The cerebellum is hypothesized to represent timing information important for organizing salient motor events during periodically performed discontinuous movements. To provide functional evidence validating this idea, we measured and manipulated Purkinje cell (PC) activity in the lateral cerebellum of mice trained to volitionally perform periodic bouts of licking for regularly allocated water rewards. Overall, PC simple spiking modulated during task performance, mapping phasic tongue protrusions and retractions, as well as ramping prior to both lick-bout initiation and termination, two important motor events delimiting movement cycles. The ramping onset occurred earlier for the initiation of uncued exploratory licking that anticipated water availability relative to licking that was reactive to water allocation, suggesting that the cerebellum is engaged differently depending on the movement context. In a subpopulation of PCs, climbing-fiber-evoked responses also increased during lick-bout initiation, but not termination, highlighting differences in how cerebellar input pathways represent task-related information. Optogenetic perturbation of PC activity disrupted the behavior by degrading lick-bout rhythmicity in addition to initiating and terminating licking bouts confirming a causative role in movement organization. Together, these results substantiate that the cerebellum contributes to the initiation and timing of repeated motor actions.

Antimicrobial peptides (AMPs) are broad spectrum antibiotics that selectively target bacteria. Here we investigate the activity of human AMP LL37 against Escherichia coli by integrating quantitative, population and single-cell level experiments with theoretical modeling. We observe an unexpected, rapid absorption and retention of a large number of LL37 peptides by E. coli cells upon the inhibition of their growth, which increases population survivability. This transition occurs more likely in the late stage of cell division cycles. Cultures with high cell density exhibit two distinct subpopulations: a non-growing population that absorb peptides and a growing population that survive owing to the sequestration of the AMPs by others. A mathematical model based on this binary picture reproduces the rather surprising observations, including the increase of the minimum inhibitory concentration with cell density (even in dilute cultures) and the extensive lag in growth introduced by sub-lethal dosages of LL37 peptides.

Efficient planning in complex environments requires that uncertainty associated with current inferences and possible consequences of forthcoming actions is represented. Representation of uncertainty has been established in sensory systems during simple perceptual decision making tasks but it remains unclear if complex cognitive computations such as planning and navigation are also supported by probabilistic neural representations. Here, we capitalized on gradually changing uncertainty along planned motion trajectories during hippocampal theta sequences to capture signatures of uncertainty representation in population responses. In contrast with prominent theories, we found no evidence of encoding parameters of probability distributions in the momentary population activity recorded in an open-field navigation task in rats. Instead, uncertainty was encoded sequentially by sampling motion trajectories randomly and efficiently in subsequent theta cycles from the distribution of potential trajectories. Our analysis is the first to demonstrate that the hippocampus is well equipped to contribute to optimal planning by representing uncertainty.

The brain plays an essential role in driving daily rhythms of behavior and metabolism in harmony with environmental light-dark cycles. Within the brain, the dorsomedial hypothalamic nucleus (DMH) has been implicated in the integrative circadian control of feeding and energy homeostasis, but the underlying cell types are unknown. Here, we identify a role for DMH leptin receptor-expressing (DMHLepR) neurons in this integrative control. Using a viral approach, we show that silencing neurotransmission in DMHLepR neurons in adult mice not only increases body weight and adiposity but also phase-advances diurnal rhythms of feeding and metabolism into the light cycle and abolishes the normal increase in dark-cycle locomotor activity characteristic of nocturnal rodents. Finally, DMHLepR-silenced mice fail to entrain to a restrictive change in food availability. Together, these findings identify DMHLepR neurons as critical determinants of the daily time of feeding and associated metabolic rhythms.

Metabolic cycles are a fundamental element of cellular and organismal function. Among the most critical in higher organisms is the Cori Cycle, the systemic cycling between lactate and glucose. Here, skeletal muscle-specific Mitochondrial Pyruvate Carrier (MPC) deletion in mice diverted pyruvate into circulating lactate. This switch disinhibited muscle fatty acid oxidation and drove Cori Cycling that contributed to increased energy expenditure. Loss of muscle MPC activity led to strikingly decreased adiposity with complete muscle mass and strength retention. Notably, despite decreasing muscle glucose oxidation, muscle MPC disruption increased muscle glucose uptake and whole-body insulin sensitivity. Furthermore, chronic and acute muscle MPC deletion accelerated fat mass loss on a normal diet after high fat diet-induced obesity. Our results illuminate the role of the skeletal muscle MPC as a whole-body carbon flux control point. They highlight the potential utility of modulating muscle pyruvate utilization to ameliorate obesity and type 2 diabetes.

C-type inactivation in potassium channels helps fine-tune long-term channel activity through conformational changes at the selectivity filter. Here, through the use of cross-linked constitutively open constructs, we determined the structures of KcsA's mutants that stabilize the selectivity filter in its conductive (E71A, at 2.25 Å) and deep C-type inactivated (Y82A at 2.4 Å) conformations. These structural snapshots represent KcsA's transient open-conductive (O/O) and the stable open deep C-type inactivated states (O/I), respectively. The present structures provide an unprecedented view of the selectivity filter backbone in its collapsed deep C-type inactivated conformation, highlighting the close interactions with structural waters and the local allosteric interactions that couple activation and inactivation gating. Together with the structures associated with the closed-inactivated state (C/I) and in the well-known closed conductive state (C/O), this work recapitulates, at atomic resolution, the key conformational changes of a potassium channel pore domain as it progresses along its gating cycle.

Several mutations identified in phospholamban (PLN) have been linked to familial dilated cardiomyopathy (DCM) and heart failure, yet the underlying molecular mechanism remains controversial. PLN interacts with sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and regulates calcium uptake, which is modulated by the protein kinase A (PKA)-dependent phosphorylation of PLN during the fight-or-flight response. Here, we present the crystal structures of the catalytic domain of mouse PKA in complex with wild-type and DCM-mutant PLNs. Our structures, combined with the results from other biophysical and biochemical assays, reveal a common disease mechanism: the mutations in PLN reduce its phosphorylation level by changing its conformation and weakening its interactions with PKA. In addition, we demonstrate that another more ubiquitous SERCA-regulatory peptide, called another-regulin (ALN), shares a similar mechanism mediated by PKA in regulating SERCA activity.

Cell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell: the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that aPBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level, we then present evidence that PBP1b, the major aPBP, contributes to cell-wall integrity by repairing cell wall defects.

Mutations in the human kinase PINK1 (hPINK1) are associated with autosomal recessive early-onset Parkinson's disease (PD). hPINK1 activates Parkin E3 ligase activity, involving phosphorylation of ubiquitin and the Parkin ubiquitin-like (Ubl) domain via as yet poorly understood mechanisms. hPINK1 is unusual amongst kinases due to the presence of three loop insertions of unknown function. We report the structure of Tribolium castaneum PINK1 (TcPINK1), revealing several unique extensions to the canonical protein kinase fold. The third insertion, together with autophosphorylation at residue Ser205, contributes to formation of a bowl-shaped binding site for ubiquitin. We also define a novel structural element within the second insertion that is held together by a distal loop that is critical for TcPINK1 activity. The structure of TcPINK1 explains how PD-linked mutations that lie within the kinase domain result in hPINK1 loss-of-function and provides a platform for the exploration of small molecule modulators of hPINK1.

Plasmodium falciparum, the causative agent of malaria, remains a global health threat as parasites continue to develop resistance to antimalarial drugs used throughout the world. Accordingly, drugs with novel modes of action are desperately required to combat malaria. P. falciparum parasites infect human red blood cells where they digest the host's main protein constituent, hemoglobin. Leucine aminopeptidase PfA-M17 is one of several aminopeptidases that have been implicated in the last step of this digestive pathway. Here, we use both reverse genetics and a compound specifically designed to inhibit the activity of PfA-M17 to show that PfA-M17 is essential for P. falciparum survival as it provides parasites with free amino acids for growth, many of which are highly likely to originate from hemoglobin. We further show that loss of PfA-M17 results in parasites exhibiting multiple digestive vacuoles at the trophozoite stage. In contrast to other hemoglobin-degrading proteases that have overlapping redundant functions, we validate PfA-M17 as a potential novel drug target.

In all domains of life, the catalysed degradation of RNA facilitates rapid adaptation to changing environmental conditions, while destruction of foreign RNA is an important mechanism to prevent host infection. We have identified a virus-encoded protein termed gp37/Dip, which directly binds and inhibits the RNA degradation machinery of its bacterial host. Encoded by giant phage фKZ, this protein associates with two RNA binding sites of the RNase E component of the Pseudomonas aeruginosa RNA degradosome, occluding them from substrates and resulting in effective inhibition of RNA degradation and processing. The 2.2 Å crystal structure reveals that this novel homo-dimeric protein has no identifiable structural homologues. Our biochemical data indicate that acidic patches on the convex outer surface bind RNase E. Through the activity of Dip, фKZ has evolved a unique mechanism to down regulate a key metabolic process of its host to allow accumulation of viral RNA in infected cells.

In meiosis, DNA double-strand break (DSB) formation by Spo11 initiates recombination and enables chromosome segregation. Numerous factors are required for Spo11 activity, and couple the DSB machinery to the development of a meiosis-specific 'axis-tethered loop' chromosome organisation. Through in vitro reconstitution and budding yeast genetics, we here provide architectural insight into the DSB machinery by focussing on a foundational DSB factor, Mer2. We characterise the interaction of Mer2 with the histone reader Spp1, and show that Mer2 directly associates with nucleosomes, likely highlighting a contribution of Mer2 to tethering DSB factors to chromatin. We reveal the biochemical basis of Mer2 association with Hop1, a HORMA domain-containing chromosomal axis factor. Finally, we identify a conserved region within Mer2 crucial for DSB activity, and show that this region of Mer2 interacts with the DSB factor Mre11. In combination with previous work, we establish Mer2 as a keystone of the DSB machinery by bridging key protein complexes involved in the initiation of meiotic recombination.

Epigenetic regulation plays extensive roles in diseases and development. Disruption of epigenetic regulation not only increases the risk of cancer, but can also cause various developmental defects. However, the question of how epigenetic changes lead to tissue-specific responses during neural crest fate determination and differentiation remains understudied. Using palatogenesis as a model, we reveal the functional significance of Kdm6b, an H3K27me3 demethylase, in regulating mouse embryonic development. Our study shows that Kdm6b plays an essential role in cranial neural crest development, and loss of Kdm6b disturbs P53 pathway-mediated activity, leading to complete cleft palate along with cell proliferation and differentiation defects in mice. Furthermore, activity of H3K27me3 on the promoter of Trp53 is antagonistically controlled by Kdm6b, and Ezh2 in cranial neural crest cells. More importantly, without Kdm6b, the transcription factor TFDP1, which normally binds to the promoter of Trp53, cannot activate Trp53 expression in palatal mesenchymal cells. Furthermore, the function of Kdm6b in activating Trp53 in these cells cannot be compensated for by the closely related histone demethylase Kdm6a. Collectively, our results highlight the important role of the epigenetic regulator KDM6B and how it specifically interacts with TFDP1 to achieve its functional specificity in regulating Trp53 expression, and further provide mechanistic insights into the epigenetic regulatory network during organogenesis.

Bone morphogenic protein (BMP) signalling contributes towards maintenance of pluripotency and favours mesodermal over neural fates upon differentiation, but the mechanisms by which BMP controls differentiation are not well understood. We report that BMP regulates differentiation by blocking downregulation of Cdh1, an event that accompanies the earliest stages of neural and mesodermal differentiation. We find that loss of Cdh1 is a limiting requirement for differentiation of pluripotent cells, and that experimental suppression of Cdh1 activity rescues the BMP-imposed block to differentiation. We further show that BMP acts prior to and independently of Cdh1 to prime pluripotent cells for mesoderm differentiation, thus helping to reinforce the block to neural differentiation. We conclude that differentiation depends not only on exposure to appropriate extrinsic cues but also on morphogenetic events that control receptivity to those differentiation cues, and we explain how a key pluripotency signal, BMP, feeds into this control mechanism. DOI: http://dx.doi.org/10.7554/eLife.01197.001.

ZMYM2 is a zinc finger transcriptional regulator that plays a key role in promoting and maintaining cell identity. It has been implicated in several diseases such as congenital anomalies of the kidney where its activity is diminished and cancer where it participates in oncogenic fusion protein events. ZMYM2 is thought to function through promoting transcriptional repression and here we provide more evidence to support this designation. Here we studied ZMYM2 function in human cells and demonstrate that ZMYM2 is part of distinct chromatin-bound complexes including the established LSD1-CoREST-HDAC1 corepressor complex. We also identify new functional and physical interactions with ADNP and TRIM28/KAP1. The ZMYM2-TRIM28 complex forms in a SUMO-dependent manner and is associated with repressive chromatin. ZMYM2 and TRIM28 show strong functional similarity and co-regulate a large number of genes. However, there are no strong links between ZMYM2-TRIM28 binding events and nearby individual gene regulation. Instead, ZMYM2-TRIM28 appears to regulate genes in a more regionally defined manner within TADs where it can directly regulate co-associated retrotransposon expression. We find that different types of ZMYM2 binding complex associate with and regulate distinct subclasses of retrotransposons, with ZMYM2-ADNP complexes at SINEs and ZMYM2-TRIM28 complexes at LTR elements. We propose a model whereby ZMYM2 acts directly through retrotransposon regulation, which may then potentially affect the local chromatin environment and associated coding gene expression.

Understanding how enhancers drive cell-type specificity and efficiently identifying them is essential for the development of innovative therapeutic strategies. In melanoma, the melanocytic (MEL) and the mesenchymal-like (MES) states present themselves with different responses to therapy, making the identification of specific enhancers highly relevant. Using massively parallel reporter assays (MPRAs) in a panel of patient-derived melanoma lines (MM lines), we set to identify and decipher melanoma enhancers by first focusing on regions with state-specific H3K27 acetylation close to differentially expressed genes. An in-depth evaluation of those regions was then pursued by investigating the activity of overlapping ATAC-seq peaks along with a full tiling of the acetylated regions with 190 bp sequences. Activity was observed in more than 60% of the selected regions, and we were able to precisely locate the active enhancers within ATAC-seq peaks. Comparison of sequence content with activity, using the deep learning model DeepMEL2, revealed that AP-1 alone is responsible for the MES enhancer activity. In contrast, SOX10 and MITF both influence MEL enhancer function with SOX10 being required to achieve high levels of activity. Overall, our MPRAs shed light on the relationship between long and short sequences in terms of their sequence content, enhancer activity, and specificity across melanoma cell states.

Mechanical stimulation, such as physical exercise, is essential for bone formation and health. Here, we demonstrate the critical role of osteocytic Cx43 hemichannels in anabolic function of bone in response to mechanical loading. Two transgenic mouse models, R76W and Δ130-136, expressing dominant-negative Cx43 mutants in osteocytes were adopted. Mechanical loading of tibial bone increased cortical bone mass and mechanical properties in wild-type and gap junction-impaired R76W mice through increased PGE2, endosteal osteoblast activity, and decreased sclerostin. These anabolic responses were impeded in gap junction/hemichannel-impaired Δ130-136 mice and accompanied by increased endosteal osteoclast activity. Specific inhibition of Cx43 hemichannels by Cx43(M1) antibody suppressed PGE2 secretion and impeded loading-induced endosteal osteoblast activity, bone formation and anabolic gene expression. PGE2 administration rescued the osteogenic response to mechanical loading impeded by impaired hemichannels. Together, osteocytic Cx43 hemichannels could be a potential new therapeutic target for treating bone loss and osteoporosis.

Sex differences in plasma growth hormone (GH) profiles, pulsatile in males and persistent in females, regulate sex differences in hepatic STAT5 activation linked to sex differences in gene expression and liver disease susceptibility, but little is understood about the fundamental underlying, GH pattern-dependent regulatory mechanisms. Here, DNase-I hypersensitivity site (DHS) analysis of liver chromatin accessibility in a cohort of 18 individual male mice established that the endogenous male rhythm of plasma GH pulse-stimulated liver STAT5 activation induces dynamic, repeated cycles of chromatin opening and closing at several thousand liver DHS and comprises a novel mechanism conferring male bias to liver chromatin accessibility. Strikingly, a single physiological replacement dose of GH given to hypophysectomized male mice restored, within 30 min, liver STAT5 activity and chromatin accessibility at 83% of the dynamic, pituitary hormone-dependent male-biased DHS. Sex-dependent transcription factor binding patterns and chromatin state analysis identified key genomic and epigenetic features distinguishing this dynamic, STAT5-driven mechanism of male-biased chromatin opening from a second GH-dependent mechanism operative at static male-biased DHS, which are constitutively open in male liver. Dynamic but not static male-biased DHS adopt a bivalent-like epigenetic state in female liver, as do static female-biased DHS in male liver, albeit using distinct repressive histone marks in each sex, namely, H3K9me3 at male-biased DHS in female liver and H3K27me3 at female-biased DHS in male liver. Moreover, sex-biased H3K36me3 marks are uniquely enriched at static sex-biased DHS, which may serve to keep these sex-dependent hepatocyte enhancers free of H3K27me3 repressive marks and thus constitutively open. Pulsatile chromatin opening stimulated by endogenous, physiological hormone pulses is thus one of two distinct GH-determined mechanisms for establishing widespread sex differences in hepatic chromatin accessibility and epigenetic regulation, both closely linked to sex-biased gene transcription and the sexual dimorphism of liver function.

The ultimate formation of a four-chambered heart allowing the separation of the pulmonary and systemic circuits was key for the evolutionary success of tetrapods. Complex processes of cell diversification and tissue morphogenesis allow the left and right cardiac compartments to become distinct but remain poorly understood. Here, we describe an unexpected laterality in the single zebrafish atrium analogous to that of the two atria in amniotes, including mammals. This laterality appears to derive from an embryonic antero-posterior asymmetry revealed by the expression of the transcription factor gene meis2b. In adult zebrafish hearts, meis2b expression is restricted to the left side of the atrium where it controls the expression of pitx2c, a regulator of left atrial identity in mammals. Altogether, our studies suggest that the multi-chambered atrium in amniotes arose from a molecular blueprint present before the evolutionary emergence of cardiac septation and provide insights into the establishment of atrial asymmetry.

Brain size and cortical folding have increased and decreased recurrently during mammalian evolution. Identifying genetic elements whose sequence or functional properties co-evolve with these traits can provide unique information on evolutionary and developmental mechanisms. A good candidate for such a comparative approach is TRNP1, as it controls proliferation of neural progenitors in mice and ferrets. Here, we investigate the contribution of both regulatory and coding sequences of TRNP1 to brain size and cortical folding in over 30 mammals. We find that the rate of TRNP1 protein evolution (ω) significantly correlates with brain size, slightly less with cortical folding and much less with body size. This brain correlation is stronger than for >95% of random control proteins. This co-evolution is likely affecting TRNP1 activity, as we find that TRNP1 from species with larger brains and more cortical folding induce higher proliferation rates in neural stem cells. Furthermore, we compare the activity of putative cis-regulatory elements (CREs) of TRNP1 in a massively parallel reporter assay and identify one CRE that likely co-evolves with cortical folding in Old World monkeys and apes. Our analyses indicate that coding and regulatory changes that increased TRNP1 activity were positively selected either as a cause or a consequence of increases in brain size and cortical folding. They also provide an example how phylogenetic approaches can inform biological mechanisms, especially when combined with molecular phenotypes across several species.