Reproduction-Related Sound Production of Grasshoppers Regulated by Internal State and Actual Sensory Environment

The interplay of neural and hormonal mechanisms activated by entero- and extero-receptors biases the selection of actions by decision making neuronal circuits. The reproductive behavior of acoustically communicating grasshoppers, which is regulated by short-term neural and longer-term hormonal mechanisms, has frequently been used to study the cellular and physiological processes that select particular actions from the species-specific repertoire of behaviors. Various grasshoppers communicate with species- and situation-specific songs in order to attract and court mating partners, to signal reproductive readiness, or to fend off competitors. Selection and coordination of type, intensity, and timing of sound signals is mediated by the central complex, a highly structured brain neuropil known to integrate multimodal pre-processed sensory information by a large number of chemical messengers. In addition, reproductive activity including sound production critically depends on maturation, previous mating experience, and oviposition cycles. In this regard, juvenile hormone released from the corpora allata has been identified as a decisive hormonal signal necessary to establish reproductive motivation in grasshopper females. Both regulatory systems, the central complex mediating short-term regulation and the corpora allata mediating longer-term regulation of reproduction-related sound production mutually influence each other’s activity in order to generate a coherent state of excitation that promotes or suppresses reproductive behavior in respective appropriate or inappropriate situations. This review summarizes our current knowledge about extrinsic and intrinsic factors that influence grasshopper reproductive motivation, their representation in the nervous system and their integrative processing that mediates the initiation or suppression of reproductive behaviors

Modulatory effects of nitric oxide and juvenile hormone on the control of reproductive behavior in female Chorthippus biguttulus

Sowohl Juvenilhormon (JH) als auch Stickstoffmonoxid (NO) beeinflussen das reproduktive Verhalten weiblicher Heuschrecken. Die vorliegende Arbeit untersucht eine mögliche Verbindung zwischen JH und NO Signalen bei der Kontrolle des weiblichen Paarungsverhaltens in der Heuschrecke Chorthippus biguttulus. Die Effekte von JH auf die Lauterzeugung und das Paarungsverhalten von Heuschreckenweibchen wurden nach der Manipulation des JH Levels analysiert. Durch neuroanatomische Studien konnte eine funktionelle Beziehung zwischen JH und NO auf zellulärer Ebene gefunden werden. Die Beobachtung des natürlichen Reproduktionsverhaltens von Ch. biguttulus Weibchen in kleinen Laborpopulationen lieferte ein verlässliches Zeitschema für den Ablauf reproduktiver Stadien. Auf die Imaginalhäutung folgt eine Periode der primären Abwehr , die nach ca. einer Woche direkt in die Periode der aktiven Paarungsbereitschaft übergeht, welche mit weiblicher Lautproduktion einhergeht, die für Männchen ein sehr attraktives Kommunikationssignal darstellt. Weibchen paaren sich mehrere Male bevor sie zur ersten Eiablage kommen. Die experimentelle Manipulation des JH Titers der Hemolymphe zeigte, dass JH benötigt wird, um Reproduktionsverhalten zu initiieren und zu erhalten, jedoch unterdrücken hohe JH Titer die weibliche Lautproduktion. Die Inhibition der NO Produktion durch systemische Applikation eines Stickstoffmonoxidsynthase (NOS) Inhibitors steigerte die weibliche Lautproduktion während sie den JH Titer der Hemolymphe absenkte. In den JH produzierenden Corpora allata wurden NOS exprimierende Zellkörper durch NADPHdiaphorase Färbung, anti NOS Immunfärbung und Immunzytochemie gegen Citrullin, ein Nebenprodukt der NO Synthese, gefunden. RFamid immunreaktive Hirn-zu-Corpora allata Projektionen aus der Pars intercerebralis und lateralis wurden als zelluläre Zielorte der NO-Abgabe in den Corpora allata identifiziert. Die Beteiligung von NO/cGMP Signalen an der Regulation der Corpora allata Funktion, NO Produktion und die Kontrolle der Hormonausschüttung durch RFamid stellt eine Parallele zur Funktion der Adenohypophyse der Vertebraten dar, wo NO unter anderem in gonadotropen Zellen produziert wird und RFamid die Ausschüttung von Gonadotropinen kontrolliert

Ensemble of conformations in non-native states of proteins by liquid NMR spectroscopy

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2005.Vita.Includes bibliographical references (p. 187-202).For a complete description of protein folding dynamics and the structure of the folded state, of unfolded and of non-native states of proteins and the kinetics of protein folding from the unfolded state to the folded state have to be determined. The focus of this PhD thesis was the development of novel NMR methodologies to study protein folding using NMR spectroscopy. This has been achieved by studying three model proteins ubiquitin, [alpha]-lactalbumin and lysozyme in their folded and especially in their unfolded states. The proteins were chosen, because [alpha]-lactalbumin and lysozyme are two proteins with nearly identical fold but different function and ubiquitin is a very stable protein without disulfide bonds. Methodologies A new NMR pulse sequence for the determination of ... and ... coupling constants in proteins was developped. The method is based on J-modulated HSQCs and can be applied to folded as well as unfolded proteins. The new coupling constants report on backbone [phi] and [psi] angles. . Residual structure and long-range interactions in unfolded proteins can be detected by a new combination of site directed non-conservative mutagenesis and NMR analysis. Identification of long-range interactions is a) based on the analysis and interpretation of R₂ relaxation rates, for which models have been derived and b) based on NMR diffusion data which directly correlate to the compactness of a given protein. A method to study laser triggered kinetics of protein folding by time-resolved photo- CIDNP NMR was developed. Two lasers were coupled into an NMR spectrometer:(cont.) one for initiation of folding by releasing ions from photo-labile chelators with dead times as low as 200ms, and one for induction of photo-CINDP NMR. The method can not only be applied to ion induced kinetics of protein folding, but is generally applicable to kinetics of biomacromolecules such as proteins and RNA that involve photo-protected cofactors as could be shown in our laboratory. Investigations on ubiquitin The newly developed NMR method for the determination of ... and ... coupling constants was applied to folded ubiquitin. Analysis revealed the dependence of the coupling constants on the backbone conformation, predominantly on [psi], making the pulse sequence and the parameterization developed here a valuable new tool for the determination of the backbone conformation in folded as well as in unfolded proteins. Unfolded ubiquitin was investigated using scalar coupling constants and ¹H,¹⁵N relaxation data. The experimental data agree well with models proposed to describe unfolded states of proteins as a statistical coil where dynamics are governed solely by segmental motions. Unfolded ubiquitin is thus a good model for a protein without detectable residual structure in its unfolded state. Investigations on lysozyme Residual structure in the unfolded states of non conservative single point mutants (A9G, W62G, W62Y, W 1 1G and W123G) of hen lysoszyme was monitored by chemical shift measurements, ¹⁵N transverse relaxation rates and particularly diffusion constants. Long- range interactions between hydrophobic clusters of unfolded lysozyme were observed. Single point mutations dramatically alter the overall compactness of the unfolded state.(cont.) Investigations on [alpha]-lactalbumin · Isotope labeled bovine [alpha]-lactalbumin (BLA) was expressed heterologously using a new construct with a His-tag and a trypsin cleavage site. The sequence and stability of the obtained BLA is identical to the wild type protein. This makes it a perfect construct to study kinetics of folding and unfolded states of BLA. ¹³C,¹⁵N isotope labeled unfolded BLA was assigned applying standard and non-standard NMR assignment experiments. Residual secondary structure was identified near the N- and the C-terminus of unfolded BLA, in regions belonging to the -domain in the folded state, suggesting a possible folding nucleus. · This unfolded state of BLA was furthermore compared to the unfolded state of human lactalbumin (HLA) and lysozyme based on residual structure and ¹⁵N relaxation data. The unfolded states vary considerably for the three proteins, which possess very similar structures in their native state. The structural ensemble in the unfolded states of proteins are determined by the primary sequence of the protein and even smallest single point mutations as found between HLA and BLA can change the conformation of the unfolded state considerably. · The Ca²⁺-triggered folding kinetics of BLA under constant denaturant (4M urea) has been investigated by laser induced release of Ca²⁺-ions from a photolabile chelator within the NMR spectrometer and subsequent photo-CIDNP signal detection. A folding intermediate possessing a tyrosine residue in a non-native conformation was detected 200ms after initiation of folding. Therefore, parts of the polypeptide chain in the [beta]-domain of BLA sample non-native conformations, while a hydrophobic core is formed.(cont.) The findings in the kinetic investigations are in line with the detected residual structure. Refolding of amino acids involved in non-native clusters in the intermediates has to proceed the correct folding and therefore constitutes a rate limiting step on the Ca²⁺-induced refolding of [alpha]- lactalbumin.by Julia Wirmer.Ph.D

Features of 80S mammalian ribosome and its subunits

It is generally believed that basic features of ribosomal functions are universally valid, but a systematic test still stands out for higher eukaryotic 80S ribosomes. Here we report: (i) differences in tRNA and mRNA binding capabilities of eukaryotic and bacterial ribosomes and their subunits. Eukaryotic 40S subunits bind mRNA exclusively in the presence of cognate tRNA, whereas bacterial 30S do bind mRNA already in the absence of tRNA. 80S ribosomes bind mRNA efficiently in the absence of tRNA. In contrast, bacterial 70S interact with mRNA more productively in the presence rather than in the absence of tRNA. (ii) States of initiation (Pi), pre-translocation (PRE) and post-translocation (POST) of the ribosome were checked and no significant functional differences to the prokaryotic counterpart were observed including the reciprocal linkage between A and E sites. (iii) Eukaryotic ribosomes bind tetracycline with an affinity 15 times lower than that of bacterial ribosomes (Kd 30 μM and 1–2 μM, respectively). The drug does not effect enzymatic A-site occupation of 80S ribosomes in contrast to non-enzymatic tRNA binding to the A-site. Both observations explain the relative resistance of eukaryotic ribosomes to this antibiotic

Influence of ground-state structure and Mg2+ binding on folding kinetics of the guanine-sensing riboswitch aptamer domain

Riboswitch RNAs fold into complex tertiary structures upon binding to their cognate ligand. Ligand recognition is accomplished by key residues in the binding pocket. In addition, it often crucially depends on the stability of peripheral structural elements. The ligand-bound complex of the guanine-sensing riboswitch from Bacillus subtilis, for example, is stabilized by extensive interactions between apical loop regions of the aptamer domain. Previously, we have shown that destabilization of this tertiary loop–loop interaction abrogates ligand binding of the G37A/C61U-mutant aptamer domain (Gswloop) in the absence of Mg2+. However, if Mg2+ is available, ligand-binding capability is restored by a population shift of the ground-state RNA ensemble toward RNA conformations with pre-formed loop–loop interactions. Here, we characterize the striking influence of long-range tertiary structure on RNA folding kinetics and on ligand-bound complex structure, both by X-ray crystallography and time-resolved NMR. The X-ray structure of the ligand-bound complex reveals that the global architecture is almost identical to the wild-type aptamer domain. The population of ligand-binding competent conformations in the ground-state ensemble of Gswloop is tunable through variation of the Mg2+ concentration. We quantitatively describe the influence of distinct Mg2+ concentrations on ligand-induced folding trajectories both by equilibrium and time-resolved NMR spectroscopy at single-residue resolution

Comprehensive determination of 3JHNHα for unfolded proteins using 13C′-resolved spin-echo difference spectroscopy

An experiment is presented to determine 3JHNHα coupling constants, with significant advantages for applications to unfolded proteins. The determination of coupling constants for the peptide chain using 1D 1H, or 2D and 3D 1H-15N correlation spectroscopy is often hampered by extensive resonance overlap when dealing with flexible, disordered proteins. In the experiment detailed here, the overlap problem is largely circumvented by recording 1H-13C′ correlation spectra, which demonstrate superior resolution for unfolded proteins. J-coupling constants are extracted from the peak intensities in a pair of 2D spin-echo difference experiments, affording rapid acquisition of the coupling data. In an application to the cytoplasmic domain of human neuroligin-3 (hNlg3cyt) data were obtained for 78 residues, compared to 54 coupling constants obtained from a 3D HNHA experiment. The coupling constants suggest that hNlg3cyt is intrinsically disordered, with little propensity for structure

Synaptic and peptidergic connectome of a neurosecretory centre in the annelid brain

This is the author accepted manuscript. The final version is available from eLife Sciences Publications via the DOI in this record.Neurosecretory centers in animal brains use peptidergic signaling to influence physiology and behavior. Understanding neurosecretory center function requires mapping cell types, synapses, and peptidergic networks. Here we use transmission electron microscopy and gene expression mapping to analyze the synaptic and peptidergic connectome of an entire neurosecretory center. We reconstructed 78 neurosecretory neurons and mapped their synaptic connectivity in the brain of larval Platynereis dumerilii, a marine annelid. These neurons form an anterior neurosecretory center expressing many neuropeptides, including hypothalamic peptide orthologs and their receptors. Analysis of peptide-receptor pairs in spatially mapped single-cell transcriptome data revealed sparsely connected networks linking specific neuronal subsets. We experimentally analyzed one peptide-receptor pair and found that a neuropeptide can couple neurosecretory and synaptic brain signaling. Our study uncovered extensive networks of peptidergic signaling within a neurosecretory center and its connection to the synaptic brain.The research leading to these results received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ European Research Council Grant Agreement 260821. The research was supported by a grant from the DFG - Deutsche Forschungsgemeinschaft (Reference no. JE 777/1)

Probing the urea dependence of residual structure in denatured human α-lactalbumin

Backbone 15N relaxation parameters and 15N–1HN residual dipolar couplings (RDCs) have been measured for a variant of human α-lactalbumin (α-LA) in 4, 6, 8 and 10 M urea. In the α-LA variant, the eight cysteine residues in the protein have been replaced by alanines (all-Ala α-LA). This protein is a partially folded molten globule at pH 2 and has been shown previously to unfold in a stepwise non-cooperative manner on the addition of urea. 15N R2 values in some regions of all-Ala α-LA show significant exchange broadening which is reduced as the urea concentration is increased. Experimental RDC data are compared with RDCs predicted from a statistical coil model and with bulkiness, average area buried upon folding and hydrophobicity profiles in order to identify regions of non-random structure. Residues in the regions corresponding to the B, D and C-terminal 310 helices in native α-LA show R2 values and RDC data consistent with some non-random structural propensities even at high urea concentrations. Indeed, for residues 101–106 the residual structure persists in 10 M urea and the RDC data suggest that this might include the formation of a turn-like structure. The data presented here allow a detailed characterization of the non-cooperative unfolding of all-Ala α-LA at higher concentrations of denaturant and complement previous studies which focused on structural features of the molten globule which is populated at lower concentrations of denaturant

Binding of aminoglycoside antibiotics to helix 69 of 23S rRNA

Aminoglycosides antibiotics negate dissociation and recycling of the bacterial ribosome’s subunits by binding to Helix 69 (H69) of 23S rRNA. The differential binding of various aminoglycosides to the chemically synthesized terminal domains of the Escherichia coli and human H69 has been characterized using spectroscopy, calorimetry and NMR. The unmodified E. coli H69 hairpin exhibited a significantly higher affinity for neomycin B and tobramycin than for paromomycin (Kds = 0.3 ± 0.1, 0.2 ± 0.2 and 5.4 ± 1.1 µM, respectively). The binding of streptomycin was too weak to assess. In contrast to the E. coli H69, the human 28S rRNA H69 had a considerable decrease in affinity for the antibiotics, an important validation of the bacterial target. The three conserved pseudouridine modifications (Ψ1911, Ψ1915, Ψ1917) occurring in the loop of the E. coli H69 affected the dissociation constant, but not the stoichiometry for the binding of paromomycin (Kd = 2.6 ± 0.1 µM). G1906 and G1921, observed by NMR spectrometry, figured predominantly in the aminoglycoside binding to H69. The higher affinity of the E. coli H69 for neomycin B and tobramycin, as compared to paromomycin and streptomycin, indicates differences in the efficacy of the aminoglycosides