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A constitutively active cryptochrome in Drosophila melanogaster

Nature Neuroscience volume 7, pages 834–840 (2004)Cite this article

Abstract

Light-activated cryptochrome (CRY) regulates circadian photoresponses in Drosophila melanogaster. Removing the carboxy (C) terminus to create CRYΔ produces, in yeast, a light-independent, constitutively active form. Here we show that flies overexpressing CRYΔ have a longer free-running period of locomotor activity, as well as altered cycling kinetics of the clock proteins timeless (TIM) and period (PER). Moreover, at the cellular level, they show a reduction in the level of TIM and in the nuclear localization of TIM and PER in two significant clusters of behavioral pacemaker cells: the large and the small ventral lateral neurons (LNvs). These effects are similar to those seen in wild-type flies under continuous light and suggest a regulatory role for the C terminus of CRY on the photosensitive, photolyase-like part of the protein.

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Figure 1: CRYΔ, but not CRY, affects the level, the oscillation and the phosphorylation status of TIM and PER in cry+ flies.
Figure 2: Expression of TIM and PER in cryb, CRY(cryb) and CRYΔ(cryb) flies.
Figure 3: CRYΔ mimics the effects of constant dim light.
Figure 4: CRYΔ flies show aberrant light responses.
Figure 5: CRYΔ and light affect the level and subcellular localization of TIM in the LNvs.

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Acknowledgements

We thank P. Emery, M. Rosbash and J.C. Hall for tim-GAL4 and UAS-cry flies, P. Taghert and F. Rouyer for pdf-GAL4 and gal1118 strains, respectively; R. Stanewsky and J.C. Hall for anti-PER antibody, M.W. Young & L. Saez for anti-TIM(307) antibody, A. Sehgal for anti-TIM(UPR41) antibody, S. Webster and H. Dircksen for anti-cPDH antibody, and M. Rosbash for anti-PDF antibody. This work was supported by a postgraduate studentship from the Biotechnology and Biological Sciences Research Council (BBSRC) to V.C., a Royal Society Wolfson Research Merit Award to C.P.K., a BBSRC David Phillips Fellowship to E.R. and a BBSRC grant to E.R. and C.P.K.

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Author notes

  1. Veryan Codd & Robert Fedic

    Present address: Department of Cardiovascular Science, University of Leicester, Glenfield Hospital, Groby Road, Leicester, LE3 9QP, UK

Authors and Affiliations

  1. Department of Biology, University of Leicester, University Road, Leicester, LE1 7RH, UK

    Stephane Dissel, Veryan Codd, Robert Fedic & Ezio Rosato

  2. Department of Genetics, University of Leicester, University Road, Leicester, LE1 7RH, UK

    Stephane Dissel, Karen J Garner & Charalambos P Kyriacou

  3. Department of Biology, University of Padua, Via Ugo Bassi 58/b, Padova, 35131, Italy

    Rodolfo Costa

  4. Institute of Entomology, Academy of Sciences of the Czech Republic, Branisovska 31, 370 05, Ceske Budejovice, Czech Republic

    Robert Fedic

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Supplementary information

Supplementary Fig. 1

Replotted data from Fig. 5b and Supplementary Fig. 2 online. These graphs show that the relationship between large and small LNvs is completely reversed in control (left panels) and CRYΔ (right panels) flies, this is true for both anti-TIM and anti-PER staining and also N+C and N/C, unlikely to be just a coincidence. The most prominent effect is seen for total TIM levels (N+C). However, the quantification of nuclear staining is likely biased by out-of-focus emission from (more strongly labelled) nearby cytoplasmic regions (see also ref 28), which increases the real N/C ratio. As this artefact is proportionally more important for lower focal emission (real staining), it follows that the quantification of nuclear signal from the large LNvs is likely the most overestimated. (GIF 87 kb)

Supplementary Fig. 2

CRYΔ affects the subcellular localisation of PER in the LNvs. (a) Localization of PER (green) within the small and large LNvs of control (tim-GAL4/+) and CRYΔ (tim-GAL4/UAS-cryΔ) adult brains at the indicated ZTs. PDF (red) was used as marker of neuronal identity and cytoplasmic localisation. (b) Quantification of the nuclear cytoplasmic ratio (N/C) of PER in the two clusters of LNvs for the two genotypes at the indicated ZTs. Quantification data are presented as mean ± s.e.m. At least three brains have been analysed for each genotype at every time point, all visible cells were imaged but only those where PDF staining showed a clear distinction between nucleus and cytoplasm were quantified. Line UAS-cryΔ14.6 was used for these experiments. Due to the high background produced by the anti-PER antibody it was not possible to image a relevant number of cells using the same detector settings, therefore it was not possible to quantify the total level of protein in this experiment. (JPG 47 kb)

Supplementary Fig. 3

Model of CRY function. (a) Under darkness a regulatory molecule (black circle) binds the C terminus, limiting CRY's ability to engage in protein-protein interactions either directly or by action of post-translational modifications. (b) Light promotes a conformational change resulting in the release of the repression (either by release of the regulatory protein or by reversal of the regulatory modification) and in binding with signalling partners (squares), also mediating the degradation of CRY. (c) The removal of the C-terminus prevents the binding of the regulator, allowing signalling to take place under darkness and consequent constant activation/degradation of CRYΔ. (d) Light changes the conformation of CRYΔ, promoting stronger interactions. (GIF 300 kb)

Supplementary Data (PDF 23 kb)

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Dissel, S., Codd, V., Fedic, R. et al. A constitutively active cryptochrome in Drosophila melanogaster. Nat Neurosci 7, 834–840 (2004). https://doi.org/10.1038/nn1285

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