Gabriella Mazzotta

Light-dependent interaction between Drosophila CRY and the clock protein PER mediated by the carboxy terminus of CRY

BACKGROUND The biological clock synchronizes the organism with the environment, responding to changes in light and temperature. Drosophila CRYPTOCHROME (CRY), a putative circadian photoreceptor, has previously been reported to interact with the clock protein TIMELESS (TIM) in a light-dependent manner. Although TIM dimerizes with PERIOD (PER), no association between CRY and PER has previously been revealed, and aspects of the light dependence of the TIM/CRY interaction are still unclear. RESULTS Behavioral analysis of double mutants of per and cry suggested a genetic interaction between the two loci. To investigate whether this was reflected in a physical interaction, we employed a yeast-two-hybrid system that revealed a dimerization between PER and CRY. This was further supported by a coimmunoprecipitation assay in tissue culture cells. We also show that the light-dependent nuclear interactions of PER and TIM with CRY require the C terminus of CRY and may involve a trans-acting repressor. CONCLUSIONS This study shows that, as in mammals, Drosophila CRY interacts with PER, and, as in plants, the C terminus of CRY is involved in mediating light responses. A model for the light dependence of CRY is discussed.

A Molecular Basis for Natural Selection at the timeless Locus in Drosophila melanogaster

Diapause is a protective response to unfavorable environments that results in a suspension of insect development and is most often associated with the onset of winter. The ls-tim mutation in the Drosophila melanogaster clock gene timeless has spread in Europe over the past 10,000 years, possibly because it enhances diapause. We show that the mutant allele attenuates the photosensitivity of the circadian clock and causes decreased dimerization of the mutant TIMELESS protein isoform to CRYPTOCHROME, the circadian photoreceptor. This interaction results in a more stable TIMELESS product. These findings reveal a molecular link between diapause and circadian photoreception.

F-ATPase of Drosophila melanogaster forms 53-picosiemen (53-pS) channels responsible for mitochondrial Ca2+-induced Ca2+ release.

Background: The Ca2+-induced Ca2+ release channel (mCrC) of Drosophila mitochondria is similar to the permeability transition pore (PTP). Results: mCrC is modulated by PTP effectors and Drosophila F-ATPase forms 53-pS channels. Conclusion: F-ATPase mediates Ca2+-induced Ca2+ release in Drosophila mitochondria. Significance: Channel formation by F-ATPases has been conserved in evolution, but species-specific features exist that may underscore different roles in different organisms. Mitochondria of Drosophila melanogaster undergo Ca2+-induced Ca2+ release through a putative channel (mCrC) that has several regulatory features of the permeability transition pore (PTP). The PTP is an inner membrane channel that forms from F-ATPase, possessing a conductance of 500 picosiemens (pS) in mammals and of 300 pS in yeast. In contrast to the PTP, the mCrC of Drosophila is not permeable to sucrose and appears to be selective for Ca2+ and H+. We show (i) that like the PTP, the mCrC is affected by the sense of rotation of F-ATPase, by Bz-423, and by Mg2+/ADP; (ii) that expression of human cyclophilin D in mitochondria of Drosophila S2R+ cells sensitizes the mCrC to Ca2+ but does not increase its apparent size; and (iii) that purified dimers of D. melanogaster F-ATPase reconstituted into lipid bilayers form 53-pS channels activated by Ca2+ and thiol oxidants and inhibited by Mg2+/γ-imino ATP. These findings indicate that the mCrC is the PTP of D. melanogaster and that the signature conductance of F-ATPase channels depends on unique structural features that may underscore specific roles in different species.

Fly cryptochrome and the visual system

Cryptochromes are flavoproteins, structurally and evolutionarily related to photolyases, that are involved in the development, magnetoreception, and temporal organization of a variety of organisms. Drosophila CRYPTOCHROME (dCRY) is involved in light synchronization of the master circadian clock, and its C terminus plays an important role in modulating light sensitivity and activity of the protein. The activation of dCRY by light requires a conformational change, but it has been suggested that activation could be mediated also by specific “regulators” that bind the C terminus of the protein. This C-terminal region harbors several protein–protein interaction motifs, likely relevant for signal transduction regulation. Here, we show that some functional linear motifs are evolutionarily conserved in the C terminus of cryptochromes and that class III PDZ-binding sites are selectively maintained in animals. A coimmunoprecipitation assay followed by mass spectrometry analysis revealed that dCRY interacts with Retinal Degeneration A (RDGA) and with Neither Inactivation Nor Afterpotential C (NINAC) proteins. Both proteins belong to a multiprotein complex (the Signalplex) that includes visual-signaling molecules. Using bioinformatic and molecular approaches, dCRY was found to interact with Neither Inactivation Nor Afterpotential C through Inactivation No Afterpotential D (INAD) in a light-dependent manner and that the CRY–Inactivation No Afterpotential D interaction is mediated by specific domains of the two proteins and involves the CRY C terminus. Moreover, an impairment of the visual behavior was observed in fly mutants for dCRY, indicative of a role, direct or indirect, for this photoreceptor in fly vision.

A CRY FROM THE KRILL

Antarctic krill (Euphausia superba) inhabit a region with strong seasonality in several parameters, such as photoperiod, light intensity, extent of sea ice, and food availability. In particular, seasonal changes in environmental light regimes have been shown to strongly influence krill metabolism, representing control signals for seasonal regulation of physiology of this key Southern Ocean species. Here, we report the identification of a cryptochrome gene, a cardinal component of the clockwork machinery in several organisms. EsCRY appears to be an ortholog of mammalian-like CRYs and clusters with the insect CRY2 subfamily. EsCRY has the canonical bipartite CRY structure, with a conserved N-terminal domain and a highly divergent C-terminus, that bears several binding motifs, some of them shared with insect CRY2 and others peculiar for EsCRY. We have evaluated the temporal expression of Escry both at mRNA and protein levels in individuals harvested from the Ross Sea at different times throughout the 24 h cycle during the Antarctic summer. We observed a daily fluctuation in abundance for Escry mRNA in the head, with high levels around 06:00 h, which is not mirrored by a cycle in the corresponding protein. Our findings represent a first step toward establishing the presence of an endogenous circadian time-keeping mechanism that might allow this organism to synchronize its physiology and behavior to the Antarctic light regimes. (Author correspondence: bru@unife.it or rodolfo.costa@unipd.it)

Systematic sequencing of mRNA from the Antarctic krill (Euphausia superba) and first tissue specific transcriptional signature.

BackgroundLittle is known about the genome sequences of Euphausiacea (krill) although these crustaceans are abundant components of the pelagic ecosystems in all oceans and used for aquaculture and pharmaceutical industry. This study reports the results of an expressed sequence tag (EST) sequencing project from different tissues of Euphausia superba (the Antarctic krill).ResultsWe have constructed and sequenced five cDNA libraries from different Antarctic krill tissues: head, abdomen, thoracopods and photophores. We have identified 1.770 high-quality ESTs which were assembled into 216 overlapping clusters and 801 singletons resulting in a total of 1.017 non-redundant sequences. Quantitative RT-PCR analysis was performed to quantify and validate the expression levels of ten genes presenting different EST countings in krill tissues. In addition, bioinformatic screening of the non-redundant E. superba sequences identified 69 microsatellite containing ESTs. Clusters, consensuses and related similarity and gene ontology searches were organized in a dedicated E. superba database http://krill.cribi.unipd.it.ConclusionWe defined the first tissue transcriptional signatures of E. superba based on functional categorization among the examined tissues. The analyses of annotated transcripts showed a higher similarity with genes from insects with respect to Malacostraca possibly as an effect of the limited number of Malacostraca sequences in the public databases. Our catalogue provides for the first time a genomic tool to investigate the biology of the Antarctic krill.

The Antarctic krill Euphausia superba shows diurnal cycles of transcription under natural conditions.

Background Polar environments are characterized by extreme seasonal changes in day length, light intensity and spectrum, the extent of sea ice during the winter, and food availability. A key species of the Southern Ocean ecosystem, the Antarctic krill (Euphausia superba) has evolved rhythmic physiological and behavioral mechanisms to adapt to daily and seasonal changes. The molecular organization of the clockwork underlying these biological rhythms is, nevertheless, still only partially understood. Methodology/Principal Findings The genome sequence of the Antarctic krill is not yet available. A normalized cDNA library was produced and pyrosequenced in the attempt to identify large numbers of transcripts. All available E. superba sequences were then assembled to create the most complete existing oligonucleotide microarray platform with a total of 32,217 probes. Gene expression signatures of specimens collected in the Ross Sea at five different time points over a 24-hour cycle were defined, and 1,308 genes differentially expressed were identified. Of the corresponding transcripts, 609 showed a significant sinusoidal expression pattern; about 40% of these exibithed a 24-hour periodicity while the other 60% was characterized by a shorter (about 12-hour) rhythm. We assigned the differentially expressed genes to functional categories and noticed that those concerning translation, proteolysis, energy and metabolic process, redox regulation, visual transduction and stress response, which are most likely related to daily environmental changes, were significantly enriched. Two transcripts of peroxiredoxin, thought to represent the ancestral timekeeping system that evolved about 2.5 billion years ago, were also identified as were two isoforms of the EsRh1 opsin and two novel arrestin1 sequences involved in the visual transduction cascade. Conclusions Our work represents the first characterization of the krill diurnal transcriptome under natural conditions and provides a first insight into the genetic regulation of physiological changes, which occur around the clock during an Antarctic summer day.

Phenotypic effects induced by knock-down of the period clock gene in Bombyx mori.

The lepidopteran Bombyx mori is an insect of considerable scientific and economic importance. Recently, the B. mori circadian clock gene period has been molecularly characterized. We have transformed a B. mori strain with a construct encoding a period double-strand RNA in order to knock-down period gene expression. We observe that this post-transcriptional silencing produces a small but detectable disruption in the egg-hatching rhythm, as well as a reduction in egg-to-adult developmental time, without altering silk production parameters. Thus we show that both circadian and non-circadian phenotypes can be altered by changing per expression, and, at a practical level, these results suggest that per knock-down may provide a suitable strategy for improving the efficiency of rearing, without affecting silk productivity.

aubergine Gene Overexpression in Somatic Tissues of auberginesting Mutants Interferes With the RNAi Pathway of a yellow Hairpin dsRNA in Drosophila melanogaster

AUBERGINE (AUB) is a member of the PPD family of proteins. These proteins are implicated in RNA interference. In this article we demonstrate that the expression of the aub gene and protein increase in aubsting mutants. We used a genetic method to test whether aubsting overexpression could interfere with proper functioning of the process of RNA interference in somatic tissues of Drosophila melanogaster. This method is based on a transgenic line bearing a construct in which a fragment of the yellow (y) gene is cloned to form an inverted repeat (y-IR) under the control of the upstream activation sequence (UAS) of the yeast transcriptional activator GAL4. The UAS-y-IR transgene and the Act5C-GAL4 driver were brought together on chromosome 3 via recombination. In the resulting strain (Act5C-y-IR), transcriptional activation by GAL4 constitutively produces a dsRNA hairpin bearing cognate sequences to the yellow gene causing continuing degradation of y mRNA resulting in yellow1 (y1) phenocopies. In this genetic background, the mutation of any factor involved in RNAi should repress degradation of y mRNA, restoring the wild-type phenotype. We employed this genetic approach to show that an increased amount of AUBERGINE interferes with the regular functioning of the somatic RNAi pathway.

Monitoring and Analyzing Drosophila Circadian Locomotor Activity

In the 1970s, the intriguing discovery of autonomous circadian rhythmicity at the behavioral level in Drosophila set the starting point for one of the most remarkably rapid advancements in the understanding of the genetic and molecular bases of a complex behavioral trait. To this end, the design of appropriate electronic devices, apt to continuously monitor behavioral activity, has proven to be fundamental to such progress. In particular, most of the mutational screens performed to date in the search for genes involved in circadian rhythmicity were based on monitoring Drosophila mutants for alterations in the circadian pattern of locomotor activity. Many different experimental paradigms, based on the use of circadian locomotor activity monitors, have been developed. Experiments can be designed to determine (1) the natural period, (2) the capacity to adapt to day-night cycles with photoperiods of differing length, and (3) the phase of the circadian activity cycles with respect to the entraining stimulus. Here we describe some of the rationale and the steps required to set up experiments to monitor circadian locomotor activity in Drosophila. Suggestions for the statistical analysis of the data obtained in such experiments are also provided.