Modulation of circuits underlying rhythmic behaviors

Summary What have we learned about behavior from neuromodulatory studies of the crustacean stomatogastric system? The emphasis of this paper has been on the analysis of one single class of behaviors (rhythmic) in terms of microcircuitry (synaptic connections between identified neurons). But in the general case, all behaviors result from the generation of spatio-temporal patterns by the central nervous system. How individual nerve cells interact with each other to produce such patterns is of fundamental interest. We know from work on simple networks that it is possible to link the circuitry of the nervous system with behavior in a precise way, and that instead of a large number of dedicated circuits, behaviors can be altered by chemically adjusting the functional properties of the neuronal elements. One circuit can be configured to perform a variety of different behaviors by activating neurons which contain neuromodulatory substances or in response to neurohormones circulating in the hemolymph. At present we know only a few of the ways neuromodulatory neurons are triggered to release their contents onto the neurons making up CPGs.The findings described here raise many questions. What are the parameters which control the distribution of neuromodulatory substances throughout the nervous system? What happens when more than one neuromodulator is present? At the cellular level, what mechanisms are involved in transforming each neuron from one functional state to another, and then how does the entire constellation of changes give rise to a new output? It is important to answer such questions in reduced networks, because there are presently no techniques available to answer them in the more complex networks of the brain. While there is no question that modulatory activity occurs in the brain, whether or not the principles which have been discovered by using simple invertebrate circuits scale up to vertebrate circuits remains an intriguing question.     

Circadian remodeling of neuronal circuits involved in rhythmic behavior

Clock output pathways are central to convey timing information from the circadian clock to a diversity of physiological systems, ranging from cell-autonomous processes to behavior. While the molecular mechanisms that generate and sustain rhythmicity at the cellular level are well understood, it is unclear how this information is further structured to control specific behavioral outputs. Rhythmic release of pigment dispersing factor (PDF) has been proposed to propagate the time of day information from core pacemaker cells to downstream targets underlying rhythmic locomotor activity. Indeed, such circadian changes in PDF intensity represent the only known mechanism through which the PDF circuit could communicate with its output. Here we describe a novel circadian phenomenon involving extensive remodeling in the axonal terminals of the PDF circuit, which display higher complexity during the day and significantly lower complexity at nighttime, both under daily cycles and constant conditions. In support to its circadian nature, cycling is lost in bona fide clockless mutants. We propose this clock-controlled structural plasticity as a candidate mechanism contributing to the transmission of the information downstream of pacemaker cells.     

New insights into trypanosomatid U5 small nuclear ribonucleoproteins

Several protozoan parasites exist in the Trypanosomatidae family, including various agents of human diseases. Multiple lines of evidence suggest that important differences are present between the translational and mRNA processing (trans splicing) systems of trypanosomatids and other eukaryotes. In this context, certain small complexes of RNA and protein, which are named small nuclear ribonucleoproteins (U snRNPs), have an essential role in pre-mRNA processing, mainly during splicing. Even though they are well defined in mammals, snRNPs are still not well characterized in trypanosomatids. This study shows that a U5-15K protein is highly conserved among various trypanosomatid species. Tandem affinity pull-down assays revealed that this protein interacts with a novel U5-102K protein, which suggests the presence of a sub-complex that is potentially involved in the assembly of U4/U6-U5 tri-snRNPs. Functional analyses showed that U5-15K is essential for cell viability and is somehow involved with the trans and cis splicing machinery. Similar tandem affinity experiments with a trypanonosomatid U5-Cwc21 protein led to the purification of four U5 snRNP specific proteins and a Sm core, suggesting U5-Cwc-21 participation in the 35S U5 snRNP particle. Of these proteins, U5-200K was molecularly characterized. U5-200K has conserved domains, such as the DEAD/DEAH box helicase and Sec63 domains and displays a strong interaction with U5 snRNA.     

Development of circuits that generate simple rhythmic behaviors in vertebrates

Neurobiologists have long sought to understand how circuits in the nervous system are organized to generate the precise neural outputs that underlie particular behaviors. Given the complexity of the nervous system in higher vertebrates this is a daunting task. Nevertheless, recent advances in developmental genetics hold out hope that studies of locomotor and respiratory circuits will provide general insight for understanding how ensembles of neurons are wired to control specific behaviors.     

Virus-host interactions: new insights from the small RNA world

RNA silencing has a known role in the antiviral responses of plants and insects. Recent evidence, including the finding that the Tat protein of human immunodeficiency virus (HIV) can suppress the host's RNA-silencing pathway and may thus counteract host antiviral RNAs, suggests that RNA-silencing pathways could also have key roles in mammalian virus-host interactions.     

Functional insights from the structural modelling of a small Fe-hydrogenase

Recently, a novel Fe-hydrogenase from a high rate of hydrogen producing Enterobacter cloacae strain IIT-BT08 was identified and partially characterized. This 147 residue protein was found to be much smaller than previously known Fe-hydrogenases, yet retaining a high catalytic activity. We predicted the structure of this protein and found it to be structurally similar to one of the two sub-domains containing the catalytic H-cluster so far jointly present in all other Fe-hydrogenases. This novel architecture allows a tentative explanation of protein function with the high rate of catalytic activity being due to a missing regulatory sub-domain, presumably allowing higher enzymatic activity at the cost of greater exposure to oxygen inactivation. This new insight may improve our understanding of the molecular and functional organization of other, more complex Fe-hydrogenases.     

New insights into Raf regulation from structural analyses

BRAF is a highly regulated protein kinase that controls cell fate in animal cells. Recent structural analyses have revealed how active and inactive forms of BRAF bind to dimers of the scaffold protein 14-3-3. Inactive BRAF binds to 14-3-3 as a monomer and is held in an inactive conformation by interactions with ATP and the substrate kinase MEK, a striking example of enzyme inhibition by substrate binding. A change in the phosphorylation state of BRAF shifts the stoichiometry of the BRAF:14-3-3 complex from 1:2 to 2:2, resulting in stabilization of the active dimeric form of the kinase. These new findings uncover unexpected features of the regulatory mechanisms underlying Raf biology and help explain the paradoxical activation of Raf by small-molecule inhibitors.     

Alternative polyadenylation: New insights from global analyses

Recent studies have revealed widespread mRNA alternative polyadenylation (APA) in eukaryotes and its dynamic spatial and temporal regulation. APA not only generates proteomic and functional diversity, but also plays important roles in regulating gene expression. Global deregulation of APA has been demonstrated in a variety of human diseases. Recent exciting advances in the field have been made possible in a large part by high throughput analyses using newly developed experimental tools. Here I review the recent progress in global studies of APA and the insights that have emerged from these and other studies that use more conventional methods.     

New insights into chemical biology from ORFeome libraries

As the genomes of many organisms have been sequenced, a variety of global analyses, called 'omics,' have been initiated. Cloning of the set of all open reading frames encoded by the genome (ORFeome) of an organism is a major challenge, which serves as an indispensable provision before one launches into the ocean of the postgenomic world. A suitable strategy for high-throughput cloning and expression of thousands of genes is crucial to success. Recently developed systems employing site-specific or homologous recombination have made it feasible to manipulate thousands of ORFs en masse. Using these technologies, several recent studies have successfully fished biologically active small molecules and target proteins out of this bountiful ocean.     

The Celtic fringe of Britain: insights from small mammal phylogeography

Recent genetic studies have challenged the traditional view that the ancestors of British Celtic people spread from central Europe during the Iron Age and have suggested a much earlier origin for them as part of the human recolonization of Britain at the end of the last glaciation. Here we propose that small mammals provide an analogue to help resolve this controversy. Previous studies have shown that common shrews (Sorex araneus) with particular chromosomal characteristics and water voles (Arvicola terrestris) of a specific mitochondrial (mt) DNA lineage have peripheral western/northern distributions with striking similarities to that of Celtic people. We show that mtDNA lineages of three other small mammal species (bank vole Myodes glareolus, field vole Microtus agrestis and pygmy shrew Sorex minutus) also form a ‘Celtic fringe’. We argue that these small mammals most reasonably colonized Britain in a two-phase process following the last glacial maximum (LGM), with climatically driven partial replacement of the first colonists by the second colonists, leaving a peripheral geographical distribution for the first colonists. We suggest that these natural Celtic fringes provide insight into the same phenomenon in humans and support its origin in processes following the end of the LGM.     

New structural insights

A signature from Vladimir Putin has finally secured the construction of a revolutionary new European X-ray research facility. Michael Gross reports.     

Two circadian timing circuits in Neurospora crassa cells share components and regulate distinct rhythmic processes

In Neurospora crassa, FRQ, WC-1, and WC-2 proteins comprise the core circadian FRQ-based oscillator that is directly responsive to light and drives daily rhythms in spore development and gene expression. However, physiological and biochemical studies have demonstrated the existence of additional oscillators in the cell that function in the absence of FRQ (collectively termed FRQ-less oscillators [FLOs]). Whether or not these represent temperature-compensated, entrainable circadian oscillators is not known. The authors previously identified an evening-peaking gene, W06H2 (now called clock-controlled gene 16 [ccg-16]), which is expressed with a robust daily rhythm in cells that lack FRQ protein, suggesting that ccg-16 is regulated by a FLO. In this study, the authors provide evidence that the FLO driving ccg-16 rhythmicity is a circadian oscillator. They find that ccg-16 rhythms are generated by a temperature-responsive, temperature-compensated circadian FLO that, similar to the FRQ-based oscillator, requires functional WC-1 and WC-2 proteins for activity. They also find that FRQ is not essential for rhythmic WC-1 protein levels, raising the possibility that this WCFLO is involved in the generation of WC-1 rhythms. The results are consistent with the presence of 2 circadian oscillators within Neurospora cells, which the authors speculate may interact with each other through the shared WC proteins.     

New structural insights into carbohydrate-protein interactions from NMR spectroscopy

Recently developed NMR methods have been applied to discover carbohydrate ligands for proteins and to identify their binding epitopes. The structural details of carbohydrate-protein complexes have also been examined by NMR, providing site-specific information on the architecture, binding selectivity and plasticity of the carbohydrate-binding sites of the proteins. New insights into the conformational behaviour of free and protein-bound glycomimetics pave the way for the design of carbohydrate-based therapeutics. Finally, recent progress towards elucidating the influence of glycosylation on peptide conformation will be of key importance to fully understanding the role of carbohydrates in the function of glycopeptides.