Attention-altering signal interactions in the multimodal courtship display of the wolf spider Schizocosa uetzi

Complex signals are common throughout the animal kingdom, consistingof one or more signals in one or more sensory modalities presentedwithin a single display. I tested an efficacy-based backup hypothesisof complex signal function using the bimodal courtship signalingwolf spider Schizocosa uetzi. This hypothesis predicts thatthe visual and vibratory courtship displays function as backupsto each other in the presence of environmental variability.I compared mating frequencies across four environmental treatmentsin which the visual and vibratory environments were manipulatedindependently in a 2 x 2 design with visual treatments of light/dark(i.e., visual signal present/absent) and vibratory treatmentsof filter paper substratum/granite substratum (i.e., vibratorysignal present/absent). Results did not match the predictionsof an efficacy-based backup hypothesis. The vibratory environmentaffected mating frequency, with more mating occurring in thevibration-present treatments compared to the vibration-absenttreatments, but the visual environment had no effect on matingfrequency. A second experiment was then conducted to test foran inter-signal interaction. Using the video-playback technique,I presented females with manipulated video sequences simultaneouswith a controlled vibratory signal to test the hypothesis thatthe presence of a vibratory signal alters a female's responseto the visual signal. In the presence of a vibratory courtshipsignal, females were more receptive to more visually ornamentedmales. This increased receptivity to increased visual ornamentationwas not seen in a previous study conducted on S. uetzi in theabsence of a vibratory signal, suggesting a potential inter-signalinteraction. In a third experiment, I tested whether a female'svisual attention was altered by the vibratory signal by examiningfemale response to a visual "predator" while exposed to allpossible combinations of male courtship signals. Females weremore likely to get caught, and thus less likely to notice apredatory visual stimulus when exposed to a courtship vibration,supporting the hypothesis that the vibratory signal alters afemale's visual attention.     

β-arrestin functionally regulates the non-bleaching pigment parapinopsin in lamprey pineal

The light response of vertebrate visual cells is achieved by light-sensing proteins such as opsin-based pigments as well as signal transduction proteins, including visual arrestin. Previous studies have indicated that the pineal pigment parapinopsin has evolutionally and physiologically important characteristics. Parapinopsin is phylogenetically related to vertebrate visual pigments. However, unlike the photoproduct of the visual pigment rhodopsin, which is unstable, dissociating from its chromophore and bleaching, the parapinopsin photoproduct is stable and does not release its chromophore. Here, we investigated arrestin, which regulates parapinopsin signaling, in the lamprey pineal organ, where parapinopsin and rhodopsin are localized to distinct photoreceptor cells. We found that beta-arrestin, which binds to stimulated G protein-coupled receptors (GPCRs) other than opsin-based pigments, was localized to parapinopsin-containing cells. This result stands in contrast to the localization of visual arrestin in rhodopsin-containing cells. Beta-arrestin bound to cultured cell membranes containing parapinopsin light-dependently and translocated to the outer segments of pineal parapinopsin-containing cells, suggesting that beta-arrestin binds to parapinopsin to arrest parapinopsin signaling. Interestingly, beta-arrestin colocalized with parapinopsin in the granules of the parapinopsin-expressing cell bodies under light illumination. Because beta-arrestin, which is a mediator of clathrin-mediated GPCR internalization, also served as a mediator of parapinopsin internalization in cultured cells, these results suggest that the granules were generated light-dependently by beta-arrestin-mediated internalization of parapinopsins from the outer segments. Therefore, our findings imply that beta-arrestin-mediated internalization is responsible for eliminating the stable photoproduct and restoring cell conditions to the original dark state. Taken together with a previous finding that the bleaching pigment evolved from a non-bleaching pigment, vertebrate visual arrestin may have evolved from a "beta-like" arrestin by losing its clathrin-binding domain and its function as an internalization mediator. Such changes would have followed the evolution of vertebrate visual pigments, which generate unstable photoproducts that independently decay by chromophore dissociation.     

Intracellular neuronal calcium sensor proteins: a family of EF-hand calcium-binding proteins in search of a function

Intracellular neuronal calcium sensors (NCS) constitute a rapidly growing family of calcium-binding proteins which belong to the superfamily of EF-hand proteins. The NCS family includes as subgroups the recoverins and GCAPs (guanylyl cyclase-activating proteins), which are primarily expressed in retinal photoreceptor cells, and the frequenins and VILIPs (visinin-like proteins), which are widely but differentially expressed in the nervous system. In this review the recent developments in elucidating the functional activities of NCS proteins on signal transduction pathways in neurons are surveyed and discussed. We will focus our attention on calcium-dependent membrane association by the so-called calcium-myristoyl switch as a possible mechanism of signal transduction and on the roles of NCS proteins in intraneuronal signaling cascades, which are best studied in the visual and olfactory systems.     

Visual and both non-visual arrestins in their "inactive" conformation bind JNK3 and Mdm2 and relocalize them from the nucleus to the cytoplasm

Arrestins bind active phosphorylated G protein-coupled receptors, terminating G protein activation. Receptor-bound non-visual arrestins interact with numerous partners, redirecting signaling to alternative pathways. Arrestins also have nuclear localization and nuclear exclusion signals and shuttle between the nucleus and the cytoplasm. Constitutively shuttling proteins often redistribute their interaction partners between the two compartments. Here we took advantage of the nucleoplasmic shuttling of free arrestins and used a "nuclear exclusion assay" to study their interactions with two proteins involved in "life-and-death" decisions in the cell, the kinase JNK3 and the ubiquitin ligase Mdm2. In human embryonic kidney 293 cells green fluorescent protein (GFP)-JNK3 and GFP-Mdm2 predominantly localize in the nucleus, whereas visual arrestin, arrestin2(Q394L) mutant equipped with the nuclear exclusion signal, and arrestin3 localize exclusively to the cytoplasm. Coexpression of arrestins moves both GFP-JNK3 and GFP-Mdm2 to the cytoplasm. Arrestin mutants "frozen" in the basal conformation are the most efficacious. Thus, arrestins in their basal state interact with JNK3 and Mdm2, suggesting that arrestins are likely "preloaded" with their interaction partners when they bind the receptor. Robust interaction of free arrestins with JNK3 and Mdm2 and their ability to regulate subcellular localization of these proteins may play an important role in the survival of photoreceptors and other neurons, as well as in retinal and neuronal degeneration.     

Non-classical protein secretion in bacteria

Background  

We present an overview of bacterial non-classical secretion and a prediction method for identification of proteins following signal peptide independent secretion pathways. We have compiled a list of proteins found extracellularly despite the absence of a signal peptide. Some of these proteins also have known roles in the cytoplasm, which means they could be so-called "moon-lightning" proteins having more than one function.     

Identification of a Post-targeting Step Required for Efficient Cotranslational Translocation of Proteins across the Escherichia coli Inner Membrane

Recent studies have shown that cytoplasmic proteins are exported efficiently in Escherichia coli only if they are attached to signal peptides that are recognized by the signal recognition particle and are thereby targeted to the SecYEG complex cotranslationally. The evidence suggests that the entry of these proteins into the secretory pathway at an early stage of translation is necessary to prevent them from folding into a translocation-incompetent conformation. We found, however, that several glycolytic enzymes attached to signal peptides that are recognized by the signal recognition particle were exported inefficiently. Based on previous studies of post-translational export, we hypothesized that the export block was due to the presence of basic residues at the extreme N terminus of each enzyme. Consistent with our hypothesis, we found that the introduction of negatively charged residues into this segment increased the efficiency of export. Export efficiency was sensitive to the number, position, and sequence context of charged residues. The importance of charge for efficient export was underscored by an in silico analysis that revealed a conserved negative charge bias at the N terminus of the mature region of bacterial presecretory proteins. Our results demonstrate that cotranslational targeting of a protein to the E. coli SecYEG complex does not ensure its export but that export also depends on a subsequent event (most likely the initiation of translocation) that involves sequences both within and just beyond the signal peptide.Since the “signal hypothesis” was proposed over 30 years ago (1), it has become clear that signal sequences are not simply generic hydrophobic peptides that earmark proteins for secretion. In bacteria, the features of a signal peptide determine the mechanism by which a given presecretory protein is targeted to the SecYEG translocation complex in the inner membrane (IM).² Whereas most or all signal peptides are recognized by the signal recognition particle (SRP) in mammalian cells, only a small fraction of Escherichia coli signal peptides are recognized by SRP. These signal peptides are typically extremely hydrophobic (2, 3), but SRP apparently can also recognize slightly less hydrophobic signal peptides that contain a highly basic N terminus (4). SRP recognizes signal peptides as they emerge from translating ribosomes and then targets ribosome-nascent chain complexes to the IM cotranslationally (5). The binding of SRP to its receptor (FtsY), which interacts with the SecYEG complex (6), leads to the release of the nascent chain in the immediate vicinity of the translocation machinery. By targeting nascent polypeptides to the SecYEG complex at an early stage of translation, SRP prevents its substrates from folding into a conformation that is incompatible with translocation through the narrow channel formed by the SecYEG complex (7). Because most signal peptides are not recognized by E. coli SRP, the majority of presecretory proteins are fully synthesized and targeted post-translationally to the IM. These proteins are maintained in a translocation-competent conformation by molecular chaperones such as SecB that keep them unfolded (or loosely folded) (8). Signal peptides themselves also appear to play a role in maintaining translocation competence (9, 10). After mediating the targeting reaction, signal peptides likely play a role in gating open the SecYEG complex to initiate translocation.Interestingly, although signal sequences are the most salient feature of presecretory proteins, they are neither completely necessary nor sufficient to mediate protein export in E. coli (11–13). A version of alkaline phosphatase that lacks a signal peptide is still exported, albeit very inefficiently (11). The export of the leaderless protein, unlike the export of wild-type alkaline phosphatase, is strictly dependent on SecB (11). Conversely, the attachment of signal peptides to cytoplasmic proteins often does not promote their export (14). In light of evidence that folding and export are competing events, these observations led to the proposal that exported proteins tend to fold slowly (or are prevented from folding by chaperones) and therefore remain translocation-competent even without a signal peptide, whereas cytoplasmic proteins fold rapidly into a conformation that is incompatible with export. Recent studies that used thioredoxin as a model protein have validated this hypothesis. Whereas the wild-type protein attached to a typical signal peptide remained trapped in the cytoplasm, four of five slow folding mutants were exported efficiently (15). Furthermore, attachment of a signal peptide that is recognized by SRP to thioredoxin led to efficient export (16). This idea was further confirmed by a report in which various DARPins (designed ankyrin Repeat proteins) were attached to different signal peptides. Most of the DARPins were exported efficiently when they were fused to signal peptides that mediate cotranslational targeting but remained in the cytoplasm when they were attached to signal peptides that are bypassed by SRP (17).Despite these observations, there are several lines of evidence suggesting that export efficiency is not simply dictated by the ability of a protein to reach the SecYEG complex before folding into a translocation-incompetent conformation. For reasons that are unclear, some DARPins are secreted inefficiently even when they are routed into the SRP pathway (17). In addition, numerous reports have indicated that the amino acid composition of the segment of post-translationally targeted presecretory proteins that lies just beyond the signal peptide cleavage site has a dramatic effect on export efficiency. Statistical analysis has shown that the first ∼5–15 residues of the mature region of most presecretory proteins produced by Gram-negative bacteria is neutral or has a net negative charge (18). Consistent with the observed sequence bias, the presence of multiple basic residues at the N terminus of the mature region often leads to accumulation of the secretory precursor, whereas conversion of the basic residues to acidic residues restores export (19–22). Because different combinations of proteins and signal peptides were used in these studies, the exact number and location of charged residues that impinge on the efficiency of export is unclear. In any case, the effect of the net charge in the region distal to the signal peptide on protein export has never been explained. Although basic residues might conceivably promote premature folding of presecretory proteins or block the cleavage of signal peptides by leader peptidase, it is also possible that they inhibit an uncharacterized post-targeting event. Even if effects on signal peptide cleavage could have been ruled out in the aforementioned studies, however, it would not have been possible to distinguish between effects on protein folding and effects on a hypothetical post-targeting step because only proteins that are targeted post-translationally were monitored.To gain further insight into the factors that govern the efficiency of protein export, we sought an explanation for the observation that the cotranslational targeting of at least some cytoplasmic proteins is insufficient to guarantee their translocation across the IM. We found that the export of several different endogenous E. coli cytoplasmic proteins required not only the attachment of a signal peptide that is recognized by SRP but also a net negative charge just past the signal peptide cleavage site. Taken together with previous results, our data show that the charge of the segment just beyond the signal peptide influences export efficiency irrespective of the mechanism by which a protein is targeted to the IM. Because proteins that are targeted cotranslationally reach the IM before they have a chance to fold, our results imply the existence of a post-targeting step (most likely the initiation of translocation) that is facilitated by acidic residues distal to the signal peptide and inhibited or delayed by basic residues. These results help to resolve a long-standing puzzle about the influence of the mature region of presecretory proteins on protein export and have significant implications for optimizing the export of cytosolic and heterologous proteins in E. coli.     

Net N-C charge imbalance may be important for signal sequence function in bacteria

The net charge distribution in a region around the signal sequence cleavage site has been calculated for samples of 41 prokaryotic and 165 eukaryotic exported proteins. The results show that prokaryotic proteins in particular have a markedly higher incidence of acidic than of basic residues in this region. The possibility that a "dipolar" structure with a positive net charge difference between the N and C-terminal regions is important for signal sequence function in bacteria is suggested, and invoked to rationalize a number of known export-defective signal sequence mutations.     

Protein targeting and integration signal for the chloroplastic outer envelope membrane.

Most proteins in chloroplasts are encoded by the nuclear genome and synthesized in the cytosol. With the exception of most quter envelope membrane proteins, nuclear-encoded chloroplastic proteins are synthesized with N-terminal extensions that contain the chloroplast targeting information of these proteins. Most outer membrane proteins, however, are synthesized without extensions in the cytosol. Therefore, it is not clear where the chloroplastic outer membrane targeting information resides within these polypeptides. We have analyzed a chloroplastic outer membrane protein, OEP14 (outer envelope membrane protein of 14 kD, previously named OM14), and localized its outer membrane targeting and integration signal to the first 30 amino acids of the protein. This signal consists of a positively charged N-terminal portion followed by a hydrophobic core, bearing resemblance to the signal peptides of proteins targeted to the endoplasmic reticulum. However, a chimeric protein containing this signal fused to a passenger protein did not integrate into the endoplasmic reticulum membrane. Furthermore, membrane topology analysis indicated that the signal inserts into the chloroplastic outer membrane in an orientation opposite to that predicted by the "positive inside" rule.     

Proteomic inventory of "anchorless" proteins on the colon adenocarcinoma cell surface

Surface proteins play important pathophysiological roles in health and disease, and accumulating proteomics-based studies suggest that several "non-membrane" proteins are sorted to the cell surface by unconventional mechanisms. Importantly, these proteins may comprise attractive therapeutic targets and novel disease markers for colon cancer. To perform a proteomics-based inventory of these so-called "anchorless" surface proteins, intact colon adenocarcinoma SW480 cells were labeled with membrane-impermeable biotin after which only soluble biotinylated proteins were isolated and identified by nanoLC-MS/MS. Computer-assisted analysis predicted that only 9 of the 97 identified surface-exposed proteins have predicted secretory signal peptides, whereas 2 other proteins have a putative transmembrane segment. Of the 9 proteins with putative signal peptides, 1 was predicted to be retained at the cell surface by a GPI-anchor, whereas 5 other proteins contained an ER-retention motif (KDEL) that should prevent them from being sorted to the cell surface. The remaining 86 soluble "surface" proteins lack known export signals and the possibility that these proteins are candidate substrates of non-classical transporters or exported by unconventional mechanisms is discussed. Alternatively, the large number of "intracellular" and ER-resident proteins may imply that biotinylation approaches are not only specific for surface proteins, but also biased against a certain subset of non-surface proteins. This underscores the importance of post-proteomic verification of proteomics-based inventories on surface-exposed proteins, which eventually should reveal to which extent non-classical export and retention mechanisms contribute to the sorting of "anchorless" proteins to the surface of colon tumor cells.     

Bipolar cells use kainate and AMPA receptors to filter visual information into separate channels

Unlike cone photoreceptors, whose light responses have a uniform time course, retinal ganglion cells are tuned to respond to different temporal components in a changing visual scene. The signals in a mammalian cone flow to three to five morphologically distinct "OFF" bipolar cells at a sign-conserving, glutamatergic synapse. By recording simultaneously from pairs of synaptically connected cones and OFF bipolar cells, I now show that each morphological type of OFF bipolar cell receives its signal through a different AMPA or kainate receptor. The characteristic rate at which each receptor recovers from desensitization divides the cone signal into temporal components. Temporal processing begins at the first synapse in the visual system.     

Linking kinetochore-microtubule binding to the spindle checkpoint

The spindle checkpoint blocks cell-cycle progression until chromosomes are properly attached to the mitotic spindle. Popular models propose that checkpoint proteins associate with kinetochores to produce a "wait anaphase" signal that inhibits anaphase. Recent data suggest that a two-state switch results from using the same kinetochore proteins to bind microtubules and checkpoint proteins. At least eight protein kinases are implicated in spindle checkpoint signaling, arguing that a traditional signal transduction cascade is integral to spindle checkpoint signaling.     

Cue-Recruitment for Extrinsic Signals after Training with Low Information Stimuli

Cue-recruitment occurs when a previously ineffective signal comes to affect the perceptual appearance of a target object, in a manner similar to the trusted cues with which the signal was put into correlation during training [1], [2]. Jain, Fuller and Backus [3] reported that extrinsic signals, those not carried by the target object itself, were not recruited even after extensive training. However, recent studies have shown that training using weakened trusted cues can facilitate recruitment of intrinsic signals [4]–[7]. The current study was designed to examine whether extrinsic signals can be recruited by putting them in correlation with weakened trusted cues. Specifically, we tested whether an extrinsic visual signal, the rotary motion direction of an annulus of random dots, and an extrinsic auditory signal, direction of an auditory pitch glide, can be recruited as cues for the rotation direction of a Necker cube. We found learning, albeit weak, for visual but not for auditory signals. These results extend the generality of the cue-recruitment phenomenon to an extrinsic signal and provide further evidence that the visual system learns to use new signals most quickly when other, long-trusted cues are unavailable or unreliable.     

Structure and mechanisms of function of visual system proteins

Recent progress in understanding visual signal transduction in retinal cells is summarized. The roles of particular proteins in activation, amplification and termination of the photoresponse are described. Detailed information on the structure and function of the photoreceptor protein rhodopsin is presented. The latest data on visual pigment sequences, rhodopsin mutations in the autosomal-dominant retinitis pigmentosa, and the results of site-directed mutagenesis of the rhodopsin molecule are summarized.     

Isolation and immunological properties of adenosine kinase

Bovine liver adenosine kinase is a 43 kDa protein that catalyzes the transfer of phosphate from GTP or ATP to adenosine. Its immunological properties were compared to other GTP-binding proteins of approximately 40 kDa, in particular those involved in signal transduction, such as Gs and Gi, the stimulatory and inhibitory regulatory proteins of adenylyl cyclase, Gt, from the visual excitation system, and Go, a similar protein of unknown function. Antibodies elicited in rabbits against adenosine kinase did not significantly cross-react with other guanyl nucleotide-binding proteins. Antibodies against the other GTP-binding proteins did not react with adenosine kinase. Thus these GTP-binding proteins do not exhibit immunological cross-reactivity.     

Use of synthetic signal sequences to explore the protein export machinery

The information for correct localization of newly synthesized proteins in both prokaryotes and eukaryotes resides in self-contained, often transportable targeting sequences. Of these, signal sequences specify that a protein should be secreted from a cell or incorporated into the cytoplasmic membrane. A central puzzle is presented by the lack of primary structural homology among signal sequences, although they share common features in their sequences. Synthetic signal peptides have enabled a wide range of studies of how these "zipcodes" for protein secretion are decoded and used to target proteins to the protein machinery that facilitates their translocation across and integration into membranes. We review research on how the information in signal sequences enables their passenger proteins to be correctly and efficiently localized. Synthetic signal peptides have made possible binding and crosslinking studies to explore how selectivity is achieved in recognition by the signal sequence-binding receptors, signal recognition particle, or SRP, which functions in all organisms, and SecA, which functions in prokaryotes and some organelles of prokaryotic origins. While progress has been made, the absence of atomic resolution structures for complexes of signal peptides and their receptors has definitely left many questions to be answered in the future.     

Prediction of protein signal sequences and their cleavage sites

Protein signal sequences play a central role in the targeting and translocation of nearly all secreted proteins and many integral membrane proteins in both prokaryotes and eukaryotes. The knowledge of signal sequences has become a crucial tool for pharmaceutical scientists who genetically modify bacteria, plants, and animals to produce effective drugs. However, to effectively use such a tool, the first important thing is to find a fast and effective method to identify the "zipcode" entity; this is also evoked by both the huge amount of unprocessed data available and the industrial need to find more effective vehicles for the production of proteins in recombinant systems. In view of this, a sequence-encoded algorithm was developed to identify the signal sequences and predict their cleavage sites. The rate of correct prediction for 1,939 secretory proteins and 1,440 nonsecretory proteins by self-consistency test is 90.14% and that by jackknife test is 90.13%. The encouraging results indicate that the signal sequences share some common features although they lack similarity in sequence, length, and even composition and that they are predictable to a considerably accurate extent.     

Calcium-binding proteins: intracellular sensors from the calmodulin superfamily.

In all eukaryotic cells, and particularly in neurons, Ca(2+) ions are important second messengers in a variety of cellular signaling pathways. In the retina, Ca(2+) modulation plays a crucial function in the development of the visual system's neuronal connectivity and a regulatory role in the conversion of the light signal received by photoreceptors into an electrical signal transmitted to the brain. Therefore, the study of retinal Ca(2+)-binding proteins, which frequently mediate Ca(2+) signaling, has given rise to the important discovery of two subfamilies of these proteins, neuronal Ca(2+)-binding proteins (NCBPs) and calcium-binding proteins (CaBPs), that display similarities to calmodulin (CaM). These and other Ca(2+)-binding proteins are integral components of cellular events controlled by Ca(2+). Some members of these subfamilies also play a vital role in signal transduction outside of the retina. The expansion of the CaM-like protein family reveals diversification among Ca(2+)-binding proteins that evolved on the basis of the classic molecule, CaM. A large number of NCBP and CaBP subfamily members would benefit from their potentially specialized role in Ca(2+)-dependent cellular processes. Pinpointing the role of these proteins will be a challenging task for further research.     

Effect of sodium nitrite on the activity of the neocortex neurons during realization of defense and inhibition conditioned reflexes

A decrease in intensity and duration of short-latency reaction components of the sensorimotor and visual cortical neurons to specific stimuli (pain reinforcement and light flashes, respectively) was observed after the administration of NO-generating sodium nitrite (11 mg/kg, subcutaneously). Activation decrease in the visual cortex took place irrespective of biological significance of the light flashes, i.e., in case when this stimulus was a signal of defensive conditioning and in case when these flashes were applied with continuous light (a conditioned inhibitor). Sodium nitrite almost did not change the late activation of sensorimotor and visual neurons in response to pain reinforcement and disinhibitory action of the latter. The results confirm the viewpoint about different neurotransmitters in "specifically modal" and "non-specific" pathways to the neocortex during learning.