein complexes within their thylakoid membranes. Here, using cryo-electron tomography, proteomics, and biophysical analyses, we elucidate the macromolecular composition, architecture, and spatial distribution of photosystem II complexes in diatom thylakoid membranes. Structural analyses reveal 2 distinct photosystem II populations: loose clusters of complexes associated with antenna proteins and compact 2D crystalline arrays of dimeric cores. Biophysical measurements reveal only 1 photosystem II functional absorption cross section, suggesting that only the former population is photosynthetically active. The tomographic data indicate that the arrays of photosystem II cores are physically separated from those associated with antenna proteins. We hypothesize that the islands of photosystem cores are repair stations, where photodamaged proteins can be replaced. Our results strongly imply convergent evolution between the red and the green photosynthetic lineages toward spatial segregation of dynamic, functional microdomains of photosystem II supercomplexes.

Multisubunit protein complexes are ubiquitous in biology and perform a plethora of essential functions. Most of the scientific literature treats such assemblies as static: their function is assumed to be independent of their manner of assembly, and their structure is assumed to remain intact until they are degraded. Recent observations of the bacterial flagellar motor, among others, bring these notions into question. The torque-generating stator units of the motor assemble and disassemble in response to changes in load. Here, we used electrorotation to drive tethered cells forward, which decreases motor load, and measured the resulting stator dynamics. No disassembly occurred while the torque remained high, but all of the stator units were released when the motor was spun near the zero-torque speed. When the electrorotation was turned off, so that the load was again high, stator units were recruited, increasing motor speed in a stepwise fashion. A model in which speed affects the binding rate and torque affects the free energy of bound stator units captures the observed torque-dependent stator assembly dynamics, providing a quantitative framework for the environmentally regulated self-assembly of a major macromolecular machine.

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) accelerates cardiac contractility. However, the mechanisms by which cMyBP-C phosphorylation increases contractile kinetics have not been fully elucidated. In this study, we tested the hypothesis that phosphorylation of cMyBP-C releases myosin heads from the inhibited super-relaxed state (SRX), thereby determining the fraction of myosin available for contraction. Mice with various alanine (A) or aspartic acid (D) substitutions of the three main phosphorylatable serines of cMyBP-C (serines 273, 282, and 302) were used to address the association between cMyBP-C phosphorylation and SRX. Single-nucleotide turnover in skinned ventricular preparations demonstrated that phosphomimetic cMyBP-C destabilized SRX, whereas phospho-ablated cMyBP-C had a stabilizing effect on SRX. Strikingly, phosphorylation at serine 282 site was found to play a critical role in regulating the SRX. Treatment of WT preparations with protein kinase A (PKA) reduced the SRX, whereas, in nonphosphorylatable cMyBP-C preparations, PKA had no detectable effect. Mice with stable SRX exhibited reduced force production. Phosphomimetic cMyBP-C with reduced SRX exhibited increased rates of tension redevelopment and reduced binding to myosin. We also used recombinant myosin subfragment-2 to disrupt the endogenous interaction between cMyBP-C and myosin and observed a significant reduction in the population of SRX myosin. This peptide also increased force generation and rate of tension redevelopment in skinned fibers. Taken together, this study demonstrates that the phosphorylation-dependent interaction between cMyBP-C and myosin is a determinant of the fraction of myosin available for contraction. Furthermore, the binding between cMyBP-C and myosin may be targeted to improve contractile function.

Metazoan cell polarity is controlled by a set of highly conserved proteins. Lethal giant larvae (Lgl) functions in apical-basal polarity through phosphorylation-dependent interactions with several other proteins as well as the plasma membrane. Phosphorylation of Lgl by atypical protein kinase C (aPKC), a component of the partitioning-defective (Par) complex in epithelial cells, excludes Lgl from the apical membrane, a crucial step in the establishment of epithelial cell polarity. We present the crystal structures of human Lgl2 in both its unphosphorylated and aPKC-phosphorylated states. Lgl2 adopts a double β-propeller structure that is unchanged by aPKC phosphorylation of an unstructured loop in its second β-propeller, ruling out models of phosphorylation-dependent conformational change. We demonstrate that phosphorylation controls the direct binding of purified Lgl2 to negative phospholipids in vitro. We also show that a coil–helix transition of this region that is promoted by phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is also phosphorylation-dependent, implying a highly effective phosphorylative switch for membrane association.

Breast cancer development is associated with increasing tissue stiffness over years. To more accurately mimic the onset of gradual matrix stiffening, which is not feasible with conventional static hydrogels, mammary epithelial cells (MECs) were cultured on methacrylated hyaluronic acid hydrogels whose stiffness can be dynamically modulated from "normal" (3,000 Pascals) via two-stage polymerization. MECs form and remain as spheroids, but begin to lose epithelial characteristics and gain mesenchymal morphology upon matrix stiffening. However, both the degree of matrix stiffening and culture time before stiffening play important roles in regulating this conversion as, in both cases, a subset of mammary spheroids remained insensitive to local matrix stiffness. This conversion depended neither on colony size nor cell density, and MECs did not exhibit "memory" of prior niche when serially cultured through cycles of compliant and stiff matrices. Instead, the transcription factor Twist1, transforming growth factor β (TGFβ), and YAP activation appeared to modulate stiffness-mediated signaling; when stiffness-mediated signals were blocked, collective MEC phenotypes were reduced in favor of single MECs migrating away from spheroids. These data indicate a more complex interplay of time-dependent stiffness signaling, spheroid structure, and soluble cues that regulates MEC plasticity than suggested by previous models.

Rac1 activation is at the core of signaling pathways regulating polarized cell migration. So far, it has not been possible to directly explore the structural changes triggered by Rac1 activation at the molecular level. Here, through a multiscale imaging workflow that combines biosensor imaging of Rac1 dynamics with electron cryotomography, we identified, within the crowded environment of eukaryotic cells, a unique nanoscale architecture of a flexible, signal-dependent actin structure. In cell regions with high Rac1 activity, we found a structural regime that spans from the ventral membrane up to a height of ∼60 nm above that membrane, composed of directionally unaligned, densely packed actin filaments, most shorter than 150 nm. This unique Rac1-induced morphology is markedly different from the dendritic network architecture in which relatively short filaments emanate from existing, longer actin filaments. These Rac1-mediated scaffold assemblies are devoid of large macromolecules such as ribosomes or other filament types, which are abundant at the periphery and within the remainder of the imaged volumes. Cessation of Rac1 activity induces a complete and rapid structural transition, leading to the absence of detectable remnants of such structures within 150 s, providing direct structural evidence for rapid actin filament network turnover induced by GTPase signaling events. It is tempting to speculate that this highly dynamical nanoscaffold system is sensitive to local spatial cues, thus serving to support the formation of more complex actin filament architectures—such as those mandated by epithelial−mesenchymal transition, for example—or resetting the region by completely dissipating.

βII-spectrin is the generally expressed member of the β-spectrin family of elongated polypeptides that form micrometer-scale networks associated with plasma membranes. We addressed in vivo functions of βII-spectrin in neurons by knockout of βII-spectrin in mouse neural progenitors. βII-spectrin deficiency caused severe defects in long-range axonal connectivity and axonal degeneration. βII-spectrin- null neurons exhibited reduced axon growth, loss of actin-spectrinbased periodic membrane skeleton, and impaired bidirectional axonal transport of synaptic cargo. We found that βII-spectrin associates with KIF3A, KIF5B, KIF1A, and dynactin, implicating spectrin in the coupling of motors and synaptic cargo. βII-spectrin required phosphoinositide lipid binding to promote axonal transport and restore axon growth. Knockout of ankyrin-B (AnkB), a βII-spectrin partner, primarily impaired retrograde organelle transport, while double knockout of βII-spectrin and AnkB nearly eliminated transport. Thus, βII-spectrin promotes both axon growth and axon stability through establishing the actin- spectrin-based membrane-associated periodic skeleton as well as enabling axonal transport of synaptic cargo.

Phosphatase PP2A expression levels are positively correlated to the clinical severity of systemic lupus erythematosus (SLE) and IL17A cytokine overproduction, indicating a potential role of PP2A in controlling T H 17 differentiation and inflammation. By generating a mouse strain with ablation of the catalytic subunit α of PP2A in peripheral mature T cells (PP2A cKO), we demonstrate that the PP2A complex is essential for T H 17 differentiation. These PP2A cKO mice had reduced T H 17 cell numbers and less severe disease in an experimental autoimmune encephalomyelitis (EAE) model. PP2A deficiency also ablated C-terminal phosphorylation of SMAD2 but increased C-terminal phosphorylation of SMAD3. By regulating the activity of RORγt via binding, the changes in the phosphorylation status of these R-SMADs reduced Il17a gene transcription. Finally, PP2A inhibitors showed similar effects on T H 17 cells as were observed in PP2A cKO mice, i.e., decreased T H 17 differentiation and relative protection of mice from EAE. Taken together, these data demonstrate that phosphatase PP2A is essential for T H 17 differentiation and that inhibition of PP2A could be a possible therapeutic approach to controlling T H 17-driven autoimmune diseases.

Myosin motors are the fundamental force-generating elements of muscle contraction. Variation in the human β-cardiac myosin heavy chain gene (MYH7) can lead to hypertrophic cardiomyopathy (HCM), a heritable disease characterized by cardiac hypertrophy, heart failure, and sudden cardiac death. How specific myosin variants alter motor function or clinical expression of disease remains incompletely understood. Here, we combine structural models of myosin from multiple stages of its chemomechanical cycle, exome sequencing data from two population cohorts of 60,706 and 42,930 individuals, and genetic and phenotypic data from 2,913 patients with HCM to identify regions of disease enrichment within β-cardiac myosin. We first developed computational models of the human β-cardiac myosin protein before and after the myosin power stroke. Then, using a spatial scan statistic modified to analyze genetic variation in protein 3D space, we found significant enrichment of disease-associated variants in the converter, a kinetic domain that transduces force from the catalytic domain to the lever arm to accomplish the power stroke. Focusing our analysis on surface-exposed residues, we identified a larger region significantly enriched for disease-associated variants that contains both the converter domain and residues on a single flat surface on the myosin head described as the myosin mesa. Notably, patients with HCM with variants in the enriched regions have earlier disease onset than patients who have HCM with variants elsewhere. Our study provides a model for integrating protein structure, large-scale genetic sequencing, and detailed phenotypic data to reveal insight into time-shifted protein structures and genetic disease.