Arrestins and spinophilin competitively regulate Na+,K+-ATPase trafficking through association with a large cytoplasmic loop of the Na+,K+-ATPase

The activity and trafficking of the Na(+),K(+)-ATPase are regulated by several hormones, including dopamine, vasopressin, and adrenergic hormones through the action of G-protein-coupled receptors (GPCRs). Arrestins, GPCR kinases (GRKs), 14-3-3 proteins, and spinophilin interact with GPCRs and modulate the duration and magnitude of receptor signaling. We have found that arrestin 2 and 3, GRK 2 and 3, 14-3-3 epsilon, and spinophilin directly associate with the Na(+),K(+)-ATPase and that the associations with arrestins, GRKs, or 14-3-3 epsilon are blocked in the presence of spinophilin. In COS cells that overexpressed arrestin, the Na(+),K(+)-ATPase was redistributed to intracellular compartments. This effect was not seen in mock-transfected cells or in cells expressing spinophilin. Furthermore, expression of spinophilin appeared to slow, whereas overexpression of beta-arrestins accelerated internalization of the Na(+),K(+)-ATPase endocytosis. We also find that GRKs phosphorylate the Na(+),K(+)-ATPase in vitro on its large cytoplasmic loop. Taken together, it appears that association with arrestins, GRKs, 14-3-3 epsilon, and spinophilin may be important modulators of Na(+),K(+)-ATPase trafficking.     

Optimization of TCR transgenic T cells for in vivo tracking of immune responses

T-cell receptor (TCR) transgenic mice have proven useful for the study of various immune parameters. Despite this, it has been suggested that transferred T cells respond differently to their endogenous counterparts at least in terms of conversion to antigen-experienced populations bearing memory cell markers. Here, we have compared the response of TCR transgenic T cells to endogenous populations within the context of infection with herpes simplex virus. We found that adoptive transfer at numbers approaching those of the endogenous virus-specific subset results in a response with similar kinetics, magnitude and memory subset conversion. This suggests that this form of optimized T-cell transfer remains a useful means of tracking antiviral immune responses.     

Cerebellar LTD and pattern recognition by Purkinje cells

Many theories of cerebellar function assume that long-term depression (LTD) of parallel fiber (PF) synapses enables Purkinje cells to learn to recognize PF activity patterns. We have studied the LTD-based recognition of PF patterns in a biophysically realistic Purkinje-cell model. With simple-spike firing as observed in vivo, the presentation of a pattern resulted in a burst of spikes followed by a pause. Surprisingly, the best criterion to distinguish learned patterns was the duration of this pause. Moreover, our simulations predicted that learned patterns elicited shorter pauses, thus increasing Purkinje-cell output. We tested this prediction in Purkinje-cell recordings both in vitro and in vivo. In vitro, we found a shortening of pauses when decreasing the number of active PFs or after inducing LTD. In vivo, we observed longer pauses in LTD-deficient mice. Our results suggest a novel form of neural coding in the cerebellar cortex.     

Multiple environmental stressors increase the realised niche breadth of a forest‐dwelling fish

Understanding the determinants of species' niche breadth is important due to the negative relationship between niche breadth and extinction probability. Species tolerant to extremely harsh abiotic conditions (e.g. low pH or hypoxia) often have relatively small realised niches due to a trade‐off between abiotic and biotic stress tolerance, and are therefore particularly vulnerable to extinction. We hypothesised that tolerance to multiple extreme conditions (e.g. low pH, hypoxia and drought) in brown mudfish Neochanna apoda, would increase their realised niche breadth because each extreme would provide additive refuge against a dominant species, the banded kokopu Galaxias fasciatus. Fish distributions were surveyed in 65 peat‐swamp‐forest streams and pools in New Zealand, which varied in dissolved oxygen, acidity and hydroperiod. Mudfish distribution was extremely patchy, and almost completely allopatric with kokopu. Allopatry was driven mainly by pool hydroperiod; mudfish occupied 88 percent of temporary pools due to their tolerance of habitat drying, whereas kokopu were absent from temporary pools. Within permanent pools, mudfish occurrence was negatively related to pool oxygen and pH, whereas kokopu occurrence was positively related to these conditions. Pool conditions were independently distributed in the landscape, suggesting that each abiotic stressor offered additive refuge for mudfish from kokopu predation/competition. Consequently, the mudfish realised niche breadth depended on the number of abiotic factors driving their allopatry with kokopu. Given the widespread negative relationships between niche breadth and species extinction probabilities, our results indicate that tolerance to multiple stressors may play an important role in insuring species persistence against the multiple drivers of global environmental change.     

Neural stem and progenitor cell fate transition requires regulation of Musashi1 function

Background

There is increasing evidence of a pivotal role for regulated mRNA translation in control of developmental cell fate transitions. Physiological and pathological stem and progenitor cell self-renewal is maintained by the mRNA-binding protein, Musashi1 through repression of translation of key mRNAs encoding cell cycle inhibitory proteins. The mechanism by which Musashi1 function is modified to allow translation of these target mRNAs under conditions that require inhibition of cell cycle progression, is unknown.

Results

In this study, we demonstrate that differentiation of primary embryonic rat neural stem/progenitor cells (NSPCs) or human neuroblastoma SH-SY5Y cells results in the rapid phosphorylation of Musashi1 on the evolutionarily conserved site serine 337 (S337). Phosphorylation of this site has been shown to be required for cell cycle control during the maturation of Xenopus oocytes. S337 phosphorylation in mammalian NSPCs and human SH-SY5Y cells correlates with the de-repression and translation of a Musashi reporter mRNA and with accumulation of protein from the endogenous Musashi target mRNA, p21^WAF1/CIP1. Inhibition of Musashi regulatory phosphorylation, through expression of a phospho-inhibitory mutant Musashi1 S337A or over-expression of the wild-type Musashi, blocked differentiation of both NSPCs and SH-SY5Y cells. Musashi1 was similarly phosphorylated in NSPCs and SH-SY5Y cells under conditions of nutrient deprivation-induced cell cycle arrest. Expression of the Musashi1 S337A mutant protein attenuated nutrient deprivation-induced NSPC and SH-SY5Y cell death.

Conclusions

Our data suggest that in response to environmental cues that oppose cell cycle progression, regulation of Musashi function is required to promote target mRNA translation and cell fate transition. Forced modulation of Musashi1 function may present a novel therapeutic strategy to oppose pathological stem cell self-renewal.

     

Dynamics of auxin-dependent Ca2+ and pH signaling in root growth revealed by integrating high-resolution imaging with automated computer vision-based analysis

Plants adapt to a changing environment by entraining their growth and development to prevailing conditions. Such 'plastic' development requires a highly dynamic integration of growth phenomena with signal perception and transduction systems, such as occurs during tropic growth. The plant hormone auxin has been shown to play a key role in regulating these directional growth responses of plant organs to environmental cues. However, we are still lacking a cellular and molecular understanding of how auxin-dependent signaling cascades link stimulus perception to the rapid modulation of growth patterns. Here, we report that in root gravitropism of Arabidopsis thaliana, auxin regulates root curvature and associated apoplastic, growth-related pH changes through a Ca2+-dependent signaling pathway. Using an approach that integrates confocal microscopy and automated computer vision-based image analysis, we demonstrate highly dynamic root surface pH patterns during vertical growth and after gravistimulation. These pH dynamics are shown to be dependent on auxin, and specifically on auxin transport mediated by the auxin influx carrier AUX1 in cells of the lateral root cap and root epidermis. Our results further indicate that these pH responses require auxin-dependent changes in cytosolic Ca2+ levels that operate independently of the TIR1 auxin perception system. These results demonstrate a methodology that can be used to visualize vectorial auxin responses in a manner that can be integrated with the rapid plant growth responses to environmental stimuli.     

Design, synthesis and structure-activity relationships of (indo-3-yl) heterocyclic derivatives as agonists of the CB1 receptor. Discovery of a clinical candidate

We report an expansion of the structure-activity relationship (SAR) of a novel series of indole-3-heterocyclic CB1 receptor agonists. Starting from the potent but poorly soluble lead, 1, a rational approach was taken in order to balance solubility, hERG activity and potency while retaining the desired long duration of action within the mouse tail flick test. This led to the discovery of compound 38 which successfully progressed into clinical development.