The recently discovered oestrogen receptor GPR30 shares structural features with the βARs, and this forms the basis for the interactions and functional overlap. GPR30 possesses protein kinase A (PKA) phosphorylation sites and PDZ binding motifs and interacts with A‐kinase anchoring protein 5 (AKAP5), all of which enable its interaction with the βAR pathways. The interactions between ERs and βARs occur downstream of the G‐protein‐coupled receptor, through the Gαs and Gαi proteins. This review presents an up‐to‐date description of ERs and βARs and demonstrates functional synergism and interactions among these receptors in cardiac cells. We explore their signalling cascades and the mechanisms that orchestrate their interactions and propose new perspectives on the signalling patterns for the GPR30 based on its structural resemblance to the βARs. In addition, we explore the relevance of these interactions to cell physiology, drugs  and cardioprotection. Furthermore, a receptor‐independent mechanism for oestrogen and its influence on the expression of βARs and calcium‐handling proteins are discussed. Finally, we highlight promising therapeutic avenues that can be derived from the shared pathways, especially the phosphatidylinositol‐3‐OH kinase (PI3K/Akt) pathway.Oestrogen receptors (ERs) and β‐adrenergic receptors (βARs) play important roles in the cardiovascular system. Moreover, these receptors are expressed in cardiac myocytes and vascular tissues. Numerous experimental observations support the hypothesis that similarities and interactions exist between the signalling pathways of ERs  and βARs (β We also discuss the ERs and βARs as coregulators of cardiac Ca2+‐handling proteins.In recent decades, the need to understand the cardiovascular functions of oestrogen and βARs has received much interest from researchers. The discovery of GPR30,22.11AR, β2AR and β3AR, are encoded by different genes.1AR and β3AR.1ARs is higher than that of β2ARs, while β3ARs show the least abundance.βARs are members of the G‐protein‐coupled receptors (GPCRs) that classically form 7 transmembrane loops, with extracellular and intracellular terminals. Three βAR subdivisions, β2.21ARs couple to the stimulatory unit of the G protein (Gαs) leading to the synthesis of cyclic adenosine monophosphate (cAMP) by adenylyl cyclase (AC) enzyme. On the other hand, β2ARs are pleiotropic receptors that couple to the Gαs, the inhibitory G protein (Gαi) and the Gβγ.2ARs couple to the Gαs, whereas at high adrenaline concentration, they switch to Gαi, a phenomenon referred to as stimulus‐mediated trafficking.2AR/Gαi pathway inhibits cAMP production, an opposing effect to β2AR/Gαs and β1AR/Gαs activation  and cardiac G‐protein‐coupled receptor kinase 2 (GRK2) phosphorylation.3ARs are less susceptible to PKA/GRK2‐mediated receptor recycling and desensitization in response to hyperstimulation.3aAR and β3bAR, were reported in Chinese hamster ovary (CHO) cells and 3T3‐L1 adipocytes.3aAR isoform coupled to the Gαi, whereas β3bAR coupled to both Gαs and Gαi. At present, there are no reports regarding the existence of the 2 isoforms in human cardiac cells. β3AR has largely been associated with metabolic functions. Nevertheless, β3AR stimulation induced positive inotropy in human atrial cells,3AR may influence chronotropic functions through the nitric oxide (NO)/guanosine 3′,5′‐monophosphate (cGMP) pathway.3AR‐NO synthase/guanylyl cyclase pathway was shown to influence the nuclear β3AR‐mediated gene transcription, suggesting a crosstalk between the surface and nuclear β3ARs.With regard to structure and function, the β2.31ARs, which constitute up to 80% of the entire cardiac βAR density of healthy human, and to a lesser extent by the β2ARs.2ARs have a higher affinity for adrenaline, while β1ARs have almost equal affinities for both noradrenaline and adrenaline.3ARs is largely associated with negative inotropy during catecholaminergic stress.βARs mediate the SNS regulation of the cardiac functions.αs, a GTP‐binding protein. This cAMP, in turn, activates PKA. What follows is the induction of intracellular rise in Ca2+ transients via the tightly regulated network of ion channels. PKA‐mediated inotropic effects are orchestrated through the phosphorylation of 2 main channels: the LTCC located in the T‐tubular network formed by sarcolemmal membrane invaginations and the RyR2 receptors on the SR membrane. Phosphorylation of LTCC allows Ca2+ entry as inward current.2+ currents further stimulate Ca2+ release from SR, the intracellular stores, by the opening of RyR2 receptors which are also phosphorylated by PKA. This phenomenon is referred to as calcium‐induced calcium release. The resultant Ca2+ transient activates the myofilament protein troponin C turning on cardiomyocyte contraction. The size of Ca2+ transients is a key determinant of the strength of the contraction.2+ uptake back to the SR by SERCA. In addition, sarcolemmal Na+/Ca2+ exchanger pump (NCX) accelerates Ca2+ extrusion, which together with SR Ca2+ uptake diminishes the Ca2+ transient resulting in relaxation.Once activated, βARs initiate cAMP synthesis by coupling to the G1AR/cAMP and provides alternative modulation of βAR activation. Indeed, both EPAC1 and EPAC2 are present in cardiomyocytes.2+ through RyR and PLB phosphorylation,Besides the classical cAMP/PKA pathway, cAMP also acts through the recently described intracellular protein named exchange protein directly activated by cAMP (EPAC).1AR activation is the multimeric protein Ca2+/calmodulin kinase II (CaMKII).2+ and intracellular levels of calmodulin.Another downstream target of β3The cardiovascular functions of oestrogen are mediated by ERs. These cellular receptors are categorized as nuclear receptors (ERα and ERβ), which modulate transcription of specific gene sets, and membrane‐bound receptor , which mediates rapid, non‐genomic actions of oestrogen. ERs are expressed in cardiomyocytes,With regard to gender, a study carried out on VSMCs of rats showed that GPR30 expression was similar in both males and females.In addition, Ma et al.44.1Similar to other steroids hormones, oestrogen signalling is initiated by the binding of 17β‐oestradiol or xenoestrogens4.24.2.11b‐adrenergic receptors.Binding of oestrogen to its receptors (membrane or nuclear) triggers 2 types of cellular effects defined by the timing of onset. (i) Part of the effects occurs through the well‐established pathway of ER‐mediated transcription of certain genes. Conventionally, this pathway is known as a genomic pathway and occurs within hours to days.4.2.2Besides the conventional receptor‐mediated mechanisms of oestrogen, experimental observations have hinted at the possibility of an alternative mechanism that does not involve membranous or nuclear ERs.4.3in vivo and ev vivo settings.2+ overload.1ARs and induced negative inotropy.Oestrogen plays several functions in the CVS. Here, we highlight some of the cell‐specific roles of oestrogen without much detail because of the limitation defined by the purpose of this review. Activation of GPR30 inhibited proliferation of rat cardiac fibroblasts and collagen synthesis in both 51ARs, β2ARs and β3ARs vs. ERα, ERβ and GPR30. We discuss various points of integration between their signalling pathways. We note 3 main pathways along which these classes of receptors interact.The crosstalk between ERs and βARs is a concept that was revealed from earlier studies.5.1αsαi/o,1AR, β2AR and GPR30 leads to coupling to Gαs protein.αs triggers the production of cAMP by AC enzyme. Subsequently, PKA and EPAC amplify the cAMP signal. In coronary arteries, GPR30 was shown to activate this pathway including the production of PKA and EPAC proteins.αs/AC and PDEs.αs/AC/cAMP pathway, it would be interesting to define whether the GPR30/Gαs/AC complex also participates in compartmentalization of cAMP signals in cardiac cells. The PKA generated downstream of GPR30 might interact with PDEs, under the direction of AKAP5, to regulate cAMP degradation as for β1ARs and β2ARs. Moreover, we speculate that GPR30's ability to activate EPAC might also influence β1AR/cAMP/EPAC‐mediated functions. Besides GPR30, activation of the ERα elevated PKA in VSMCs of aortic tissue further illustrating oestrogen involvement in the GPCR/Gαs/cAMP signalling pathway.Similar to βARs, GPR30 possesses PKA phosphorylation sites and PDZ binding motifs and associates with A‐kinase anchoring proteins (AKAPs).1AR, β2AR and GPR30 possess type I PDZ binding motifs: ‐ESKV, ‐DSLL and ‐SSAV respectively.1AR (‐ESKV) motif was shown to be a determinant factor for its coupling to Gαs and not Gαi. Induced disruption of this motif permitted β1AR/Gαi coupling.2AR (‐DSLL) motif plays a role in its coupling to Gαi.αs or Gαi. In addition to the PDZ motifs, β2AR phosphorylation by PKA influences its ability to bind Gαs or Gαi.αs or Gαi may be dependent on cell/tissue type, collectively, these observations raise the possibility that PKA phosphorylation or disruption of its PDZ motif would have implications on its coupling to Gαs and Gαi as is the case for β1ARs and β2ARs. This possibility calls for further inquiry to the conditions under which GPR30 couples to Gαi and Gαs, and the proteins that interact with its PDZ motif.Additional interactions are possible due to the structural resemblance between GPR30 and βARs. βARs interact with cellular proteins through PDZ motifs located at their C‐terminus regions. The PDZ motifs give a bearing on the localization and signalling of βARs. Of note, β1ARs facilitated its recycling by enhancing PKA phosphorylation of the receptor.1ARs. Therefore, an association of GPR30 with AKAP5 and possibly other unidentified proteins could be a mechanism through which oestrogen participates in the regulation of β1AR density. Further research should be carried out to characterize the interactions of GPR30 with AKAPs and their implications on cellular functions.One functional implication of this motif draws from the recent observation that GPR30 inhibited βAR‐mediated production of cAMP in response to isoproterenol stimulation in HEK293 cells.1AR, β2AR and GPR30 are capable of coupling to Gαs, their cellular effects are not similar. For instance, activation of β1ARs,2ARs and ERβ do not. Importantly, the observation that GPR30 activates cAMP through Gαs pathway challenges previous findings that oestrogen induced negative inotropy at both cardiomyocyte and organ levels. Therefore, the effects of GPR30/Gαs/cAMP pathway may not be identical to the classical βAR/Gαs/cAMP pathway, especially in cardiomyocytes. However, as we reported, oestrogen may tilt the activation of β2AR/Gαs or β2AR/Gαi pathways in certain disease conditions, as in stress‐induced cardiomyopathy.Although β5.22ARs and β3ARs, couple to the Gαi subunit.2ARs exposes cardiomyocytes to cell death in ischaemic conditions.2AR/Gαi pathway triggered anti‐apoptotic signals through the PI3K/Akt pathway.2ARs. In addition to the GPR30, oestrogen activates the ERα, which directly binds the p85 alpha regulatory component of the PI3K.As mentioned earlier, GPR30, like β2ARs enables switching of its coupling from Gαs to Gαi under extreme catecholamine stimulation,2ARs act as a switch that coordinates synthesis of cAMP and indicates cross‐communication between β1AR and β2AR signalling. Moreover, AKAP5 tethering of PKA allows it to phosphorylate β2ARs.αs or Gαi. Although we appreciate that such signal trafficking is intricately complicated and may involve different mechanisms, further research is necessary to determine whether this phenomenon can be replicated in adult cardiomyocytes. Unlike β2ARs, the conditions under which GPR30 activates Gαs or Gαi are not clearly understood. Perhaps a possible hint as to when GPR30 activates Gαi comes from the observation that under stress conditions, both β2ARs and GPR30 activate Gαi/PI3K/Akt pathway to confer cardioprotection.αs or Gαi in cardiomyocytes need further investigation.In addition, PKA phosphorylation of the β1ARs, β2ARs and GPR30, β3ARs lack PKA phosphorylation sites.3ARs, there is a great deal of variability with respect to their ability to activate both Gαs and Gαi. Some β3AR splice variants were shown to display dual coupling to Gαs and Gαi in other cell types, although not in cardiac cells.3ARs act through Gαi/o to suppress contractility via induction of NO‐cGMP pathway under chronic catecholaminergic stimulation.3AR/NO/cGMP pathway was enhanced in the presence of β1AR blocker, which was interpreted to be beneficial in chronic volume‐overloaded heart.αs and Gαi act as pivots around which both ER and βAR signalling pathways converge. β2AR and GPR30 signal through the Gαs/AC/cAMP and Gαi/PI3K/Akt pathways in the cardiovascular system. Similarly, both GPR30 and ERα signalling cascades interact through the PI3K/Akt pathway  and, in addition, that this role of oestrogen was mediated by the ERα.1AR expression.1AR levels are affected by both ERα and GPR30. Furthermore, it was shown that oestrogen not only suppressed the expression of β1ARs but also increased sensitivity to catecholamines.1ARs by oestrogen may be a cardioprotective strategy against the adverse effects associated with hyperstimulation of β1ARs.Our studies6.22ARs. We further showed that oestrogen in combination with testosterone enhanced the cardiac expression of β2ARs in OVX rats.2AR mRNA and protein were upregulated in female hearts but not male hearts in response to the arteriovenous fistula procedure.1AR expression and upregulates β2AR expression in cardiac cells.In female rat models of I/R and heart failure, oestrogen, acting through the GPR306.33AR expression in cardiac tissues is lacking. However, variations in expression of β3ARs in adipose tissues have been linked to oestrogen levels. One group reported that oestrogen elevated the expression of β3ARs in murine adipocytes in culture,3ARs in brown adipose tissue of female rats in vivo.Currently, information on direct effects of oestrogen on β72+ levels in cardiac cells are coregulated by both ERs and βARs. Numerous studies provide compelling evidence that oestrogen influences the expression levels of Ca2+‐handling proteins, whose functions are primarily under the regulation of βARs (Figure 2+ handling.2+‐handling proteins channels are summarized in Table Intracellular Cas Figure .144 In a2+‐handling proteins. ERα, ERβ and GPR30 mediate these roles of oestrogen.2+ regulation and contractility. Furthermore, through ERα,2+ sensitivity.2+ sensitivity, indicating that oestrogen deficiency may play a role in cardiac disorders by lowering the myofilament sensitivity to Ca2+.These studies were carried out in different animal species, disease models, tissue/cell types, age groups, in vivo and ex vivo and using different oestrogen types. Moreover, the observed changes in protein expression due to OVX were reversed by oestrogen replacement.88.12+ channel blockers are 2 mainstays for the treatment of cardiovascular disease.1ARs/β2ARs have been reported, which may be attributed to oestrogen.2+ handling,2+ blockers may vary with gender or age groups based on the interplay between ERs and βARs. As demonstrated, carvedilol, a non‐selective β‐blocker, protected against myocardial contractile dysfunction caused by oestrogen deficiency.2AR‐Gαi/β‐arrestin pathways.2+ blockers and β‐blockers respectively. Consequently, men and women may respond differently to these classes of drugs. The crosstalk may inform the decisions regarding the choice of antihypertensive drugs to patients with consideration to age and gender.Interactions between ER and βAR pathways could have broad implications in the clinical context. β‐Blockers and Ca8.21AR activation promotes CaMKII‐induced apoptosis,2AR, ERα and GPR30 activation seems to act in a manner that promotes anti‐apoptosis through the Gi/PI3K/Akt pathway. Considerations for strategies targeting the PI3K/Akt pathway will provide a feasible avenue for cardioprotection. For instance, the ability of ERα binding to the alpha subunit of PI3K seems attractive, as it is more specific and avoids various points of integration between ER and βAR signalling cascades discussed above. Activation of Akt pathway will protect against mitochondria‐associated apoptosis induction. Moreover, Akt was shown to act as a surrogate molecular ligand for ERα that induced expression of oestrogen‐regulated cardioprotective genes in breast cancer cells.2+ channel blocker if appropriate technologies are applied to enhance its specificity. We anticipate that the therapeutic value of this manoeuvre could be a potential target for Ca2+‐related pathologies such as arrhythmia treatments.The functional synergism between ERs and βARs provides therapeutic avenues for cardioprotection. For example, while β92ARs to the Gαs and Gαi proteins. On this basis, oestrogen pathways play into the network of the βAR signalling cascades. Furthermore, GPR30 and βARs show similarities with regard to their ability to associate with AKAPs, PDZ motif‐binding proteins and possession of PKA and CaMKII binding sites. Despite the signalling pathways discussed in this review, the functions of the GPR30 and other ERs remain incompletely understood. Further research is required to uncover the identities of signalling molecules that orchestrate their functions.This review demonstrates the expression patterns and functions of ERs and βARs in cardiovascular tissues. The data sets reviewed above show some inconsistencies with regard to tissue‐specific expression of the ERs. For instance, it is not clear whether ERβ is expressed in adult cardiomyocytes, and the cellular localization of GPR30 is not clear. Therefore, further studies are warranted to resolve these observations. In addition, the reviewed data sets strongly support the hypothesis that ERs and βARs function as collaborative partners in modulating the physiology of the cardiovascular system. The recently described oestrogen receptor GPR30 mimics the dual coupling of the β2+ channel blockers. Oestrogen influences the expression of βARs and Ca2+‐handling proteins, which could compromise the efficacy of the drugs in a gender‐dependent manner. This perspective requires further evaluation at the clinical level. In addition, the concept of direct oestrogen binding to the LTCC might be of great clinical relevance as oestrogen might be used to design Ca2+ channel blockers. This opens a window of research into the receptor‐independent pathways for oestrogen. Furthermore, future research should exploit the therapeutic potential of the cardioprotective PI3K/Akt pathway that is activated downstream of both ERs and βARs.The crosstalk between the ERs and βARs could have implications on drugs that target these receptors, especially β‐blockers and CaThe authors report no conflict of interests.


In Escherichia coli, two tyrosine-family recombinases, XerC and XerD, bind to dif and carry out two pairs of sequential strand exchange reactions. However, what makes the reaction unique among site-specific recombination reactions is that the first step, XerD-mediated strand exchange, relies on interaction with the very C-terminus of the FtsK DNA translocase. FtsK is a powerful molecular motor that functions in cell division, co-ordinating division with clearing chromosomal DNA from the site of septation and also acts to position the dif sites for recombination. This is a model system for unlinking, separating and segregating large DNA molecules. Here we describe the molecular detail of the interaction between XerD and FtsK that leads to activation of recombination as deduced from a co-crystal structure, biochemical and in vivo experiments. FtsKγ interacts with the C-terminal domain of XerD, above a cleft where XerC is thought to bind. We present a model for activation of recombination based on structural data.Bacterial chromosomes are most often circular DNA molecules. This can produce a topological problem; a genetic crossover from homologous recombination results in dimerization of the chromosome. A chromosome dimer is lethal unless resolved. A site-specific recombination system catalyses this dimer-resolution reaction at the chromosomal site  The majority of bacterial species have circular DNA genomes. Prior to cell division each circular chromosome must be entirely replicated, unlinked and segregated to ensure that each daughter cell inherits a full genome complement. During or following replication DNA repair processes involving homologous recombination can produce a chromosomal crossover, and any odd number of these events between the circular DNA molecules results in a chromosome dimer2dif located in the replication terminus region578Escherichia coli dif site consists of two 11 bp sites that are an imperfect inverted repeat, separated by a 6 bp spacer, or central regiondif sites bound by XerCD can then be synapsed by protein-protein interactions between the two dif-XerCD complexes forming a pseudo-tetrameric structure11131415dif)2 is not by itself catalytically competent; it is not until FtsK is present that cleavage by XerD occurs12Most bacteria encode two tyrosine family recombinases XerC and XerD which bind to the specific chromosomal site dif sitesThe FtsK protein is a powerful DNA translocase that is involved in co-ordinating cell division with DNA unlinking and segregation to ensure that the closing septum does not trap or guillotine chromosomal DNA182122232526dif sites; FtsKγ binds specifically to 8 bp sequences termed KOPS that are highly skewed on each chromosome arm to point towards the dif site2829dif siteThe FtsKγ domain also plays another role in orienting the FtsK motor to translocate toward dif synapse to promote recombination.Here we present the structure of the FtsKγ domain interacting with the XerD recombinase and provide biochemical evidence to support the involvement of amino acids at the interaction surfaces in the activation of recombination. A mutant in XerD that disrupts the interaction with FtsKγ is severely impaired for recombination activity on plasmid substrates and is also incapable of supporting chromosome dimer resolution in cells. Further, we demonstrate that a 9 amino acid stretch from FtsKγ that encompasses the interaction surface is sufficient for activation of recombination. It is of note that the same region of FtsKγ that binds KOPS-DNA (between helices 2 and 3) is also responsible for the protein-protein interaction with XerD to activate recombination. Finally, we also provide structure-based models to explain how the FtsKγ-XerD interaction can influence the XerCD-dif requires the interaction of XerD with the γ domain of FtsK. To understand how this interaction can lead to activation of recombination, a fusion protein between the C-terminal domain of XerD, which contains the catalytic residues required for DNA cleavage and strand exchange, and FtsKγ was produced (XerDC–γ) and crystallized. The full length XerD-FtsKγ fusion protein has previously been shown to be a functional, self-activating, recombinase protein at dif sitesC-FtsKγ fusion protein was solved by molecular replacement and refined to a resolution of 2.3 Å (E. coli FtsKγ domain (2VE8) as search models30C–γ complex, but the flexible linker joining the two protein domains was not visible in the density. Details of the linker sequence and all amino acids for which there is insufficient electron density are shown in The activation of recombination at of 2.3 Å , using tC domain and the FtsKγ domain are structurally very similar to the previously determined individual proteins, with no major conformational changes revealed in either protein domain; overall the XerDC domain only varied by an average RMSD of 0.93 Å on matched Cα atoms, while the RMSD between matched Cα atoms for the FtsKγ domain was 0.77 Å. The density observed for FtsKγ was weaker than that for XerDC and increasingly diminished for atoms that were distal from its interaction with XerD, resulting in the final refined model having chain breaks in this region. However, the density for the interface between the two binding partners was sufficient to facilitate an in depth examination of the key molecular contacts .Both the XerDC is a 563 Å2 interface that involves two helices on the surface of XerDC .in vitro recombination reactions in the presence of XerC and a model plasmid containing two dif sites. The relative levels of recombination were again compared to wild-type proteins  shows that these are interactions which contribute greatly to the required association of these two proteins.Since the mutants in XerD for both in vitro and in vivo recombination data supports the interaction surface seen in the crystal structure being vital for the activation of recombination.Taken together the dif as well as, or even more efficiently than, a XerD-FtsKγ fusion : of the 5 amino acids found to be important for activation of XerD there are 3 identical and 1 similar in the H. influenzae protein whereas the L. lactis XerS (there is only a single Xer recombinase in this species) has only 1of the 5 amino acids identical to E. coli . Again, this is mirrored in the cross-species activities seen; E. coli FtsK can activate H. influenzae XerDE. coli FtsKγ alone cannot activate XerS from L. lactisA number of studies have used inter-species recombination assays using Xer recombinases and FtsKs from different organisms both for dif complexes has been observed in the absence of the FtsKγ domain1112dif synapse to the D* state must require interaction with DNA or with XerC, or likely both, in addition to the FtsKγ interaction. Indeed, this makes sense mechanistically; catalysis is only activated within the properly assembled XerCD-dif synapse reducing the occurrence of inappropriate XerD-mediated cleavage of the DNA.Synapsis between two XerCD-C–γ active-site structure to that of activated Cre or λ Int, it is clear that two key amino-acids of the XerD active-site are inappropriately positioned for catalysis13in vitro studiesBy comparing the XerDdif synapses along with data that shows that the very C-termini of both XerC and XerD are important for this reciprocal control of partner activation3611Another important feature of the Cre and λ Int synapses is the cyclic interaction between monomers obtained by donation of their C-termini to the adjacent monomer13loxP synaptic structure . We can, therefore, be confident that this is an important site of interaction between XerC and XerD, and that the FtsKγ domain is positioned to modulate this interaction.By modelling the structure of XerD onto the active structures of Cre or λ Int the XerD M and N helices can be re-arranged to produce an active-site close to those seen for other tyrosine recombinasestructure . There itructure , and amiion site . The Pyrdif, and this mutation is synergistic with mutation that alters E184, which directly contacts FtsKγ  +2 amino acids (SR) from the linking XbaI restriction enzyme site followed by FtsK residues 1261 to 1329) was amplified from the full length XerD-FtsKγThe XerDxerD-ftsKγ mutants were made by a two-step overlap PCR process, using mutagenic DNA primers and Phusion DNA polymerase (sequences available on request), and cloned into pBAD24. Selected mutant sequences were subcloned into pBAD24-XerD by PCR from the relevant mutant fusion using XerD primers as described previouslyxerC–ftsKγ-peptide fusion sequence was cloned by cutting the pBad24 XerC-FtsKγ fusionC-FtsKγ fusion overproduction and purification using Ni2+ resin was as previously described2, 1 mM DTT, using a VivaSpin 6 centrifugal concentrator  to a final concentration of ~9 mg/ml.XerDin vitro recombination assays were overproduced and purified as previouslyAll other proteins and fusion proteins for 2. Data was collected on MX2 beamline at the Australian Synchrotron, using a wavelength of 0.9184 ÅInitial hits were identified using 200 nl protein drops, using commercial screens (Molecular Dimensions) mixed by Mosquito nano-litre robot (TTP Labtech). Following initial screening, trigonal bipyramidal crystals were obtained by 2μl + 2μl hanging drop vapour diffusion in a solution of 100 mM Bicine (pH9), 10% (w/v) polyethylene glycol (8000 g/mol) over 2–5 days. Crystals were washed in paratone-N and flash frozen in liquid NE. coli XerD (PDB: 1A0P) and E. coli FtsKγ (PDB: 2J5P) as search models . The program PHASERC-FtsKγ in the asymmetric unit. The structure was built using Arp/WarpThe structure was solved by molecular replacement using the known structures of All structural alignments and structure figures were produced using PyMol .In vivo recombination assays using XerD-γ fusion proteins were carried out as previously published17E. coli strain GR51 (AB1157 xerC ftsK)xerC-γ fusion gene along with plasmid resolution reporter, pRB10, a pSC101 derivative  carrying two directly repeated dif sites, flanking the KmR gene cassette. pRB10 is almost identical to the pFX142 reporter used previously except that pRB10 lacks the duplication of restriction enzyme sites surrounding the two dif sites found in pFX142. Transformants were grown in LB with selection; following 16 h incubations plasmid DNA was recovered from the cultured cells and examined by agarose gel electrophoresis followed by SYBR green staining. Levels of parental and recombinant sized plasmid were quantified using ImageQuant software  and percentage recombination calculated.In vitro recombination, using the 2x dif reporter plasmid pSI56, was as previously describedlac::tetO180 GmR) and WX31 ΔxerD containing the relevant expression vector was prepared and grown in LB at 37 °C to stationary phase (~20 generations)3Assays were carried out essentially as previously published1734Accession codes: Coordinates and structure factors for the XerDC-γ complex have been deposited in the Protein Data Bank under the accession code 5DCF.How to cite this article: Keller, A. N. et al. Activation of Xer-recombination at dif: structural basis of the FtsKγ–XerD interaction. Sci. Rep.6, 33357; doi: 10.1038/srep33357 (2016).


We observed not only stable polarization variants, but also meta-stable polarization variants, which can reduce the charge accumulated at domain boundaries. We considered the number of neighboring domains that are in contact, in order to analyze the complexity of the ferroelectric domain structure. Comparison of the ferroelectric domains from the patterned and unpatterned regions showed that the elastic relaxation induced by removal of the film surrounding the mesas led to a reduction of the average number of neighboring domains, indicative of a decrease in domain complexity. We also found that the rectangular BFO patterns with high aspect ratio had a simpler domain configuration and enhanced piezoelectric characteristics than square-shaped mesas. Manipulation of the ferroelectric domains by controlling the aspect ratio of the patterned BFO thin film mesas can be useful for nanoelectronic applications.We have studied the ferroelectric domains in (001) BiFeO Among various materials candidates, the lead zirconate titanate O3, PZT) family is the most studied for applications in nonvolatile memories and piezoelectric actuators3. An increased piezoelectric response was reported for PZT nanostructures due to the removal of the clamping effect and the enhanced mobility of domain walls5. PZT, however, has a critical drawback because the toxicity of lead creates environmental and safety issues6. Recently, the lead-free ferroelectric BiFeO3 (BFO) has received great attention due to its superior thin-film ferroelectric properties and high ferroelectric Curie temperature 10. In applying BFO to data storage technology, thin, nanostructured BFO films have been studied as a way of reducing operational voltage and increasing circuit density12. The effect of lateral size on nanostructured BFO was studied in films patterned using focused-ion-beam (FIB) milling with a sacrificial layer, and it was discovered that ferroelectricity was preserved in structures with sizes down to 250 nm15. Moreover, Johann et al. reported enhancement of the piezoelectric response in the BFO nanostructures16.Ferroelectric oxide materials are of great interest because of a demand for next-generation memory devices, actuators, energy harvesters, transducers, and microelectromechanical (MEMS) systems applications19. However, the formation of the domain boundaries in BFO and their effects on the piezoresponse at the nanoscale are not clear. Therefore, constructing accurate ferroelectric domain maps is important for understanding not only the position of the domain boundaries and domain configuration but also the mechanism of polarization switching and electrical properties. The most common approach is to use piezoresponse force microcopy (PFM), however, constructing three-dimensional PFM images of polarization domains has been a great challenge because of sliding of the tip and cantilever buckling and an asymmetric tip apex, in addition to sample issues such as surface morphology and surface adsorbates22. Previously, Park et al. introduced angle-resolved PFM (AR-PFM) which provides clearer information on the in-plane polarization directions than conventional PFM method26. More recently, this method was also extended to study charged domain boundaries and the effect of poling on domain structures27.BFO thin films also have disadvantages such as high leakage currents, for which oxygen vacancies and the unusual local electronic transport behavior at ferroelectric domain walls are the main causeHere we present direct observation using AR-PFM of domain boundaries and intermediate polarization variants in epitaxially-grown BFO thin films patterned into mesas, and we discuss the correlation between the resulting domain configurations and piezoresponse switching behavior. To observe the effect of the elastic relaxation induced by FIB patterning, we imaged the ferroelectric domains of BFO thin films patterned with various aspect ratios. We introduced a new parameter called ‘number of neighboring domains’ (NND) to analyze the complexity of the ferroelectric domain map. We claim that the domain structures of patterned BFO mesa structures are less complicated than that of unpatterned BFO films based on the NND analysis. Moreover, high aspect ratio of BFO mesas have an ordered domain configuration and enhanced piezoelectric characteristics.3 (SRO) thin film heterostructure grown on a (001)-oriented STO substrate. The BFO film was patterned into mesas using combined electron-beam lithography and focused ion-beam (FIB) patterning with a removable tungsten mask for protecting from Ga implantation and knock-on damage from the ion-beam tails12. Figure AR-PFM images were obtained of an epitaxial (35 nm) BFO/(70 nm) SrRuOVarious studies have reported that BFO polarization vectors can adopt one of eight thermodynamically-stable variants along the <111> crystallographic directions. By also considering the polarization components determined using VPFM  nanostructures, the effective clamping stress changes when the length over thickness is below 100, and beyond ratios of 100:1 it remains constant28. In the system being studied here, the length over thickness of the BFO mesas ranges from 14 to 29, which is small enough so that clamping can be relieved sufficiently by removing the surrounding materials. In order to explore the effect of elastic relaxation on the ferroelectric domains, we conducted AR-PFM imaging of patterned and unpatterned regions of the BFO thin film as shown in Fig. D at a pixel position i:In the case of PbOi,D will increase or remain constant as shown in Fig. x, which is the percentage of all pixel areas for which the value of NND is x.We carried out this analysis at each pixel across our image, as a way of mapping the domain boundaries and of determining the complexity of the domain pattern. As the diameter of the circle increases NNDD analysis showed similar trends for all values of D that we explored, decreasing exponentially as shown in Fig. D to 20 nm because the tip radius of curvature was <25 nm. Figure 2 patterned BFO mesa and the unpatterned region of the BFO film shown in Fig. The percentage area for the NND12.Figure In order to investigate the complexity of the domain boundaries, we compared the ratio of the percentage area with NND ≥ 5 or more to the percentage area with NND ≥ 2 by defining the complex node density:Figure To investigate the effect of size and aspect ratio of the patterned mesas on the elastic relaxation, we analyzed the AR-PFM domain maps and NND maps of the mesas with respect to domain orientation, as shown in Figs 2 and 1.0 × 0.5 μm2  BFO films epitaxially grown on SRO/STO substrate using AR-PFM. We imaged the in-plane ferroelectric domains with angular increment of 30° and determined the degree of charging at the domain boundaries. We observed meta-stable polarization variants which the angle is not restricted to the <111> directions, which decrease the degree of charging at the domain boundaries. We compared the domain complexity of patterned and unpatterned BFO using the NND, and found out that patterned BFO had simpler domain structures due to the removal of the film surrounding the mesa structure of BFO. In addition, we found that the wider mesas showed simpler domain configuration and a better piezoresponse characteristics such as low coercive voltage or high remanent piezoresponse. Therefore, the patterning of thin films to mesa structures can be useful for nanoelectronic applications because of reducing the NND distribution.3 (SRO) thin film heterostructure was grown on a (001)-oriented STO substrate and the BFO film was patterned into mesas ranging in size from 500 nm to 1.0 μm, using a combined electron-beam lithography and focused ion-beam (FIB) patterning procedure described elsewhere12. The details of how the ferroelectric domain maps were constructed are described in supplementary material. Nanoworld EFM-PtIr coated probes were used with an ac modulation voltage  for the PFM measurements in MFP-3D AFM of Asylum. Local piezoresponse loop was measured by PSIA XE-100 with lock-in amplifier (Standard Research Systems) using the same probes .An epitaxial (35 nm) BFO/(70 nm) SrRuOSupplementary Information


The hexameric type II AAA ATPase (ATPase associated with various activities) p97  is critically involved in a variety of cellular activities including pathways such as DNA replication and repair which both involve chromatin remodeling, and is a key player in various protein quality control pathways mediated by the ubiquitin proteasome system as well as autophagy. Correspondingly, p97 has been linked to various pathophysiological states including cancer, neurodegeneration, and premature aging. p97 encompasses an N-terminal domain, two highly conserved ATPase domains and an unstructured C-terminal tail. This enzyme hydrolyzes ATP and utilizes the resulting energy to extract or disassemble protein targets modified with ubiquitin from stable protein assemblies, chromatin and membranes. p97 participates in highly diverse cellular processes and hence its activity is tightly controlled. This is achieved by multiple regulatory cofactors, which either associate with the N-terminal domain or interact with the extreme C-terminus via distinct binding elements and target p97 to specific cellular pathways, sometimes requiring the simultaneous association with more than one cofactor. Most cofactors are recruited to p97 through conserved binding motifs/domains and assist in substrate recognition or processing by providing additional molecular properties. A tight control of p97 cofactor specificity and diversity as well as the assembly of higher-order p97-cofactor complexes is accomplished by various regulatory mechanisms, which include bipartite binding, binding site competition, changes in oligomeric assemblies, and nucleotide-induced conformational changes. Furthermore, post-translational modifications (PTMs) like acetylation, palmitoylation, phosphorylation, SUMOylation, and ubiquitylation of p97 have been reported which further modulate its diverse molecular activities. In this review, we will describe the molecular basis of p97-cofactor specificity/diversity and will discuss how PTMs can modulate p97-cofactor interactions and affect the physiological and patho-physiological functions of p97. Through specific structural elements, like for example additional domains and insertions as well as different oligomeric arrangements, they act as molecular motors by using conformational changes induced by ATP hydrolysis to perform mechanical work on many different substrates  UBAIn analogy to the FPR loop of UBX proteins, the crystal structure of the UBXL domain of the DUB OTU1  in complex with the N domain also features a loop (YPP motif) inserting into the hydrophobic pocket . All three arginines are important, with the highly conserved last arginine (Arg636 in gp78) being pivotal, and they engage in several electrostatic interactions as well as hydrophobic interactions via the aliphatic part of their side chains with the N domain. The crystal structure of the N domain in complex with the VBM of RHBDL4, a protein crucial for the retro-translocation of polyubiquitylated substrates in the ERAD pathway  are important to maintain the N domain interaction by contributing the majority of the ionic and hydrogen bonded interactions in the interface. However, the RHBDL4 VBM-N domain structure revealed a novel binding mode, which unexpectedly combined the two types of p97-cofactor specificities observed in the UBX and VIM interactions. Specifically, the RF motif in RHBDL4 VBM corresponds to the FPR motif in UBX  corresponds to the RRR motif in the gp78 VIM (RRx2h3AAx2Rh)  Figure .The binding pocket formed in the Nn and Nc lobes provides a sterically unopposed interface for the interaction of the various p97 cofactor proteins. Proteomic studies identified phosphorylation, ubiquitylation, and mono-methylation sites in the VIM/VBM binding motifs of different proteins 0−1GxGx2L . The initial crystal structure of full-length p97 in complex with the SHP motif of UFD1 revealed that the motif adopts a mostly extended, yet slightly bent conformation, and binds at the periphery of the C-terminal α+β subdomain  of the N domain, in direct vicinity of the ND1 linker  found to be involved in UBX/UBXL and VIM/VBM interaction, thus suggesting that the rigidification of this region upon SHP binding may affect binding of other cofactors.The SHP binding motif has been identified as an additional binding element in several UBX proteins and in UFD1, the latter typically forming a stable heterodimer with the UBXL protein NPL4 Figure . In contThe interaction of the UFD1 SHP binding motif could be regulated by phosphorylation. Proteomic studies indicate that both serine residues (Ser229 and Ser231) located in the SGSG motif are phosphorylated, whereas no possible PTMs for the DER1 motif have been identified so far  to an orientation where they are located above the D1 ring  , ubiquitylation (38 identified sites) and acetylation (24 identified sites)  was demonstrated to accumulate at sites of DNA double-strand breaks (DSBs)  family] stimulates the ATPase activity of p97 , which plays important roles in cell survival and phosphorylates p97 at Ser352 (buried), Ser746 (buried), and Ser748   ThrCovalent ubiquitin-protein conjugates are introduced in a series of three consecutive enzymatic steps  are attached to lysine residues of target proteins via an isopeptide bond through the sequential action of E1, E2, and E3 enzymes  can tri-methylate p97 on Lys315  and sulfonic acids (SO3H) on cysteine residues are induced.Reactive oxygen/nitrogen species (ROS/RNS) have been found to act as important physiological modulators of intracellular signaling pathways, but are also causative of aging, cancer, neurodegenerative disorders, and cardiovascular diseases  is (are) still unknown and different models have been proposed Figure : (i) TheThe threading model Figure : In thisThe D2 in-out model Figure : Here suThe side access model Figure : SubstraThe translocation-independent or disassembly model Figure . At leasMost likely, the mechanism by which p97 unfolds/disassembles target proteins depends on the fate of the substrate (Barthelme and Sauer, A common function of p97 is its ATP-dependent extraction or disassembly of ubiquitylated substrates from chromatin, membranes, and protein complexes in many diverse cellular functions that maintain cellular homeostasis, contribute to genomic stability and govern important signaling pathways Figure . The keyPH primarily wrote this review with input from HS. Both authors approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, it is unclear whether the C2A domain mediates other Munc13 functions in addition to this inactivation–activation switch. Here, we utilize mutations that modulate the homodimerization and heterodimerization states to define additional roles of the Munc13 C2A domain. Using electron microscopy and electrophysiology in hippocampal cultures, we show that the C2A domain is critical for additional steps of vesicular release, including vesicle docking. Optimal vesicle docking and priming is only possible when Munc13 heterodimerizes with RIM via its C2A domain. Beyond being a switching module, our data suggest that the Munc13-RIM heterodimer is an active component of the vesicle docking, priming and release complex.The presynaptic active zone protein Munc13 is essential for neurotransmitter release, playing key roles in vesicle docking and priming. Mechanistically, it is thought that the C 2A domain of Munc13 is known to be required for synaptic vesicle priming. Here the authors show new implications of the C2A domain of Munc13, through its dynamic interaction with RIM, in orchestrating a wide range of modulatory operations that shape vesicle docking, priming and neurotransmitter release.The interaction between RIM and the C Caenorhabditis elegans, Drosophila melanogaster and mammalian synapses have identified several AZ protein families that play a key role in vesicle docking and priming, including Munc13  release occurs at a specialized presynaptic release site, known as the active zone (AZ), and depends on the availability of docked and primed fusion-competent vesicles. The processes of docking, priming and fusion rely on the function of specific proteins at the AZ. Loss-of-function studies in c13 refs , 5, 6, CC. elegans and D. melanogaster are of particular interest because they are absolutely crucial for both of these steps in the synaptic vesicle (SV) cycle. In the mammalian central nervous system, the Munc13 protein family comprises of four members: the most prominently expressed Munc13-1, the two Munc13-2 isoforms bMunc13-2 and ubMunc13-2, and Munc13-3 (ref. Munc13-1/2 double knockout (DKO) lack of a measurable ready releasable pool (RRP) and show no spontaneous or Ca2+-dependent evoked release1unc-13 mutant in C. elegansD. melanogaster dunc-13 mutantC. elegans21734Among these proteins, Munc13s and their homologues in 3-3 ref. . Neurons1 domain, a Ca2+/phospholipid binding C2B domain, a MUN domain and a C2C domain at the very C-terminal end. Biophysical studies show that the Munc13 C terminus is crucial for membrane fusion in reconstitution experiments that include the SNAREs, Munc18-1, NSF  and α-SNAP (α-soluble NSF attachment protein) because Munc13 and Munc18-1 orchestrate SNARE complex assembly in an NSF/α-SNAP-resistant manner20211C2B region preceding the MUN domain and the C2C domain is critical for neurotransmitter release as they may help bridge the SV and plasma membrane, that in turn facilitates opening of syntaxin-1  binding Cn-1 ref. . In addi. 1 refs , 28, 29 C. elegans, the long and short Unc13 isoforms differ in their regulation of both vesicle location relative to Ca2+ channels and neurotransmitter release kinetics322A domain that is not present in bMunc13-2 and Munc13-3. Biochemical studies have demonstrated that the C2A domain can form tight homodimeric complexes2A domain interactions, two distinct roles have been proposed for this domain: the recruitment of Munc13 to the presynaptic active zone2A disrupted the Munc13 homodimer and activated vesicle priming. Remarkably, either expression of an N-terminally truncated Munc13 or a Munc13 with a C2A domain point mutation that inhibits Munc13 homodimerization in RIM1/2 conditional DKO bypassed the requirement of RIM in vesicle priming, suggesting that RIM only interacts with Munc13 to disrupt the homodimer, and indicating that the active form of Munc13 is the monomer. These findings further implied that the Munc13/RIM complex does not play a role in priming itself and that the Munc13 C2A domain merely acts as an inactivation–activation switch. This model is surprising given that classical electron microscopy (EM) studies of aldehyde-fixed synapses lacking all RIM1 and RIM2 isoforms12unc-10 mutant in C. elegans2172A domain and the individual functions of Munc13 monomers, Munc13/Munc13 homodimers and Munc13/RIM heterodimers, we performed a structure–function analysis of the role of the C2A domain of Munc13-1 in docking, priming and vesicular release in mammalian central synapses.The Munc13 N terminus, on the other hand, varies considerably between the Munc13 isoformsMunc13-1/2 DKO neurons342A domain and the RIM ZF domain211122A domain with RIM. To test this hypothesis, we performed gain-of-function rescue experiments in Munc13-1/2 DKO cultures from murine hippocampal neurons. Using lentiviral gene transfer, we expressed three different mutants of Munc13-1. The first was Munc13-1 with a complete truncation of the N terminus (Munc13-1 del 1–520), corresponding to the mutant published previously372A domain function within the N terminus: one that specifically eliminated the C2A domain and the subsequent α-helix (Munc13 del 1–150), and another that incorporated the C2A domain but eliminated the intermediate region spanning from the end of the C2A domain to the region preceding the C1 domain (Munc13-1 del 151–520) (Munc13-1/2 DKO neurons rescued with a full-length Munc13-1 protein (Munc13-1 wild type (WT)) served as our WT-positive control and untransfected Munc13-1/2 DKO neurons served as negative controls. All Munc13-1 deletion mutants were found at presynaptic glutamatergic compartments, as indicated by the green fluorescent protein (GFP) signal detected at the vesicular glutamate transporter 1 (VGLUT1)-positive synapses  studies using cryofixation techniques on 151–520) . Munc13-synapses . Fluoressynapses  showed tT levels . We alsoneurites . The eff neurons .2A domain in vesicle docking, we performed TEM on high-pressure frozen samples from the Munc13-1/2 DKO and Munc13-1/2 DKO rescued with Munc13-1 WT or with the Munc13-1 C2A domain-deleted mutants. EM analysis was performed on 40 nm sections and the number of synaptic vesicles in physical contact with the plasma membrane at active zones was counted. In line with previous studies34Munc13-1/2 DKO active zones were essentially devoid of docked vesicles (0.2±0.04 docked vesicle/AZ), while the active zones of the Munc13-1 WT-rescued synapses showed approximately 2 docked synaptic vesicles (1.62±0.11 docked vesicle/AZ). However, synapses from DKO rescued with the two Munc13-1 mutants that lacked the C2A domain, that is, the C2A domain (del 1–150) and the N-terminus (del 1–520) truncations, showed a 42% reduction in the number of docked SVs. In contrast, deletion of the intermediate region of the N terminus that does not eliminate the C2A domain (del 151–520) showed the same docking activity as the Munc13-1 WT  only moderately altered RRP size without affecting the AP-evoked EPSC. The Munc13-1 mutant that lacked the entire N terminus (del 1–520) caused a 50% reduction in RRP size, similar in extent to the reduction in vesicle docking, but only a mildly altered AP-evoked response. However, the mutant that lacked the C2A domain and the subsequent α-helix (del 1–150) showed a severe 80% loss of primed vesicles and evoked synaptic output (As shown previouslyc output .Pvr), while truncation of the C2A domain (del 1–150) caused a reduction in Pvr. Deleting both the C2A domain and the intermediate region from the N terminus (del 1–520) exhibited a relative increase in Pvr but did not reach statistical significance  led to an increase in vesicular release probability , including the C2A domain and the subsequent α-helix, is not functionally equivalent to the truncation of the entire N terminus (amino acids 1–520)  . This ra2A monomers can interact either with another Munc13 C2A domain to form a homodimer or alternatively with RIM to form a heterodimeric complex2A domain of Munc13 in vesicle docking, priming and release probability, we asked whether the Munc13 C2A domain functions differentially depending on its interaction partner. Indeed, while the Munc13 homodimer is thought to inhibit priming, disruption of the Munc13 homodimer via RIM binding to the C2A domain activates priming2A domain interactions on vesicle docking, priming and release probability, we generated a set of point mutants in the Munc13-1 C2A domain in which homodimerization and/or RIM heterodimerization is disrupted  and the subsequent α-helix (E137K) based on the crystal structure of the Munc13-1 C2A domain bound to the RIM ZF domain2A domain and the additional α-helix (residues 3–150) binds robustly to the RIM ZF domain, while the Munc13-1 fragment (3–150) with the double point mutation  exhibited no detectable binding  and Munc13-1  failed to pull down flag-tagged Munc13-1, demonstrating that they do not form C2A/C2A homodimers . As expected, Munc13-1 WT and the Munc13-1 (K32E) that favours the heterodimerization state efficiently co-immunoprecipitated RIM-1α. However, the two Munc13-C2A/ZF-RIM heterodimerization-disrupting mutants  and ) exhibited a major disruption to RIM binding  helps to study the role of Munc13-1 in the Munc13/RIM heterodimeric state, while the double point mutation  probes the function of the Munc13-1 as a homodimer. The triple point mutation  represents the monomeric state .Munc13-1/2 DKO synapses expressing the set of Munc13-1 C2A domain mutant variants generated  showed full rescue of vesicle priming, demonstrated by the measurement of the RRP size , which arrests Munc13-1 in its monomeric state led to 50% reduced RRP size and AP-evoked EPSC amplitudes without affecting Pvr  mutant, which is unable to interact with RIM to disassemble the C2A/C2A homodimers is deficient in vesicle priming and exhibit a tendency to facilitate  with the heterodimeric Munc13-1 (K32E) mutant demonstrate that Munc13 monomers are less efficient in priming and neurotransmitter release compared with Munc13-1/RIM heterodimers  affects the regulation by the C2A domain.Our previous electrophysiological analysis of the Munc13-1 point mutants that promote heterodimerization revealed a novel regulation of docking via the Munc13 CMunc13-1/2 DKO neurons and DKO neurons rescued with Munc13-1 WT or Munc13-1 C2A point mutants to PDBu. The number of docked synaptic vesicles was quantified within each group and compared with each corresponding PDBu-untreated group. PDBu treatment did not cause a change in the number of docked SVs in Munc13-1/2 DKO. The same effect was observed on DKO synapses rescued with Munc13-1 WT or the Munc13-1 C2A homodimerization- and heterodimerization-disrupting mutants , which tend to have high initial Pvr , Munc13-1 (del 1–520), Munc13-1  and Munc13-1  mutants, are the result of a local reduction of Munc13 translocation at the AZ, given the proposed role of RIM in recruitment of, Munc13 to the active zone2A/RIM-ZF domains. Indeed, RIM protein was still detected in the co-immunoprecipitation assays that utilized the mutant described above , the Munc13-1 monomer that can bind to RIM, with the triple point mutant , the Munc13-1 monomer that lacks RIM binding activity  is a novel negative regulator of vesicular release probability, as deletion of residues 1–150 impairs release more strongly than deletion of residues 1–520  but lacking the exons 48, 49 and 53. The complementary DNAs (cDNAs) were subsequently cloned into a lentiviral shuttle vector under the control of a human synapsin-1 promoter. To enable identification of infected cells, Munc13-1 rescue proteins were fused with fluorescent protein tagged Venus. Concentrated lentiviral particles were prepared as previously describedMunc13-1 full-length, truncation, deletions and point mutants rescue construct were generated from a rat Munc13-1/2 DKO mice on a FVB/N background were used for all studies. Animal Welfare Committee of Charité—Universitätsmedizin and the Berlin state government agency for Health and Social Services approved all protocols for animal maintenance and experiments (license no. T 0220/09). Microislands of growth-permissive substrate (collagen and poly-D-lysine) were printed onto 30 mm round glass coverslips precoated with agarose type 4, using a stamp containing 200 × 200 μm spaced dots. Astrocytes were plated at a density of 5,000 cells per cm2 onto the microdot-coated coverslips to allow them to grow onto the growth-permissive substrate. Hippocampi were dissected from embryonic day 18.5 Munc13-1/2 DKO mouse and enzymatically treated with 25 units per ml of papain for 45 min at 37 °C. After enzyme digestion, solution was exchanged for an inactivation solution containing albumin, trypsin inhibitor and 5% fetal calf serum. Hippocampi were mechanically dissociated and the neuron suspension was plated onto the astrocyte microislands at a final density of 300 cells per cm2. Neurons were incubated at 37 °C for 13–16 days to mature before initiating the experiments. For rescue experiments neurons were infected 24 h after plating with lentiviral vectors.Munc13-1/2 DKO or DKO hippocampal neurons infected with the individual rescue constructs were plated at a density of 50,000–100,000 cells onto 6 mm sapphire disks for 13–16 days. Neuronal cultures were frozen using a high-pressure freezer (HPM 100 Leica) under a pressure of 2,100 bar by mounting it into a sandwich support with extracellular solution containing 15% Ficoll. The cryofixation was achieved within milliseconds allowing simultaneous immobilization of all macromolecular components. After freezing, samples were transferred into cryovials containing 1% glutaraldehyde, 1% osmium tetroxide, 1% ddH2O in anhydrous acetone and processed in an AFS2 automated freeze-substitution device (Leica) with the following temperature program: 5 h at −90 °C, from −90 °C to −20 °C (5 °C per h), 12 h at −20 °C and from −20 °C to 20 °C (10 °C per h). After freeze substitution, samples were en bloc stained in 0.1% uranyl acetate for 1 h and infiltrated in increasing concentration of epoxy resin  diluted in acetone at room temperature. Subsequently, samples were flat embedded in Epon and baked for 48 h at 60 °C to allow Epon polymerization. Approximately 200–300 μm square areas containing neurons were randomly selected and sectioned at 40–50 nm thickness using an Ultracut UCT ultramicrotome (Leica) equipped with a diamond knife (Diatome ultra 45). Ultrathin sections were collected on formvar-coated (0.5%) 200 mesh copper grids (EMS) and counterstained with 2% uranyl acetate and lead citrate . Sections were examined in a FEI Tecnai G20 TEM operated at 80–120 keV and digital images were taken with a Veleta 2 K × 2 K CCD camera (Olympus). EMs  containing a single synaptic profile were collected blindly at 135,000 × magnification (pixel size 0.38 nm). Data were analysed blindly by using an analysis program developed for ImageJ and Matlab as reported by Watanabe et al.Munc13-1/2 DKO hippocampal neuronal cultures were used for each data set.Munc13-1/2 DKO neurons at 13–16 days in vitro. Synaptic currents were monitored at room temperature using a Multiclamp 700B amplifier (Axon Instrument). The series resistance was compensated by 70% and only cells with series resistances of <10 MΩ were analysed. Data were acquired using pClamp 10 software (Axon Instrument) at 10 kHz and filtered using a low-pass Bessel filter at 3 kHz. Borosilicate glass pipettes with a resistance between 2 and 3.5 MΩ were used and filled with a solution containing the following (in mM): 136 KCl, 17.8 HEPES, 1 EGTA, 4.6 MgCl2, 4 Na2ATP, 0.3 Na2GTP and 12 creatine phosphate; 50 U ml−1 phosphocreatine kinase; adjusted to pH 7.4 with osmolarity at ∼300 mOsm. Neurons were continuously perfused with standard extracellular solution including the following (in mM): 140 NaCl, 2.4 KCl, 10 HEPES, 10 glucose, 2 CaCl2 and 4 MgCl2; adjusted to pH 7.4 with osmolarity at ∼300 mOsm. AP-evoked EPSCs were triggered by 2 ms somatic depolarization from −70 to 0 mV. To determine the size of the RRP, 500 mM sucrose was applied directly onto the isolated neuron for 5 s using a fast-flow systemPvr ) calculations, the ratio of EPSC charge to RRP charge was determined. Paired-pulse stimulation protocol contained two inductions of an AP at an interval of 100 ms (10 Hz). The paired-pulse ratio was calculated by dividing the amplitude of the second EPSC by amplitude of the first. For phorbol ester experiments, we first determined the RRP size under resting condition. One minute later, EPSC amplitudes were monitored at 0.2 Hz for 30 s. without PDBu application and 30 s under application of 1 μM of PDBu to determine the degree of EPSC potentiation. Immediately after the application of PDBu, the RRP size was again measured. Data were analysed offline using Axograph X (Axograph Scientific).Synaptic function was assayed by whole-cell voltage clamp from autaptic hippocampal 2 and 1 mM TCEP as describedITC experiments were performed on a VP-ITC system  at 20 °C in a buffer containing 50 mM Tris (pH 7.3), 150 mM NaCl, 5 mM MgClMunc13-1 WT and C2A domain mutants were constructed in pEGFP-N1 (CLONTECH Laboratories). Transgenes were cloned between the cytomegalovirus promoter and the SV40 polyA sequences. Munc13-1-FLAG was generated from rat Unc13a by PCR amplification with a reverse primer that harbours a 3xFLAG sequence (Sigma-Aldrich). The RIM-1α cDNA was a gift from Dr Thomas C Südhof at the Stanford University and RFP sequence was fused to the C terminus.HEK293 expression vectors for Munc13-1 with 3xFLAG fused to the C terminus and Munc13-1 WT or C2A mutants plasmids with C-terminal GFP tag to detect C2A–C2A interaction or (2) RIM-1α with RFP fused to the C terminus and C-terminally GFP-tagged Munc13-1 WT or C2A domain mutants to assess RIM/Munc13 interaction. After 48 h, cells were washed twice with phosphate-buffered saline (PBS) and lysed at 4 °C with a NP-40 cell lysis buffer containing: 50 mM Tris-HCl (pH 7.9), 150 mM NaCl, 1% NP-40, 0.5% deoxycholate and 5 mM EDTA and protease inhibitor cocktail-complete mini.HEK293TN (BioCat GmbH) cells were transiently double transfected at 80% confluency with: (1) Next, 250 μg of the double transfected lysates were incubated for 30 min at room temperature with protein G-Dynabeads  bound to the antibody (Ab) Rabbit anti-GFP (Abcam 6556) or control Ab Rabbit IgG . The Dynabeads-Ab-Antigene (Ag) c were washed three times with washing buffer and eluted with 25 μl elution buffer. Final 25 μl eluted Dynabeads-Ab-Ag complexes were mixed with 25 μl sample buffer (Laemmli 2 × concentrated containing 0.2 M dithiothreitol) and boiled for 10 min at 70 °C. Dynabeads were removed after boiling and 25 μl of the immunoprecipitates and 25 μg protein from the double transfected lysates were used for the SDS–polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes. After blocking with 5% skim milk in Tris-buffered saline with Tween-20, blots were incubated with mouse monoclonal ANTI-FLAG M2 (Sigma F1804), Living Colors A.v. monoclonal Ab (JL-8) (Clontech 632380) or rabbit polyclonal anti RIM1/2 (SYSY 140203) Abs. After washing the blots with Tris-buffered saline with Tween-20, the immune reactive proteins were detected by ECL  with horseradish peroxidase-coupled secondary Ab (Jackson ImmunoResearch Laboratories). Western blot images  have beeMunc13-1/2 DKO or DKO hippocampal neurons infected with the different rescue constructs were plated at a density of 25,000 cells and fixed in 4% paraformaldehyde in PBS at 16 days in vitro. After fixation, neurons were permeabilized with PBS–Tween 20, blocked with serum in PBS–Tween 20 and incubated overnight with rabbit polyclonal antibody against GFP  and guinea pig polyclonal antibody VGLUT1 . Primary antibodies were labelled with Alexa Fluor 488 Affinipure donkey anti-rabbit IgG and Alexa Fluor 647 Affinipure donkey anti-guinea pig IgG . Coverslips were mounted with Mowiol 4–88 antifade medium (Polysciences Europe). Fixed neurons were imaged on a confocal laser-scanning microscope Leica TCS SP8 with identical settings used for all samples. Neuronal cultures were visualized using a 63 × oil immersion objective. Images were acquired using Leica Application Suite X (LAsX) software at 1,024 × 1,024 pixels resolution using a z-series projection of 8–10 images with 0.3 μm depth intervals. Ten independent neurons per group for each cultured and three different cultures were imaged and analysed using ImageJ software.post hoc test. For the data in P values were calculated by comparison with the non-PDBu-treated group using the unpaired Student's t-test: Mann–Whitney. Data were acquired and analysed in a blinded fashion and differences between data sets were considered significant at P<0.05.For the statistical analysis data were collected from at least three independent hippocampal cultures to minimize culture-to-culture variability. To minimize variability among the electrophysiological data sets, an approximately equal number of autaptic neurons were recorded from all experimental groups per day. Electrophysiological data from the different experimental groups during 3–4 consecutive days of recording were normalized to the mean value of the control group. Data are expressed as mean±s.e.m. Statistical comparison was performed in all figures except for The data that support the findings of this study are available from the corresponding author on reasonable request.How to cite this article: Camacho, M. et al. Heterodimerization of Munc13 C2A domain with RIM regulates synaptic vesicle docking and priming. Nat. Commun.8, 15293 doi: 10.1038/ncomms15293 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures and Supplementary Table.


Text CorrectionIn the original article, there was an error. In the text, the diameters of the zones of inhibition given for the strains (MG1655 and Δ10) were interchanged.A correction has been made to Results and Discussion, Sub-section Relative Hypersensitivity of MG1655 and Δ10 Strains to Ciprofloxacin and Ampicillin, Paragraph one:In light of our observations, we were curious about the degree of sensitivity to various antibiotics. We determined the sensitivity of the MG1655 and Δ10 strains to various antibiotics by disk diffusion method, as it is highly sensitive and quantifiable. We observed that the zone of inhibition of MG1655 with ciprofloxacin (10 μg) was 3 cm (averages) while that of Δ10 strain was 3.6 cm . With ampicillin (10 μg), the zones of inhibition for MG1655 and Δ10 strain were 2.1 cm and 2.45 cm, respectively. With nalidixic acid, the zones of inhibition for MG1655 and Δ10 strain were 1.78 and 1.98 cm, respectively. We did not find any significant difference with the other antibiotics at the concentrations used .The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Assessing the physical connections and allosteric communications in multi-domain nuclear receptor (NR) polypeptides has remained challenging, with few crystal structures available to show their overall structural organizations. Here we report the quaternary architecture of multi-domain retinoic acid receptor β–retinoic X receptor α (RARβ–RXRα) heterodimer bound to DNA, ligands and coactivator peptides, examined through crystallographic, hydrogen–deuterium exchange mass spectrometry, mutagenesis and functional studies. The RARβ ligand-binding domain (LBD) and DNA-binding domain (DBD) are physically connected to foster allosteric signal transmission between them. Direct comparisons among all the multi-domain NRs studied crystallographically to date show significant variations within their quaternary architectures, rather than a common architecture adhering to strict rules. RXR remains flexible and adaptive by maintaining loosely organized domains, while its heterodimerization partners use a surface patch on their LBDs to form domain-domain interactions with DBDs. Nuclear receptors (NR) are multidomain proteins, which makes their crystallization challenging. Here the authors present the crystal structure of the retinoic acid receptor β–retinoic X receptor α (RARβ–RXRα) heterodimer bound to DNA, ligands and coactivator peptides, which shows that NR quaternary architectures are variable. Their polypeptides consist of a variable N-terminal domain (NTD), a central DNA-binding domain (DBD), and a 12-helical ligand-binding domain (LBD) located at their C-terminus3. Physical connections and the routes for allosteric communications between domains have remained difficult to assess, given the relatively few examples of crystal structures involving multi-domain NR complexes. Employing full-length or multi-domain NR complexes bound to DNA, crystal structures have been reported to date only for the peroxisome proliferator-activated receptor γ–retinoic X receptor α (PPARγ–RXRα) heterodimer 4, the hepatocyte nuclear factor 4α (HNF-4α) homodimer (PDB code: 4IQR)5, and the liver X receptor β (LXRβ)–RXRα heterodimer (PDB code: 4NQA)6. In some cases, multi-domain NRs have been studied by alternate biophysical techniques and those data were interpreted to suggest possible structural models. For example, RXRα was studied heterodimerized with RARα, PPARγ2 and vitamin D nuclear receptor (VDR) using small-angel X-ray scattering, fluorescent studies and/or small-angle neutron scattering7. These interpretations were further used to suggest the existence of a common architecture of NR heterodimers on DNA direct repeats7. However, the crystallographic models have not been supportive of these structural models, and have been showing instead that the quaternary structures of RXR heterodimers consist of significant variations without a commonly adopted organization10. To understand larger NR complexes with full-length coactivators, the quaternary structure of an active complex of DNA-bound estrogen receptor α (ERα), steroid receptor coactivator 3 (SRC-3/NCOA3), and a secondary coactivator (p300/EP300) has been described based on cryoelectron microscopy (cryo-EM)11.Nuclear receptors (NRs) are a family of transcription factors that respond to lipophilic ligands and control a variety of metazoan gene programs12. The six mammalian NRs for retinoids are encoded by distinct genes, but they share close structural homology at the level of their LBDs and DBDs13.Retinoids bind to a subclass of NRs in mammals consisting of three retinoic acid receptors (RARα/β/γ) and three retinoid X receptors (RXRα/β/γ). Through heterodimerization between an RAR and an RXR, productive and functional transcription factors are formed within this subclass of NRs. Ligand binding modulates the transcriptional activities of these receptor heterodimers by altering the protein surface conformations at each of their LBDs and shaping preferences for coactivators and corepressors. Specifically, ligand-binding repositions helix-12 of the LBDs into an active conformation that fosters the binding of LXXLL motifs of coactivators, including those belonging to members of the steroid receptor coactivator (SRC or p160) family. The repertoire of retinoid actions through these NRs includes a variety of essential biological processes such as embryogenesis, organogenesis, cell growth, differentiation, and apoptosis14. The retinoic acid receptors exhibit high-affinity binding to endogenous molecules that include all-trans retinoic acid  and 9-cis retinoic acid . Ligand binding to RARs leads to modulation of gene programs that are particularly relevant in embryonic development and pattern formation15. In adult organisms, RAR ligands can directly impact cellular differentiation, and synthetic ligands that bind and regulate RXRs and RARs have been developed as therapeutic agents for cancers  and inflammatory diseases (severe acne and psoriasis)16.The RAR and RXR proteins are among the most intensively studied for their structural properties, but most structural characterizations to date have focused on isolated LBD domains, and have not been successful with their heterodimeric multi-domain polypeptide complexesTo directly visualize how different domains of RAR and RXR polypeptides physically interact within and between their polypeptides, we conducted crystallographic studies using multi-domain recombinant RARβ and RXRα proteins. Both receptors’ ligands, including REA for RARβ and 9CR for RXRα, their idealized DNA response element (RARE) and coactivator peptides were included to visualize how all these components are configured and interact within the overall complex. We further utilized hydrogen–deuterium exchange mass spectrometry (H/D-ex MS) to determine if signals at one domain can register their effects across the heterodimer to distal domains, and further relied on functional transcriptional studies and mutagenesis to validate our understanding of domain–domain junctions. Finally, we sought here to understand the quaternary architecture of RARβ–RXRα heterodimer in the wider context of other recently reported crystal structures of multi-domain NR complexes, to assess their level of overall variations and to pinpoint specific sites where domain–domain junctions are formed in each case.Escherichia coli. Using extensive crystallization screens that utilized various sized DBD–hinge–LBD segments of each isotype of RXRs and RARs, and a variety of known RAR ligands, we successfully obtained crystals of only the RARβ–RXRα heterodimer on a DR1 DNA in the presence of REA and 9CR retinoic acids, and with LXXLL synthetic peptides derived from SRC-2′s second (middle) NR box. The crystal structure was solved at 3.5 Å resolution by molecular replacement using known RAR–RXR DBD and LBD dimeric structures (PDB codes: 1DSZ17 and 1XDK18) as the search models  when ligands were switched from agonist REA to antagonist BMS-189453  that show altered H/D-ex MS patterns when we switched ligands between REA and BMS-189453  as compared to antagonists (KD of 12–16 nM)  Fig. . A possinM) Fig. .ANGPTL4 (DR1) and CYP26A1 (DR5), both of which are target genes of RARβ–RXRα  abolished the transcriptional repression seen with wild-type RARβ on DR1, as expected Fig. . Then ex22.Interestingly, these same mutations positioned specifically at the DBD–LBD interface of RARβ also compromised the transcriptional activation from the DR5 reporter Fig. . This fiKD of 35 nM for DR1 binding and 24 nM for DR5 binding, when WT RARβ and RXRα proteins were used together. As a reference point, we observed that single point mutation located precisely at the RARβ’s DNA-binding helix (E99A) known to contact the DNA major groove within a half-site reduced the binding affinities for both DR1 (to 80 nM) and DR5 (to 143 nM). When we measured the effects of mutations located at the DBD–LBD interface (double and triple mutations), we also observed losses in the heterodimer’s DNA affinities (three to tenfold)  Fig. , suggestThe RARβ–RXRα heterodimer described here represents the fourth distinct multi-domain NR complex studied to date using X-ray crystallography. To better understand the common features within the quaternary architectures of all these NR complexes, we directly compared the crystal structures of RARβ–RXRα, PPARγ–RXRα, HNF-4α homodimer, all of which were on DR1 DNA, and that of LXRβ–RXRα on DR4 DNA Fig. . With re9. Importantly, clear variations are observed in the relative locations of DBDs and LBDs within each complex  domains located at the N-termini of these receptors, which have not been successfully visualized in any of the NR crystal structures to date. Future co-crystal and cryo-EM studies focused on more complete protein–protein and protein–DNA complexes will be needed to fully understand the wider spectrum of molecular components and interactions underlying NR functions.Moving forward, significant gaps still remain in our detailed understandings of how pairs of protein coactivators and other transcriptional components in their full-length forms assemble on these receptors. Given the limitations in crystallographically observing more elaborate and larger scale multi-protein NR structures, cryo-EM studies may prove to be increasingly useful. Cryo-EM studies recently conducted on the estrogen receptorall-trans retinoic acid), 9CR (9-cis retinoic acid) and the SRC-2 LXXLL peptide (EKHKILHRLLQDSY).Human RARβ  and RXRα  were cloned into the vectors pET28a (N-His tagged) and pET20b (No tag), respectively. PCR primer sequences used in this study are summarized in Supplementary Table −1) and reservoir solution containing 0.1 M MES (pH 6.5), 25% (v/v) PEG300. Before flash frozen in liquid nitrogen, crystals were soaked in reservoir plus 30% (v/v) glycerol as the cryoprotectant. Diffraction data were collected at the Argonne National Laboratory SBC-CAT 19ID beamline at 100 K, and processed using the HKL3000 program23.Crystallization of the RARβ–RXRα–DNA complex was carried out using the hanging drop vapor diffusion method at 4 °C, by mixing equal volume of protein–DNA complex  as the search models. Further manual model building was facilitated using Coot25, combined with the structure refinement using phenix.refine26. The diffraction data and final statistics are summarized in Table 27, are 95%/0% (favored/outliers). All the structural figures were prepared using PyMOL (http://www.pymol.org).The structure of RARβ–RXRα–DNA complex was solved by molecular replacement with the program PhaserAGGTCAAAGGTCAGAAAG-3′ for DR1, and 5′-AAACTAGGTCACCGAAAGGTCAGAAAG-3′ for DR5) with the unlabeled reverse strands  in the buffer consisting of 10 mM Tris (pH 7.5), 1 mM EDTA and 2 mM MgCl2. 2 nM DNA was incubated with purified proteins for 30 min for the binding assay. Final protein concentrations were varied by serial dilution in binding buffer . The fluorescence polarization signals were recorded using black 96-well plates on FlexStation 3 (Molecular Devices)and converted to “fraction bound”, before fitting the curves sigmoidally in GraphPad Prism 7 to get the KD values28.The fluoresceinated double-strand DNA was prepared by annealing 6-FAM 5′-labeled forward strands (5′-AAACTANGPTL4 (DR1) reporter, HEK293T cells  were seeded in 24-well plates, and 1 day later transfected with 100 ng of human RXRα, 100 ng of luciferase reporter, 1 ng of pRL (control Renilla luciferase) and 100 ng of pCMV-Tag4-RARβ  by using 0.6 μL jetPRIME regent (Polyplus-transfection SA) for each well. For the experiments using CYP26A1 (DR5) reporter, HEK293T cells were prepared similarly and transfected with 100 ng of luciferase reporter, 1 ng of pRL (control Renilla luciferase) and 200 ng of pCMV-Tag4-RARβ  by the same regent. After 6 h transfection, medium was refreshed with 1 μM REA; and another 24 h later, luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega). Final data were normalized by the relative ratio of firefly and Renilla luciferase activity. All experiments were repeated independently for at least twice.Full-length human RARβ was cloned into the pCMV-Tag4 vector, and the subsequent site-directed mutagenesis was confirmed by DNA sequencing. For the experiments using 30, in which enzymatic digestion, peptide separation and MS analysis was fully automated. To initiate the HDX reaction, 6 μl of pre-chilled protein stock solution at 5.4 µM  was diluted into 18 μl D2O buffer , and incubated at 0 °C for 30, 100, 300, 1000, 3000, and 10,000 s. At indicated times, 6 μl of ice cold acidic buffer  Glycerol, pH 2.4) was added to quench the exchange reaction. Quenched samples were frozen on dry ice and passed over an in-house made immobilized pepsin column (16 μl bed volume) on ice in H2O contain 0.05% (v/v) trifluoroacetic acid at a flow rate of 20 μl min−1 for pepsin digestion. The proteolytic products were collected on a C18 trap column for desalting  and eluted for separation on a Michrom Magic C18  with a 30 min linear acetonitrile gradient (6.4–38.4%). The effluent was subject to an OrbiTrap Elite mass spectrometer (Thermo Fisher Scientific) for MS analysis. The instrument settings were optimized for HDX analysis as previously reported31. Peptide identification was performed using Proteome Discoverer (Thermo Fisher Scientific) and deuteration level of each peptide was determined by using HDEXaminer . In addition, HDX analysis was also carried out on non-deuterated and fully deuterated samples to correct back-exchange32. A peptide coverage map for the RARβ protein used in the HDXMS studies and individual uptake curves (including the regions highlighted in Fig. Hydrogen–deuterium exchange experiments were performed using our in-house deuterium exchange system33, and were subsequently processed by the ESPrint 3.0 program34 for figures.The protein sequence alignments were generated on the Clustal Omega severCoordinates and structure factors have been deposited in the Protein Data Bank under accession code PDB 5UAN. Other data are available from the corresponding author upon reasonable request.Supplementary InformationPeer Review file


Paedotherium minor, Glyptodontidae and Proscelidodon sp.Large cylindrical sediment-filled structures interpreted as mammal burrows occur within the loess-paleosol sequence of the late Miocene Cerro Azul Formation of central Argentina. A total of 115 burrow fills from three localities were measured. They are typically shallowly dipping, subcylindrical, unbranched structures with rounded ends and lacking enlargements. The horizontal diameter of the burrows range between 0.15 and 1.50 m, with most of the burrows in the interval of 0.39 to 0.98 m. Geometric morphometric analysis of transverse cross-sections support their distinct subcircular and elliptical  shapes. Burrow fills are typically laminated in the lower part and massive in the upper part. The laminated intervals reflect pulses of flowing water entering the abandoned burrow during moderate rains, whereas massive intervals reflect mass flow input of dense sediment-water mixtures during heavy rains that produced sheet floods. Approximately 1% of the burrows contained fragmentary, disarticulated and weathered mammal bones that were introduced in the open burrow by currents along with other sedimentary particles. Analysis of the tetrapod burrow fossil record suggests that Miocene burrows, including those studied herein, reflect a remarkable increase in the average size of the fossorial fauna. We conclude that large late Miocene mammals dug burrows essentially as a shelter against environmental extremes and to escape predation. The simple architecture of the burrows suggests that the producers essentially foraged aboveground. Several mammal groups acquired fossorial habits in response to cold and seasonally dry climatic conditions that prevailed during the late Miocene in southern South America. The considerable range of horizontal diameters of the studied burrows can be attributed to a variety of producers, including dasypodids, the notoungulate  Fossil vertebrate burrows are relatively common biogenic structures and the oldest convincing evidence of tetrapod burrows are Early Permian (Asselian-Artinskian) lysophorid amphibian burrows from Kansas, USA . Most prDaimonelix ix e.g., . Also inix e.g., . The maiix e.g., . Pliocenix e.g., . These aix e.g., . The smaix e.g., .A trend towards larger diameter burrows is evident from the early Miocene to the late Pleistocene see . Pre-MioIn this context, the tetrapod burrows from the late Miocene Cerro Azul Formation are the largest pre-Pleistocene tetrapod burrows and can help to understand the reasons for acquisition of burrowing habits in large Cenozoic mammals from South America. These structures have been partially and briefly described , but a dThe studied localities with late Miocene burrows are distributed in a latitudinal belt of approximately 25 km : SalinasAnother locality of the Cerro Azul Formation, a roadcut in national road 154 , has been previously described as having vertebrate burrows . HoweverStandard measurements in burrows were horizontal diameter (Dh), vertical diameter (Dv), preserved length, mean azimuth of burrow fill and inclination of fill laminae. The mean azimuth was measured using a compass and considering the burrow fill boundary and dominant plunge of laminated fill. When changes of dip direction or inclination were observed in a single burrow fill they were recorded separately. Burrow diameter  was measured orthogonal to the main axis of the structure. The horizontal diameter was obtained from almost all types of exposures , whereas the vertical diameter was mostly an apparent value, except for the rare transverse sections, where it can be considered the true vertical diameter. At each location, GPS coordinates were recorded; the burrow fills were photographed and sketched.n = 24) were used for a 2-D geometric morphometric analysis. From field photographs, the outline of the burrow boundary was sketched in Corel Draw™. These images were used to build a file with the TPSutil software. Burrow cross section outlines were oriented with respect to the top and bottom of the hosting bed and treated as symmetrical outlines. For each image a total of 16 type II landmarks  are housed at the Museo Histórico Regional de Guaminí “Coronel Marcelino E. Freyre” under the acronym MHG-P. Field work was approved by the Dirección Provincial de Museos y Preservación Patrimonial, under the Project “Vertebrados del Mioceno tardío-Plioceno en el área de las lagunas encadenadas del oeste de la provincia de Buenos Aires. Aportes a la bioestratigrafía del Cenozoico tardío de la Región Pampeana”, permit nr. 2015-3-P-156-2.At each locality, a detailed sedimentary log was measured and samples of host rock and burrow fill were collected for petrographic analysis. Also, a selected locations a detailed sedimentologic log of the burrow fill was measured. Mammal bones found The Cerro Azul Formation outcrops are located in the north-western, central, and eastern part of La Pampa province and adjacent western Buenos Aires province, Argentina . The unin = 5), which is very close to the average for the formation , siltstone to fine- grained sandstone with pervasive carbonate cementation, both in the form of nodules and rhizoliths. Rhizoliths are small to medium sized and commonly 1–30 mm in diameter. The lower horizon can be up to more than 5 m thick and contain relicts of primary sedimentary structures, like tangential cross-bedding or horizontal bedding, as seen in the Salinas Grandes de Hidalgo section . MudstonThe section at Laguna Chillhué also contains similar paleosols  and diffA total of 115 fossil burrows were measured from three localities: 53 from Salinas Grandes de Hidalgo, 59 from Laguna La Paraguaya, and three from Laguna Chillhué see . The stun = 112) and the frequency distribution suggests a roughly normal distribution where three subpopulations can be distinguished  ranges from 0.15 to 1.50 m ( nguished . The sman = 78), a number of morphologies can be distinguished  Thenguished , 7B. Somnguished , 7D. Thenguished 7F, whicIn a few cases, the distal portion of burrow showed a lateral expansion of up to 23% of the Dh, commonly having a subhorizontal lamination , 7H. Othn = 24 from all localities) suggest a distinction between elliptical  and subcircular cross-sections. Elliptical cross-sections are more common (n = 18) and the corresponding Dh ranges from 0.39 to 1.50 m  with a flat bottom and convex top. The subcircular cross-sectional shape (n = 6) is represented in the intermediate subpopulation with a Dh ranging from 0.39 to 0.56 m, and an average Dv/Dh ratio of 0.88.The analysis of the well defined cross-sectional shape of burrows  belongs to the intermediate subpopulation with a range from 37 to 439 kg, whereas the remaining estimates comes from the large subpopulation (n = 7) with a range 708 to 1,623 kg. Burrows with subcircular cross-section from the intermediate subpopulation, are linked with a producer having body mass from 92.84 to 186.0 kg.Body mass estimates of the producers of the burrow on the basis of the cross-sectional area  , 9C. TheThe vertebrate burrows are easily spotted in the field because of the distinctive laminated structure of the infilling sediments that contrasts with the structureless host rock. The filling is composed of an alternation of laminated claystone and siltstone with massive fine-grained sandstone and siltstone containing floating claystone intraclasts. Laminated intervals are a few millimeters to about 50 mm thick, whereas massive intervals tend to be thicker. Most of the burrow fills display a laminated interval in the lowermost part of the fill, with the upper part massive, especially in the Salinas Grandes de Hidalgo locality –10C. A fRosellichnus isp. In the paleosol adjacent to the fills we found Taenidium barretti, Attaichnus kuenzelli and Coprinisphaera isp.Only the ichnofossils found within or very close to the burrow fills are considered. We found within the fills abundant rhizoliths and rare smaller vertebrate burrows, vertebrate footprints and ?Calcareous rhizoliths, including rhizocretions and root casts, are abundant and were recorded in all studied localities. Rhizoliths are arranged in vertical, oblique and subhorizontal position . RhizoliA single small burrow  that cutA partially eroded burrow fill from Laguna La Paraguaya (# 708) exposed an internal bedding plane of the filling showing closely spaced elliptical depressions with a noticeable marginal rim . These aRosellichnus (A group of five subcircular rings in the upper part of a burrow fill (# 659A from SG) is tentatively identified as a cluster of bee cells and compared with the ichnogenus llichnus . The preAttaichnus kuenzelii were identified, in some cases very close, but no cross-cutting relationship were seen. A few specimens of Taenidium barretti also occur at this locality, in the form of subcylindrical burrows, 12 mm wide and 80 mm long with an average meniscus thickness of 2.2 mm. At Laguna La Paraguaya we also found two specimens of cemented and compact spherical chambers (diameter 18.7–23.0 mm) with a large emergence hole (10–11 mm) assigned to Coprinisphaera isp. , Doellotatus sp., Eosclerocalyptus sp., Mesotheriinae indet., Gyptodontidae indet. (three specimens), and undeterminate mammals (two specimens). For details about the taxonomy and illustrations of mammal remains, see Eosclerocalyptus sp.  appeared disarticulated but associated within a single burrow fill. The fossil remains display different degree of weathering and corrosion, as well as biostratinomic fractures.Mammal bones within the burrow fills are scarce (only 1% of burrows contained fossil remains) and usually appear disarticulated and poorly preserved, in some cases with signs of abrasion . The fosptus sp. , and remThe studied fossil burrows are unbranched and display a significant variation in the horizontal diameter, which ranges from 0.15 m to 1.5 m . The simDoellotatus sp. and one of the specimens of Paedotherium minor and the considerably small size of the animals   of the burrow e.g., . The rem 1.50 m) . In consThe mammals with fossorial habits recorded in the Cerro Azul Formation include xenarthrans, notoungulates and rodents e.g., . Among tPaedotherium minor, the dasypodids Doellotatus, Chorobates, Proeuphractus, and Chasicotatus; and the rodent Lagostomus. Paedotherium (Hegetotheriidae) is a medium-sized rodent-like ungulate native to South America. This taxon is very common in the Cerro Azul Formation, both in La Pampa and Buenos Aires provinces , with a body mass ranging from 1 to 13 kg, the likely candidates are the notoungulate es e.g., . Articules e.g.,  and a moes e.g., .Doellotatus, Chasicotatus, Proeuphractus and Chorobates  is well known for its digging adaptations and for living in communal burrow systems ; Macrochorobates and Macroeuphractus ; and Proscelidodon . The fossil remains found in this size range that are likely candidates are those of Glyptodontidae and Proscelidodon sp. , with an estimated body mass ranging from 37 to 438 kg, the likely candidates are the Mesotheriinae ; odon sp. . There ailveyrai . These silveyrai  (Table 1ilveyrai .n = 4). In our case study, glyptodonts are considered good candidates for constructing the subcircular burrows of the intermediate subpopulation, which are 0.39–0.56 m wide and display an average Dv/Dh ratio of 0.88. Regarding the large dasypodids Macrochorobates and Macroeuphractus, the available body mass estimates suggest a range of 10 to 100 kg  forelimb  and an extrapolated body mass of 700–1,600 kg, the more likely producer is Doellotatus, Chasicotatus, Proeuphractus and Chorobates) on the basis of body mass, the fossorial habit and architecture of modern dasypodid burrows and, secondarily, Paedotherium minor. (2) For the intermediate and large subpopulations, the Glyptodontidae and Mylodontidae (Proscelidodon sp.) are good candidates as these were the largest representatives of the late Miocene burrowing fauna of the Cerro Azul Formation. The Glyptodontidae were generalized diggers, like modern dasypodids, and exhibited a carapace fitting especially the subcircular burrows. Proscelidodon sp. is also a likely candidate of the elliptical and larger burrows. For the intermediate subpopulation, probably the large dasipodids (Macrochorobates and Macroeuphractus) and Mesotheriinae should be considered.To summarize, the studied fossil burrows can be attributed to several producers, according to their horizontal diameter. The more likely producers of the studied fossil burrows are: (1) for the small subpopulation, the smaller dasypodids (en masse pouring of the sediment-water mixture at high angle (30°) produced a massive and poorly sorted sediment fill, whereas en masse pouring at a low angle (5°) produced thicker graded beds  found inside the fill. These trace fossils suggest that partially filled burrows with a surface communication were explored or reoccupied by other tetrapods and used by bees to nest. Alternatively, the bee cells may be constructed after the complete filling of the burrow in the soil profile. Among the presumed producers of burrows of intermediate size, the outline and size of the footprints match those of Pleistocene glyptodonts but are quite different from those of ground sloths  and Belize , and was compared with the Celliforma ichnofacies  were produced by small dasypodids and, secondarily, by the notoungulate  4.Proscelidodon sp.) are the best candidates. The Glyptodontidae are related to the subcircular burrows of intermediate size and Proscelidodon sp. would be the producer of the elliptical and largest burrows.For the dominant burrows exhibiting an intermediate , and large horizontal diameter , the Glyptodontidae and Mylodontidae . During filling, the abandoned burrows were visited or reoccupied by other tetrapods and used by bees to nest,After abandonment, the burrows received sediments from the surface that progressively filled the structure. The filling process was produced dominantly by several pulses of sediment laden currents related to moderate rains (well laminated fill) and  7.In general, it is not considered likely that well laminated burrow fills will contain remains of the producer, whereas massive fills have a greater chance of containing remains of the tetrapod that dug the burrow. 8.The main factor explaining the appearance of large mammal burrows in the late Miocene are environmental changes, including the appearance of open environments with low vegetation and semiarid and seasonal climate.10.7717/peerj.4787/supp-1Article S1Plot of average horizontal diameter of fossil burrows vs age of the hosting rock. Table containing raw data for Click here for additional data file.10.7717/peerj.4787/supp-2Article S2Compressed .TPS file.Click here for additional data file.10.7717/peerj.4787/supp-3Article S3Systematic assignation of bone remains found inside burrow fills. Credit: María Cristina Cardonatto.Click here for additional data file.10.7717/peerj.4787/supp-4Figure S1Proscelidodon sp. (GHUNLPam 18807-1). (B) fragmented metacarpal of Proscelidodon sp. (GHUNLPam 18807-2). (C) Gliptodontidae indet. (MGH-P126/41). (D) Eosclerocalyptus sp. (MGH-P126/34). (E–F) Glyptodontidae indet. (MGH-P126/37). (G) Doellotatu s sp. (MGH-P126/40). (H) Paedotherium minor (MGH-P126/39). (I) Mesotheriinae indet. (MGH-P126/42). Scale bar: 1 cm. Image credit: María Cristina Cardonatto.(A) Click here for additional data file.10.7717/peerj.4787/supp-5Table S1References: SC: slightly curved, SI: sinuous, C: “C” shaped, E: elliptical, S: subcircular, PC: planoconvex, * approximate value,  minimum value. Dh, horizontal diameter; Dv, vertical diameter. Body mass estimation after Wu et al. (2015). Wu NC, Alton LA, Clemente CJ, Kearney MR, White CR. 2015. Morphology and burrowing energetics of semi-fossorial skinks (Liopholis spp.). The Journal of Experimental Biology, 218: 2416–2426. Credit: María Cristina Cardonatto.Click here for additional data file.