Calmodulin Enhances Cryptochrome Binding to INAD in Drosophila Photoreceptors

Light is the main environmental stimulus that synchronizes the endogenous timekeeping systems in most terrestrial organisms. Drosophila cryptochrome (dCRY) is a light-responsive flavoprotein that detects changes in light intensity and wavelength around dawn and dusk. We have previously shown that dCRY acts through Inactivation No Afterpotential D (INAD) in a light-dependent manner on the Signalplex, a multiprotein complex that includes visual-signaling molecules, suggesting a role for dCRY in fly vision. Here, we predict and demonstrate a novel Ca2+-dependent interaction between dCRY and calmodulin (CaM). Through yeast two hybrid, coimmunoprecipitation (Co-IP), nuclear magnetic resonance (NMR) and calorimetric analyses we were able to identify and characterize a CaM binding motif in the dCRY C-terminus. Similarly, we also detailed the CaM binding site of the scaffold protein INAD and demonstrated that CaM bridges dCRY and INAD to form a ternary complex in vivo. Our results suggest a process whereby a rapid dCRY light response stimulates an interaction with INAD, which can be further consolidated by a novel mechanism regulated by CaM

Calmodulin enhances cryptochrome binding to INAD in Drosophila photoreceptors

Light is the main environmental stimulus that synchronizes the endogenous timekeeping systems in most terrestrial organisms. Drosophila cryptochrome (dCRY) is a light-responsive flavoprotein that detects changes in light intensity and wavelength around dawn and dusk. We have previously shown that dCRY acts through Inactivation No Afterpotential D (INAD) in a light-dependent manner on the Signalplex, a multiprotein complex that includes visual-signaling molecules, suggesting a role for dCRY in fly vision. Here, we predict and demonstrate a novel Ca2+-dependent interaction between dCRY and calmodulin (CaM). Through yeast two hybrid, coimmunoprecipitation (Co-IP), nuclear magnetic resonance (NMR) and calorimetric analyses we were able to identify and characterize a CaM binding motif in the dCRY C-terminus. Similarly, we also detailed the CaM binding site of the scaffold protein INAD and demonstrated that CaM bridges dCRY and INAD to form a ternary complex in vivo. Our results suggest a process whereby a rapid dCRY light response stimulates an interaction with INAD, which can be further consolidated by a novel mechanism regulated by CaM

F-ATPase ofDrosophila melanogasterForms 53-Picosiemen (53-pS) Channels Responsible for Mitochondrial Ca2+-induced Ca2+Release

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 byMg(2+)/gamma-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

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

Inactivation of six genes from chromosomes VII and XIV of Saccharomyces cerevisiae and basic phenotypic analysis of the mutant strains.

Within the frame of the EUROFAN project, aimed at the functional analysis of the novel ORFs revealed by the systematic sequencing of the Saccharomyces cerevisiae genome, we have inactivated six ORFs encoding putative proteins with unknown function in the two S. cerevisiae strains FY1679 and W303-1B. Five ORFs are located on chromosome VII (YGR250c, YGR251w, YGR260w, YGR262c, YGR263c) and one on chromosome XIV (YNL234w). The genes have been inactivated in the FY1679 strain by a strategy that makes use of deletion cassettes containing the kanMX4 module, which confers resistance to geneticin to yeast cells, and short flanking regions homologous to the target locus (SFH). Tetrad dissection of heterozygous mutants and basic phenotypic analysis of the spores revealed that ORF YGR251w is an essential gene, while the disruption of YGR262c causes a severe slow-growth phenotype. Deletion of the remaining ORFs did not give rise to a detectable phenotype in the mutant strains. For each ORF we have cloned, in the pUG7 plasmid, a replacement cassette that possesses long flanking regions homologous to the target locus (LFH) and, in the pRS416 plasmid, the cognate wild-type gene. The LFH replacement cassettes were used to inactivate the respective genes in the W303-1B strain. This work has been performed in the framework of the B0 Consortium of the EUROFAN I project