From the Cover: PNAS Plus: The CentO satellite confers translational and rotational phasing on cenH3 nucleosomes in rice centromeres

Plant and animal centromeres comprise megabases of highly repeated satellite sequences, yet centromere function can be specified epigenetically on single-copy DNA by the presence of nucleosomes containing a centromere-specific variant of histone H3 (cenH3). We determined the positions of cenH3 nucleosomes in rice (Oryza sativa), which has centromeres composed of both the 155-bp CentO satellite repeat and single-copy non-CentO sequences. We find that cenH3 nucleosomes protect 90–100 bp of DNA from micrococcal nuclease digestion, sufficient for only a single wrap of DNA around the cenH3 nucleosome core. cenH3 nucleosomes are translationally phased with 155-bp periodicity on CentO repeats, but not on non-CentO sequences. CentO repeats have an ∼10-bp periodicity in WW dinucleotides and in micrococcal nuclease cleavage, providing evidence for rotational phasing of cenH3 nucleosomes on CentO and suggesting that satellites evolve for translational and rotational stabilization of centromeric nucleosomes.Centromeres, the chromosomal domains that attach to spindle microtubules to segregate eukaryotic chromosomes in mitosis and meiosis, are DNA elements bound by special nucleosomes that contain a centromere-specific variant of histone H3 (cenH3). In most plants and animals, cenH3 nucleosomes are found on centromeric DNA that comprises megabases of tandemly repeated “satellite” sequences. Despite this apparent preference for repetitive DNA, a fully functional centromere, called a neocentromere, can occasionally form by assembling cenH3 nucleosomes on a single-copy DNA sequence that was not previously part of a centromere, indicating that centromere specification is epigenetic in plants and animals (for reviews, see refs. 1–4).The tandem arrays of highly repeated satellite sequences that compose most plant and animal centromeres can differ dramatically between closely related species (5), and even between different chromosomes (6–8), suggesting that satellite arrays undergo rapid evolution through expansions, contractions, gene conversions, and transpositions. Monomers of satellite repeats range in length from 5 bp in Drosophila to 1,419 bp in cattle although more than half of described monomers in 282 species have lengths between 100 and 200 bp, often regarded as approximately the length of nucleosomal DNA (6, 9). The cenH3 nucleosomes typically occupy only a portion of the satellite repeats, often in discontinuous blocks (7, 10–12), and the same or similar repeats often underlie flanking pericentromeric heterochromatin composed of conventional nucleosomes. Some of these repeats, for example African green monkey α-satellite DNA, have long been known to position conventional nucleosomes, resulting in arrays of regularly spaced nucleosomes, said to be translationally phased (13–15). Nucleosomes can occupy multiple alternative translational phases on the same satellite (16, 17). Translationally phased nucleosomal arrays have also been observed on satellites in cucumber and in several cereal species, where phasing varies among repeats and chromosomal regions (18, 19).Recently deep-sequencing technology has been applied to centromeres treated with micrococcal nuclease (MNase), which preferentially digests linker DNA between nucleosomes, to determine the positioning of cenH3 nucleosomes on satellite repeats. In human cultured cells, substantial translational phasing of CENP-A, the human cenH3, was reported on α-satellite (20). In maize, a similar approach mapped CENH3 (the name used for plant cenH3s) on the 156-bp maize centromeric satellite CentC and on two retrotransposon-derived centromeric sequences, CRM1 and CRM2 (21). Evidence for translational phasing of CENH3 on CentC and CRM1 was lacking, but 190-bp phasing was observed on CRM2. CentC was shown to have a strong periodicity of AA or TT dinucleotides about every 10 bp, which corresponds to one turn of the DNA double helix. This periodicity is thought to favor a particular orientation of the DNA toward the nucleosome core particle, based on DNA bendability, and is known as rotational phasing of nucleosomes (22–24).Rice has centromeres characterized by the 155-bp satellite sequence CentO, which is related to maize CentC (25, 26). Although some rice centromeres have megabases of CentO satellites, other evolutionarily new centromeres have little CentO, so CENH3 nucleosomes are found on both CentO and non-CentO sequences (12). For example, Cen8 is comprised of mostly non-CentO sequences and has a CentO array (CentO_8) that is spanned by a sequenced BAC (27). Centromeres like Cen8 are thought to represent an intermediate stage in centromere evolution between rare neocentromeres that form on unique sequences and mature centromeres populated by megabase-sized arrays of satellites (7, 12). Cen8 therefore presents an opportunity to compare the organization of CENH3 nucleosomes on CentO and non-CentO sequences. To that end, we used an antibody to rice CENH3 (27) to perform chromatin immunoprecipitation (ChIP) of CENH3 nucleosomes digested with MNase and sequenced the bound DNA (ChIP-Seq) to determine the positions of CENH3 nucleosomes on rice centromeres. We analyzed the sizes and positions of CENH3 nucleosomal DNA fragments on both CentO and non-CentO sequences to address the role of satellites in organizing centromeric chromatin and analyzed the sequence features of these fragments to look for evidence of nucleosome positioning signals.     

Sequential de novo centromere formation and inactivation on a chromosomal fragment in maize

The ability of centromeres to alternate between active and inactive states indicates significant epigenetic aspects controlling centromere assembly and function. In maize (Zea mays), misdivision of the B chromosome centromere on a translocation with the short arm of chromosome 9 (TB-9Sb) can produce many variants with varying centromere sizes and centromeric DNA sequences. In such derivatives of TB-9Sb, we found a de novo centromere on chromosome derivative 3-3, which has no canonical centromeric repeat sequences. This centromere is derived from a 288-kb region on the short arm of chromosome 9, and is 19 megabases (Mb) removed from the translocation breakpoint of chromosome 9 in TB-9Sb. The functional B centromere in progenitor telo2-2 is deleted from derivative 3-3, but some B-repeat sequences remain. The de novo centromere of derivative 3-3 becomes inactive in three further derivatives with new centromeres being formed elsewhere on each chromosome. Our results suggest that de novo centromere initiation is quite common and can persist on chromosomal fragments without a canonical centromere. However, we hypothesize that when de novo centromeres are initiated in opposition to a larger normal centromere, they are cleared from the chromosome by inactivation, thus maintaining karyotype integrity.The centromere is an important chromosomal region responsible for correct chromosome segregation during cell division. Centromeres are found in the primary constriction region on the chromosome, upon which the kinetochore complex assembles to produce a platform for spindle binding (1). Centromere function is conserved among different species, and several epigenetic markers of active centromeres have been found, including a histone H3 variant referred to as CENH3 in plants (2, 3) or CENP-A in animals (4–6) and phosphorylation of histone H2A at Thr133 in plants (7). Correct loading of CENH3 to the centromere region is a key component of kinetochore assembly (8). Centromeric DNA sequences have experienced rapid evolution (9, 10), and arrangements of DNA sequence in centromere regions differ in species and even in different chromosomes of an individual organism (11). Myriad repeat sequences exist in the centromeres of higher plants. In maize (Zea mays), there are two major types of centromere specific DNA sequences: the simple satellite repeat sequence CentC (12) and centromeric retrotransposon of maize (CRM) (3). Many epigenetic features have been identified in centromeric regions, including DNA methylation levels, histone variants, histone modifications, and RNA components (11). Both epigenetic elements and DNA sequences take part in centromere formation and maintenance, but it is still unknown how genetic and epigenetic factors work together in this process.The centromere is one of the most complex regions on the chromosome, and complete DNA sequencing through the centromeric region is difficult to obtain due to their highly repetitive nature. Centromere sizes, defined by CENP-A/CENH3 binding regions, range from 125 bp in Saccharomyces cerevisiae to 500–1,500 kb in humans and mice (11). In plants, centromere sizes can range to several megabases (Mb) with many repetitive transposable elements, which makes it difficult to study centromere structure and function. For example, the sizes of centromere 2 and 5 in maize are roughly 2 and 7 Mb, respectively.Previous work sought misdivision derivatives of the B chromosome centromere using a translocation between the supernumerary chromosome and the short arm of chromosome 9 (9S) to reduce the size of the centromere for functional studies (13–15). B chromosomes are extra chromosomes that have been found in many plants, animals, and fungi. In maize, a reciprocal translocation between a B chromosome and the short arm of chromosome 9 produced two chromosomes referred to as B-9 and 9-B (13), together referred to as TB-9Sb. Chromosome 9-B contains the long arm of the B chromosome and most of chromosome 9, including its centromere. Correspondingly, chromosome B-9 contains part of the short arm of chromosome 9 and the other part of the B chromosome with the active B centromere. The translocation breakpoint is near Wx1, which is located on 9-B (16). The B centromere of B-9 can undergo misdivision during meiosis, producing many derivatives (14). The first misdivision derivative was a pseudoisochromosome, and subsequently, many telocentric chromosomes and isochromosomes were derived by additional misdivisions (13, 17). Misdivision events can be recognized in crosses of TB-9Sb onto a tester via a fusion-breakage cycle recognized by the behavior of the C1 color marker on the B-9 chromosome. The cycle continues during endosperm development to produce a mosaic phenotype but is “healed” in the embryo, which when grown and analyzed cytologically will reveal the nature of the new chromosomes formed (13, 17). This type of screen was used to assemble a large collection of misdivisions to examine the structural features of the B centromere (18). Centromere sizes of these derivatives were changed and progressively reduced. In these previous studies, molecular analysis of centromere size relied on studying the B centromere-specific DNA repeat before the maize centromere elements, CentC and CRM, were known. The B-specific repeat allows this centromere to be studied against the background of the other centromeres; it surrounds and is interspersed within the active core of the B centromere (15, 19).New functional centromeres formed at ectopic locations rather than native centromeric regions on the chromosomes are called de novo centromeres. Many de novo centromeres have been found in human patients and other organisms (20, 21). There are reports of de novo centromeres in plants, such as barley (Hordeum vulgare) (22), oat (Avena sativa)-maize addition lines (23), and maize (24, 25). The conditions for de novo centromere formation remain unclear (26). Recent research revealed that many de novo centromeres prefer to form near native centromeric regions or in the heterochromatic regions, such as the pericentromere and telomere (27, 28). There are also de novo centromeres in human formed far from native centromeres (20). We have previously described two de novo centromeres in maize: one is near the position of the native centromere (25) and the other is distal to the site of the corresponding native centromere (24). Specific chromatin environments may be required for centromere formation, but the major elements are as yet unknown.DNA sequence alone is insufficient to direct centromere formation, and dicentric chromosomes containing two centromeres are good examples. To be stable, structurally dicentric chromosomes must have one inactive and one active centromere; otherwise, two active centromeres will lead to chromosome breakage during cell division. In maize, many dicentric chromosomes have been reported from B-A translocation chromosome derivatives (29). Dicentric chromosomes can be produced through the process of the chromosome type breakage-fusion-bridge (BFB) cycle, and the inactive centromeres can be reactivated by intrachromosomal recombination (30). The DNA sequences of the active and inactive centromeres of dicentric chromosomes are essentially identical, but the centromere activity states are completely different. We screened several misdivision derivatives using FISH probes specific to maize centromere sequences, CentC and CRM, as well as probes specific to the B centromere repeat sequence (B-repeat) to gain further insight into the nature of the centromeres in this collection. We discovered that one such chromosome, derivative 3-3, lacks detectable CentC and CRM signals, but still has a functional centromere that is not associated with the B-repeat sequence. The results of chromatin immunoprecipitation sequencing (ChIP-seq) using maize CENH3 antibody revealed that a 288-kb region on 9S is involved in the de novo centromere formation. The functional B centromere of progenitor telo2-2 is deleted from derivative 3-3. Further, new derivatives of derivative 3-3 had been selected (31) but there was no change in the B-specific repeat patterns. Here, we found that the de novo centromere of 3-3 has become inactive in all of its derivatives, and in each case a shift to a new de novo centromere position occurred; one of these contains only a 200-kb CENH3 binding region within 9S. The other two are apparently in B chromosome sequences. Thus, sequential de novo centromere formation and exchange of centromere activity occurred in chromosome 3-3 and its derivatives, providing new insight into centromere formation and maintenance.These results help formulate the nature of de novo centromere formation. In all of the examples now documented in maize, the size range is within a few hundred kilobases. In contrast, normal maize centromeres, as noted above, are typically several megabases. The regular occurrence of de novo centromeres found here and previously (24, 25) indicates that they are capable of being formed regularly on chromosomal fragments that are structurally acentric; however, they do not persist in normal chromosomes. The reason might reside in the previous observation in maize (30) and wheat (32) that in functional dicentrics the smaller centromere becomes inactive in a tug of war between large and small. However, in the absence of a normal centromere, the present work illustrates that de novo centromeres can persist. Thus, in normal chromosomes, if a de novo is initiated, it will be as quickly inactivated in opposition to the much larger preexisting centromere; the chromosome will not be affected, and will seldom change structure over evolutionary time despite such a high rate of de novo formation. This hypothesis also suggests that a selective pressure will be placed on the normal centromeres to expand to a size that can regularly inactivate de novo centromeres based on their initial size at formation.     

Reconstitution of a prokaryotic minus end-tracking system using TubRC centromeric complexes and tubulin-like protein TubZ filaments

Segregation of DNA is a fundamental process during cell division. The mechanism of prokaryotic DNA segregation is largely unknown, but several low-copy-number plasmids encode cytomotive filament systems of the actin type and tubulin type important for plasmid inheritance. Of these cytomotive filaments, only actin-like systems are mechanistically well characterized. In contrast, the mechanism by which filaments of tubulin-like TubZ protein mediate DNA motility is unknown. To understand polymer-driven DNA transport, we reconstituted the filaments of TubZ protein (TubZ filaments) from Bacillus thuringiensis pBtoxis plasmid with their centromeric TubRC complexes containing adaptor protein TubR and tubC DNA. TubZ alone assembled into polar filaments, which annealed laterally and treadmilled. Using single-molecule imaging, we show that TubRC complexes were not pushed by filament polymerization; instead, they processively tracked shrinking, depolymerizing minus ends. Additionally, the TubRC complex nucleated TubZ filaments and allowed for treadmilling. Overall, our results indicate a pulling mechanism for DNA transport by the TubZRC system. The discovered minus end-tracking property of the TubRC complex expands the mechanistic diversity of the prokaryotic cytoskeleton.Eukaryotes use microtubules for the segregation of replicated DNAs during the fundamental processes of mitosis and meiosis (1). Some prokaryotes use mitosis-like machineries based on different cytomotive filament systems to maintain and segregate plasmid DNA (2, 3). For example, the ParMRC actin-like system, composed of ATPase protein ParM, adaptor protein ParR, and centromeric DNA parC, pushes plasmids to the cell poles by insertional elongation of bipolar ParM spindles, leading to efficient DNA transport and segregation (4–6).Bacillus thuringiensis (7), Bacillus anthracis (8), and Bacillus cereus (9) maintain their large, low-copy-number virulence plasmids using tubZRC loci. TubZRC loci encode the tubulin-like GTPase protein TubZ, adaptor protein TubR, and tubC centromeric DNA repeats, which are located directly upstream of the tubZ and tubR genes on the plasmids.For B. thuringiensis pBtoxis plasmid, it has been shown that the TubZRC maintenance system (referred as “TubZRC”) functions via TubZ filaments whose formation and dynamics are both essential for plasmid stability (10). However, TubZRC alone maintains synthetic plasmids only under artificial selective pressure (7, 11), and replication of pBtoxis occurs without the dnaA-box but not without TubZRC, prompting others to propose a role for the TubZRC system in plasmid replication (12). In vitro, TubZ assembles into two- and four-stranded polymers (13–16), and structural studies suggested that the centromeric TubRC complex forms a ring-like structure (17), interacting with the long C-terminal TubZ extensions (18). Thus, it was proposed that the TubRC complex tracks growing ends of TubZ filaments in analogy to the centromeric complex that follows growing filament ends of the actin-like partitioning systems (5, 6, 19). In bulk assays, TubRC has been reported to enhance TubZ filament formation, possibly indicating a switch in dynamic behavior (17, 20).To uncover how TubR protein and tubC DNA harness TubZ filament dynamics for DNA transport, we probed the ability of the three TubZRC components encoded on plasmid pBtoxis of B. thuringiensis to self-organize in vitro. We used exclusively untagged, full-length proteins, and molecules were labeled with small chemical dyes for detection. Single-filament dynamics of TubZ have not been described before in vitro. Hence, we first characterized growth and shrinkage of individual filaments, because treadmilling has been described for TubZ filaments in cells (10), in contrast to the dynamic instability that is a hallmark of microtubules and ParM filaments (21, 22).     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

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

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

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.     

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

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