Putative Partners Research Articles

The Biological Properties and Potential Interacting Proteins of d-Alanyl-d-alanine Ligase A from Mycobacterium tuberculosis.

(1) Background: d-alanine-d-alanine ligase (DdlA), an effective target for drug development to combat against Mycobacterium tuberculosis (Mtb), which threatens human health globally, supplies a substrate of d-alanyl-d-alanine for peptidoglycan crosslinking by catalyzing the dimerization of two d-alanines. To obtain a better understanding of DdlA profiles and develop a colorimetric assay for high-throughput inhibitor screening, we focused on explicating and characterizing Tb-DdlA. (2) Methods and Results: Rv2981c (ddlA) was expressed in Escherichia coli, and the purified Tb-DdlA was identified using (anti)-polyhistidine antibody followed by DdlA activity confirmation by measuring the released orthophosphate via colorimetric assay and the yielded d-alanyl-d-alanine through high performance thin layer chromatography (HP-TLC). The kinetic assays on Tb-DdlA indicated that Tb-DdlA exhibited a higher affinity to ATP (KmATP: 50.327 ± 4.652 μmol/L) than alanine (KmAla: 1.011 ± 0.094 mmol/L). A colorimetric assay for Tb-DdlA activity was developed for high-throughput screening of DdlA inhibitors in this study. In addition, we presented an analysis on Tb-DdlA interaction partners by pull-down assay and MS/MS. Eight putative interaction partners of Tb-DdlA were identified. (3) Conclusions: Our dataset provided a valuable resource for exploring Tb-DdlA biology, and developed an easy colorimetric assay for screening of Tb-DdlA inhibitors.

A Complex of RIM2alpha and RIM-Binding Protein 2 Stabilizes Slow Voltage-Dependent Inactivation of Cochlear Inner Hair Cell Cav1.3 L-Type Ca2+ Channels

Cav1.3 L-type Ca2+-channels (LTCCs) in cochlear inner hair cells (IHCs) exhibit a unique slow current inactivation to enable tonic sound-induced neurotransmitter release. The slow Ca2+-dependent inactivation can be explained by the calmodulin-competing binding of CaBP2, whereas the molecular mechanism underlying slow voltage-dependent inactivation (VDI) remains unexplained. Here we investigated if slow VDI is stabilized by the formation of a large signaling complex of Cav1.3 with the presynaptic scaffolding proteins RIM and RIM-binding-protein (RBP). The expression of the putative interaction partners in IHCs was investigated via nested-PCR. RBP2 interaction with the C-terminus of the full length (Cav1.3L) or short Cav1.3 isoform (Cav1.3S) was determined using GST-pulldowns and co-immunoprecipitations. The functional impact of coexpressed RIM and/or RBP on VDI of Cav1.3 Ba2+ currents (15 mM,β3 or β2a,α2δ1) was evaluated via whole-cell patch-clamp recordings in tsA201-cells. We detected RIM2α and RBP2+3 transcripts in mature IHCs and showed for the first time that β2e is the predominant IHC β2-splice variant. RBP2 could only interact with the full-length Cav1.3 C-terminus. Using β3, coexpression of RIM2α and RBP2 significantly slowed Cav1.3L VDI to a similar extent than observed in IHCs, while RIM2α or RBP2 alone had less (RIM2α) or no effect (RBP2). This effect was splice variant-specific because when co-expressed with Cav1.3S RBP2 even antagonized the slowing of VDI by RIM2α. The palmitoylated β2a-isoform slowed VDI by its own preventing an additional effect by RIM2α and/or RBP2 (β2e is currently investigated). We show that different mechanisms contribute to the slow VDI of IHCs. RIM2α/RBP2 stabilize slow VDI in Cav1.3L-β3 LTCC complexes while membrane-associated β2 variants (β2a and most probably also β2e) generate slow VDI without further modulation by RIM/RBP. Supported by Austrian Science Fund (FWFP27809,W11010,F44150).

The cell fate determinant Scribble is required for maintenance of hematopoietic stem cell function.

Cell fate determinants influence self-renewal potential of hematopoietic stem cells. Scribble and Llgl1 belong to the Scribble polarity complex and reveal tumor-suppressor function in drosophila. In hematopoietic cells, genetic inactivation of Llgl1 leads to expansion of the stem cell pool and increases self-renewal capacity without conferring malignant transformation. Here we show that genetic inactivation of its putative complex partner Scribble results in functional impairment of hematopoietic stem cells (HSC) over serial transplantation and during stress. Although loss of Scribble deregulates transcriptional downstream effectors involved in stem cell proliferation, cell signaling, and cell motility, these effectors do not overlap with transcriptional targets of Llgl1. Binding partner analysis of Scribble in hematopoietic cells using affinity purification followed by mass spectometry confirms its role in cell signaling and motility but not for binding to polarity modules described in drosophila. Finally, requirement of Scribble for self-renewal capacity also affects leukemia stem cell function. Thus, Scribble is a regulator of adult HSCs, essential for maintenance of HSCs during phases of cell stress.

The heterologous expression of Arabidopsis PAP2 induces anthocyanin accumulation and inhibits plant growth in tomato.

Anthocyanins are naturally occurring secondary metabolites, responsible for the color of many plants. The Arabidopsis thaliana MYB90/PAP2 (production of anthocyanin pigment 2) was introduced into tomato to study its effect on anthocyanin accumulation. The transgenic tomato displayed much greater anthocyanin accumulation than wild type in all plant organs, but the organs were not fully purple in color except for the stamen. The expression of anthocyanin biosynthetic genes and an anthocyanin-related basic helix-loop-helix (bHLH) gene SlAN1 was significantly increased in the transgenic line, suggesting that ectopic expression of AtPAP2 increases the expression of anthocyanin-related structural and regulatory genes to enhance anthocyanin content. Yeast two-hybrid assays revealed that the endogenous MYB protein SlAN2 interacted with two putative bHLH partners, SlAN1 and SlJAF13, while AtPAP2 only interacted with SlJAF13, which may be why AtPAP2 transgenic plants showed limited anthocyanin accumulation in fruits. In addition to anthocyanin accumulation, the transgenic tomato plants were significantly smaller in size, and the length of primary roots and number of lateral roots were obviously decreased. The expression of lignin biosynthetic genes was downregulated in transgenic tomato plants, which may be the reason for the inhibited growth. The lateral organ boundaries-domain (LBD) genes, which regulate lateral root organogenesis in the auxin signaling pathway, were downregulated in transgenic tomato roots, which may partly account for the disturbed lateral root formation in the transformants. Taken together, the results demonstrate that heterologous expression of transcription factor AtPAP2 not only resulted in anthocyanin accumulation but also inhibited plant growth in tomato.

Analysis of ORP2-knockout hepatocytes uncovers a novel function in actin cytoskeletal regulation.

ORP2 is implicated in cholesterol transport, triglyceride metabolism, and adrenocortical steroid hormone production. We addressed ORP2 function in hepatocytes by generating ORP2-knockout (KO) HuH7 cells by CRISPR-Cas9 gene editing, followed by analyses of transcriptome, F-actin morphology, migration, adhesion, and proliferation. RNA sequencing of ORP2-KO cells revealed >2-fold changes in 579 mRNAs. The Ingenuity Pathway Analysis (IPA) uncovered alterations in the following functional categories: cellular movement, cell-cell signaling and interaction, cellular development, cellular function and maintenance, cellular growth and proliferation, and cell morphology. Many pathways in these categories involved actin cytoskeleton, cell migration, adhesion, or proliferation. Analysis of the ORP2 interactome uncovered 109 putative new partners. Their IPA analysis revealed Ras homolog A (RhoA) signaling as the most significant pathway. Interactions of ORP2 with SEPT9, MLC12, and ARHGAP12 were validated by independent assays. ORP2-KO resulted in abnormal F-actin morphology characterized by impaired capacity to form lamellipodia, migration defect, and impaired adhesion and proliferation. Rescue of the migration phenotype and generation of typical cell surface morphology required an intact ORP2 phosphoinositide binding site, suggesting that ORP2 function involves phosphoinositide binding and transport. The results point at a novel function of ORP2 as a lipid-sensing regulator of the actin cytoskeleton, with impacts on hepatocellular migration, adhesion, and proliferation.-Kentala, H., Koponen, A., Kivelä, A. M., Andrews, R., Li, C., Zhou, Y., Olkkonen, V. M. Analysis of ORP2-knockout hepatocytes uncovers a novel function in actin cytoskeletal regulation.

Visualization of RMRs (Receptor Membrane RING-H2) Dimerization in Nicotiana benthamiana Leaves Using a Bimolecular Fluorescence Complementation (BiFC) Assay.

The bimolecular fluorescent complementation (BiFC) is a fluorescent complementation method largely used to investigate protein-protein interaction in living cells. This method is based on the ability of two nonfluorescent fragments to assemble forming a native fluorescent reporter with the same spectral properties of the native reporter. Such fragments are fused to putative protein partners that in case of interaction will bring the two halves in close proximity, allowing for the reconstitution of an active fluorescent reporter. The BiFC has been used to investigate protein-protein interaction in a number of different organisms, including plants. In plant cells, many essential pathways of protein trafficking and subcellular localization necessitate the intervention of several protein units organized in multisubunit complexes. It is well known that vacuolar sorting of many secretory soluble proteins require the intervention of specific transmembrane cargo receptors able to interact forming dimers. In this chapter we describe a BiFC method for the efficient visualization of RMR (Receptor Membrane RING-H2) type 2 dimerization in agro-infiltrated Nicotiana benthamiana leaves. Furthermore, this relatively simple method represents an optimal strategy to test protein-protein interaction using any other putative protein partners of interest in plant cells.

Analysis of Direct Interaction between Viral DNA-binding Proteins by Protein Pull-down Co-immunoprecipitation Assay.

This protocol analyzes the direct interaction between two DNA-binding proteins by pull-down co-immunoprecipitation. One of the proteins is overexpressed in E. coli as HA-tagged recombinant protein and cell-free extracts are immunoprecipitated in HA-affinity resin. Cell extracts are treated with nuclease to degrade DNA and RNA, which rules out nucleic acid-mediated indirect interaction. Then, a second immunoprecipitation step is performed using the purified putative partner protein. Co-immunoprecipitated proteins can be detected either by Coomassie Blue staining and/or Western blotting (WB) if a specific antibody is available. Moreover, many DNA/RNA binding proteins are highly electropositive, which can hinder WB under standard conditions, as has been shown in histones and histone-like proteins. In this case, we show that the high isoelectric point of the putative partner results in a poor transfer. Tips to troubleshot WB transfer of highly electropositive DNA-binding proteins are provided.

Contribution of non-circadian neurons to the temporal organization of locomotor activity.

ABSTRACTIn the fruit fly, Drosophila melanogaster, the daily cycle of rest and activity is a rhythmic behavior that relies on the activity of a small number of neurons. The small ventral lateral neurons (sLNvs) are considered key in the control of locomotor rhythmicity. Previous work from our laboratory has showed that these neurons undergo structural remodeling on their axonal projections on a daily basis. Such remodeling endows sLNvs with the possibility to make synaptic contacts with different partners at different times throughout the day, as has been previously described. By using different genetic tools to alter membrane excitability of the sLNv putative postsynaptic partners, we tested their functional role in the control of locomotor activity. We also used optical imaging to test the functionality of these contacts. We found that these different neuronal groups affect the consolidation of rhythmic activity, suggesting that non-circadian cells are part of the circuit that controls locomotor activity. Our results suggest that new neuronal groups, in addition to the well-characterized clock neurons, contribute to the operations of the circadian network that controls locomotor activity in D. melanogaster.

Begomoviral Movement Protein Effects in Human and Plant Cells: Towards New Potential Interaction Partners.

Geminiviral single-stranded circular DNA genomes replicate in nuclei so that the progeny DNA has to cross both the nuclear envelope and the plasmodesmata for systemic spread within plant tissues. For intra- and intercellular transport, two proteins are required: a nuclear shuttle protein (NSP) and a movement protein (MP). New characteristics of ectopically produced Abutilon mosaic virus (AbMV) MP (MPAbMV), either authentically expressed or fused to a yellow fluorescent protein or epitope tags, respectively, were determined by localization studies in mammalian cell lines in comparison to plant cells. Wild-type MPAbMV and the distinct MPAbMV: reporter protein fusions appeared as curled threads throughout mammalian cells. Co-staining with cytoskeleton markers for actin, intermediate filaments, or microtubules identified these threads as re-organized microtubules. These were, however, not stabilized by the viral MP, as demonstrated by nocodazole treatment. The MP of a related bipartite New World begomovirus, Cleome leaf crumple virus (ClLCrV), resulted in the same intensified microtubule bundling, whereas that of a nanovirus did not. The C-terminal section of MPAbMV, i.e., the protein’s oligomerization domain, was dispensable for the effect. However, MP expression in plant cells did not affect the microtubules network. Since plant epidermal cells are quiescent whilst mammalian cells are proliferating, the replication-associated protein RepAbMV protein was then co-expressed with MPAbMV to induce cell progression into S-phase, thereby inducing distinct microtubule bundling without MP recruitment to the newly formed threads. Co-immunoprecipitation of MPAbMV in the presence of RepAbMV, followed by mass spectrometry identified potential novel MPAbMV-host interaction partners: the peptidyl-prolyl cis-trans isomerase NIMA-interacting 4 (Pin4) and stomatal cytokinesis defective 2 (SCD2) proteins. Possible roles of these putative interaction partners in the begomoviral life cycle and cytoskeletal association modes are discussed.

No mutual mate choice for quality in zebra finches: Time to question a widely held assumption.

Studies of mate choice typically assume that individuals prefer high quality mates and select them based on condition-dependent indicator traits. In species with biparental care, mutual mate choice is expected to result in assortative mating for quality. When assortment is not perfect, the lower quality pair members are expected to compensate by increased parental investment to secure their partner (positive differential allocation). This framework has been assumed to hold for monogamous species like the zebra finch (Taeniopygia guttata), but progress has been hampered by the difficulty to define individual quality. By combining multiple measures of causes (inbreeding, early nutrition) and consequences (ornaments, displays, fitness components) of variation in quality into a single principal component, we here show that quality variation can be quantified successfully. We further show that variation in quality indeed predicts individual pairing success, presumably because it reflects an individual's vigor or ability to invest in reproduction. However, despite high statistical power, we found no evidence for either assortative mating or for positive differential allocation. We suggest that zebra finch ornaments and displays are not sufficiently reliable for the benefits of choosiness to exceed the costs of competition for the putative best partner. To assess the generality of these findings unbiased quantification of signal honesty and preference strength is required, rather than selective reporting of significant results.

Loss of the nutrient sensor TAS1R3 leads to reduced bone resorption.

The taste receptor type 1 (TAS1R) family of heterotrimeric G protein-coupled receptors participates in monitoring energy and nutrient status. TAS1R member 3 (TAS1R3) is a bi-functional protein that recognizes amino acids such as L-glycine and L-glutamate or sweet molecules such as sucrose and fructose when dimerized with TAS1R member 1 (TAS1R1) or TAS1R member 2 (TAS1R2), respectively. It was recently reported that deletion of TAS1R3 expression in Tas1R3 mutant mice leads to increased cortical bone mass but the underlying cellular mechanism leading to this phenotype remains unclear. Here, we independently corroborate the increased thickness of cortical bone in femurs of 20-week-old male Tas1R3 mutant mice and confirm that Tas1R3 is expressed in the bone environment. Tas1R3 is expressed in undifferentiated bone marrow stromal cells (BMSCs) in vitro and its expression is maintained during BMP2-induced osteogenic differentiation. However, levels of the bone formation marker procollagen type I N-terminal propeptide (PINP) are unchanged in the serum of 20-week-old Tas1R3 mutant mice as compared to controls. In contrast, levels of the bone resorption marker collagen type I C-telopeptide are reduced greater than 60% in Tas1R3 mutant mice. Consistent with this, Tas1R3 and its putative signaling partner Tas1R2 are expressed in primary osteoclasts and their expression levels positively correlate with differentiation status. Collectively, these findings suggest that high bone mass in Tas1R3 mutant mice is due to uncoupled bone remodeling with reduced osteoclast function and provide rationale for future experiments examining the cell-type-dependent role for TAS1R family members in nutrient sensing in postnatal bone remodeling.

Identification of novel Ack1-interacting proteins and Ack1 phosphorylated sites in mouse brain by mass spectrometry.

Ack1 (activated Cdc42-associated tyrosine kinase) is a non-receptor tyrosine kinase that is highly expressed in brain. This kinase contains several protein-protein interaction domains and its action is partially regulated by phosphorylation. As a first step to address the neuronal functions of Ack1, here we screened mouse brain samples to identify proteins that interact with this kinase. Using mass spectrometry analysis, we identified new putative partners for Ack1 including cytoskeletal proteins such as Drebrin or MAP4; adhesion regulators such as NCAM1 and neurabin-2; and synapse mediators such as SynGAP, GRIN1 and GRIN3. In addition, we confirmed that Ack1 and CAMKII both co-immunoprecipitate and co-localize in neurons. We also identified that adult and P5 samples contained the phosphorylated residues Thr 104 and Ser 825, and only P5 samples contained phosphorylated Ser 722, a site linked to cancer and interleukin signaling when phosphorylated. All these findings support the notion that Ack1 could be involved in neuronal plasticity.

Disclosing 3' UTR cis-elements and putative partners involved in gene expression regulation in Leishmania spp.

To identify putative cis-elements involved in gene expression regulation in Leishmania, we previously conducted an in silico investigation to find conserved intercoding sequences (CICS) in the genomes of L. major, L. infantum, and L. braziliensis. Here, the CICS databank was explored to search for sequences that were present in the untranslated regions (UTRs) of groups of genes showing similar expression profiles during in vitro differentiation. Using a selectable marker as a reporter gene, flanked by either an intact 3' UTR or a UTR lacking the conserved element, the regulatory role of a CICS was confirmed. We observed that the pattern of modulation of the mRNA levels was altered in the absence of the CICS. We also identified putative CICS RNA-binding proteins. This study suggests that the publicly available CICS database is a useful tool for identifying regulatory cis-elements for Leishmania genes and suggests the existence of post-transcriptional regulons in Leishmania.

Critical periods regulating the circuit integration of adult-born hippocampal neurons.

The dentate gyrus (DG) in the adult brain maintains the capability to generate new granule neurons throughout life. Neural stem cell-derived new-born neurons emerge to play key functions in the way information is processed in the DG and then conveyed to the CA3 hippocampal area, yet accumulating evidence indicates that both the maturation process and the connectivity pattern of new granule neurons are not prefigured but can be modulated by the activity of local microcircuits and, on a network level, by experience. Although most of the activity- and experience-dependent changes described so far appear to be restricted to critical periods during the development of new granule neurons, it is becoming increasingly clear that the surrounding circuits may play equally key roles in accommodating and perhaps fostering, these changes. Here, we review some of the most recent insights into this almost unique form of plasticity in the adult brain by focusing on those critical periods marked by pronounced changes in structure and function of the new granule neurons and discuss how the activity of putative synaptic partners may contribute to shape the circuit module in which new neurons become finally integrated.

An “on-matrix” digestion procedure for AP-MS experiments dissects the interplay between complex-conserved and serotype-specific reactivities in Dengue virus-human plasma interactome

The interactions between the four Dengue virus (DENV) serotypes and plasma proteins are crucial in the initial steps of viral infection to humans. Affinity purification combined with quantitative mass spectrometry analysis, has become one of the most powerful tools for the investigation on novel protein-protein interactions. Using this approach, we report here that a significant number of bait-interacting proteins do not dissociate under standard elution conditions, i.e. acid pH and chaotropic agents, and that this problem can be circumvented by using the “on-matrix” digestion procedure described here. This procedure enabled the identification of 16 human plasma proteins interacting with domain III from the envelope protein of DENV serotypes 1, 3 and 4 that would have not been detected otherwise and increased the known DIIIE interactors in human plasma to 59 proteins. Selected Reaction Monitoring analysis evidenced DENV interactome in human plasma is rather conserved although significant differences on the reactivity of viral serotypes with specific proteins do exist. A comparison between the serotype-dependent profile of reactivity and the conservation pattern of amino acid residues suggests an evolutionary selection of highly conserved interactions with the host and other interactions mediated for surface regions of higher variability. SignificanceFalse negative results on the identification of interacting proteins in pull-down experiments compromise the subsequent interpretation of results and the formulation of a working hypothesis for the derived future work. In this study we demonstrate the presence of bait-interacting proteins reluctant to dissociate under elution conditions of acid pH and presence of chaotropics. We propose the direct proteolytic digestion of proteins while still bound to the affinity matrix (“on-matrix” digestion) and evaluate the impact of this methodology in the comparative study of the interactome of the four serotypes of Dengue virus mediated by the domain III of the viral envelope glycoprotein. Fifty nine proteins were identified as putative interaction partners of Dengue virus (IPs) either due to direct binding or by co-isolation with interacting proteins. Collectively the IPs identified from the pull-down with the recombinant domain III proteins representing the four viral serotypes, 29% were identified only after “on-matrix” digestion which demonstrate the usefulness of this method of recovering bait-bound proteins. Results highlight a particular importance of “on-matrix” digestion procedure for comparative studies where a stronger interaction with one of the interest baits could prevent a bound protein to elute under standard conditions thus leading to misinterpretation as absent in the interactome of this particular bait. The analysis of the Interaction Network indicates that Dengue virus interactome mediated by the domain III of the envelope protein is rather conserved in the viral complex suggesting a key role of these interactions for viral infection thus making candidates to explore for potential biomarkers of clinical outcome in DENV-caused disease. Interestingly, some particular IPs exhibit significant differences in the strength of the interaction with the viral serotypes representing interactions that involve more variable regions in the surface of the domain III. Since such variable regions are the consequence of the interaction with antibodies generated by human immune response; this result relates the interaction with proteins from human plasma with the interplay of the virus and the human immune system.

Variations on a theme in fruit development: the PLE lineage of MADS-box genes in tomato (TAGL1) and other species.

This article focuses on the role of TOMATO AGAMOUS-LIKE 1 (TAGL1) on a wide range of ripening functions in tomato. We also examine orthologs of this gene in related species that produce different fruit types and discuss some evolutionary implications. TOMATO AGAMOUS-LIKE 1 (TAGL1) is a MADS-box transcription factor gene that belongs to the PLENA (PLE) lineage within the AGAMOUS (AG) clade. The most well-studied genes in this lineage are the SHATTERPROOF (SHP) genes in Arabidopsis, known to be involved in dehiscence zone formation during silique development. In tomato, TAGL1 has been shown to control several aspects of tomato fruit ripening. Most notably, carotenoid synthesis seems to be controlled by TAGL1, likely via the ethylene synthesis and signaling pathway and in combination with RIPENING INHIBITOR (RIN). In addition, TAGL1 regulates genes involved in cell cycle regulation, flavonoid and lignin biosynthesis, and cuticle development. We discuss many of the genes in these different pathways that are likely controlled by TAGL1, directly or indirectly. We also examine the relationship of TAGL1 with known and putative interaction partners. PLE lineage genes have also been examined in other species such as Antirrhinum, Petunia, and Nicotiana and provide an interesting example of conservation and diversification of function in species that produce very different types of fleshy and dry fruits. The control of lignification may be a common mechanism for this group of genes. Lastly, we discuss future work needed to elucidate the TAGL1 regulatory pathway in tomato and to help better understand the functional diversification of genes in this lineage in related species.

Novel Immunity Proteins Associated with Colicin M-like Bacteriocins Exhibit Promiscuous Protection in Pseudomonas.

Bacteriocins related to colicin M, acting via cleavage of the cell wall precursor lipid II, have been characterized in γ- and β-proteobacteria. Depending on the species, immunity is provided by either an inner membrane-anchored periplasmic protein or by an integral membrane protein. In Pseudomonas however, the immunity partner of colicin M-like bacteriocins remains unknown. Based on an in silico analysis in pseudomonad genomes, we here identify a gene encoding a putative immunity partner that represents a novel type of integral membrane protein (PmiA, Pseudomonas colicin M-like immunity type A). By heterologous expression of pmiA genes in susceptible strains, we show that immunity to colicin M-like bacteriocins is indeed provided by the cognate PmiA. Sequence homology among PmiA proteins is essentially absent, except for a short motif with a conserved periplasm-exposed aspartate residue. However, PmiA's protective function is not abolished by changing this acidic residue to the uncharged alanine. Immunity by PmiAs appears promiscuous to the extent that PmiA homologs from a clade sharing <40% pairwise amino acid identity, equally provide protection against the bacteriocin linked to the original PmiA. This study shows that multiple immunity factors have evolved independently to silence lipid II-targeting enzymatic bacteriocins. Their relaxed bacteriocin immunization capacity contrasts to the strict specificity of immunity proteins shielding the enzymatic domain of nuclease bacteriocins. The nature of associated immune functions needs consideration when using such natural protein antibiotics or designing novel variants.

Androgen Stimulates Growth of Mouse Preantral Follicles In Vitro: Interaction With Follicle-Stimulating Hormone and With Growth Factors of the TGFβ Superfamily

Androgens are essential for the normal function of mature antral follicles but also have a role in the early stages of follicle development. Polycystic ovary syndrome (PCOS), the most common cause of anovulatory infertility, is characterized by androgen excess and aberrant follicle development that includes accelerated early follicle growth. We have examined the effects of testosterone and dihydrotestosterone (DHT) on development of isolated mouse preantral follicles in culture with the specific aim of investigating interaction with follicle-stimulating hormone (FSH), the steroidogenic pathway, and growth factors of the TGFβ superfamily that are known to have a role in early follicle development. Both testosterone and DHT stimulated follicle growth and augmented FSH-induced growth and increased the incidence of antrum formation among the granulosa cell layers of these preantral follicles after 72 hours in culture. Effects of both androgens were reversed by the androgen receptor antagonist flutamide. FSH receptor expression was increased in response to both testosterone and DHT, as was that of Star, whereas Cyp11a1 was down-regulated. The key androgen-induced changes in the TGFβ signaling pathway were down-regulation of Amh, Bmp15, and their receptors. Inhibition of Alk6 (Bmpr1b), a putative partner for Amhr2 and Bmpr2, by dorsomorphin resulted in augmentation of androgen-stimulated growth and modification of androgen-induced gene expression. Our findings point to varied effects of androgen on preantral follicle growth and function, including interaction with FSH-activated growth and steroidogenesis, and, importantly, implicate the intrafollicular TGFβ system as a key mediator of androgen action. These findings provide insight into abnormal early follicle development in PCOS.

Dendritic Arborization Patterns of Small Juxtaglomerular Cell Subtypes within the Rodent Olfactory Bulb.

Within the glomerular layer of the rodent olfactory bulb, numerous subtypes of local interneurons contribute to early processing of incoming sensory information. Here we have investigated dopaminergic and other small local juxtaglomerular cells in rats and mice and characterized their dendritic arborization pattern with respect to individual glomeruli by fluorescent labeling via patching and reconstruction of dendrites and glomerular contours from two-photon imaging data. Dopaminergic neurons were identified in a transgenic mouse line where the expression of dopamine transporter (DAT) was labeled with GFP. Among the DAT+ cells we found a small short-axon cell (SAC) subtype featuring hitherto undescribed dendritic specializations. These densely ramifying structures clasped mostly around somata of other juxtaglomerular neurons, which were also small, non-dopaminergic and to a large extent non-GABAergic. Clasping SACs were observed also in wild-type mice and juvenile rats. In DAT+ SAC dendrites, single backpropagating action potentials evoked robust calcium entry throughout both clasping and non-clasping compartments. Besides clasping SACs, most other small neurons either corresponded to the classical periglomerular cell type (PGCs), which was never DAT+, or were undersized cells with a small dendritic tree and low excitability. Aside from the presence of clasps in SAC dendrites, many descriptors of dendritic morphology such as the number of dendrites and the extent of branching were not significantly different between clasping SACs and PGCs. However, a detailed morphometric analysis in relation to glomerular contours revealed that the dendrites of clasping SACs arborized mostly in the juxtaglomerular space and never entered more than one glomerulus (if at all), whereas most PGC dendrites were restricted to their parent glomerulus, similar to the apical tufts of mitral cells. These complementary arborization patterns might underlie a highly complementary functional connectivity. The morphometric approach may serve to differentiate also other subtypes of juxtaglomerular neurons, help to identify putative synaptic partners and thus to establish a more refined picture of glomerular network interactions during odor sensing.

People Are More Generous to a Partner Who Pays Attention to Them.

People use relatively low-cost signals to maintain close relationships, in which they engage in costlier exchanges of tangible support. Paying attention to a partner allows an individual to communicate his or her interest in the relationship with the partner. Previous studies have revealed that when Person A pays attention to Person B, B's feeling of intimacy toward A increases. If social attention strengthens the bond between A and B, it is predicted that A's attention will also increase B's generous behavior toward A. This study tested this prediction. Participants first engaged in a collaborative task using computers. In the task, the putative partner (a computer program) either paid or did not pay attention to participants (high attention condition vs. low attention condition). In the control condition, the partner could not choose when to pay attention to participants. They then played three rounds of the dictator game with the partner. Confirming the previous finding, perceived intimacy was highest in the high attention condition, in the middle in the control condition, and lowest in the low attention condition. More importantly, participants in the high attention condition decided to give more resources to their partner than those in the low attention condition (but the difference between the high attention condition and the control condition was not significant). In addition, self-reported intimacy was positively correlated with the resource allocated to the partner. The results of this study demonstrated that social attention fosters a partner's generosity.