Functional Analyses of Four Cryptochromes From Aquatic Organisms After Heterologous Expression in Drosophila melanogaster Circadian Clock Cells

Chenghao Chen; T Katherine Tamai; Min Xu; Libero Petrone; Paola Oliveri; David Whitmore; Ralf Stanewsky

doi:10.1177/07487304241228617

Functional Analyses of Four Cryptochromes From Aquatic Organisms After Heterologous Expression in Drosophila melanogaster Circadian Clock Cells

J Biol Rhythms. 2024 Mar 28:7487304241228617. doi: 10.1177/07487304241228617. Online ahead of print.

Authors

Chenghao Chen^{1

2}, T Katherine Tamai^{1

3}, Min Xu¹, Libero Petrone⁴, Paola Oliveri⁴, David Whitmore^{1

5}, Ralf Stanewsky^{1

6}

Affiliations

¹ Department of Cell and Developmental Biology, University College London, London, UK.
² Department of Neurobiology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.
³ Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.
⁴ Department of Genetics, Evolution and Environment, University College London, London, UK.
⁵ Australian Institute of Tropical Health & Medicine, James Cook University, Townsville, QLD, Australia.
⁶ Institute for Neuro- and Behavioral Biology, University of Münster, Münster, Germany.

PMID: 38544471
DOI: 10.1177/07487304241228617

Abstract

Cryptochromes (Crys) represent a multi-facetted class of proteins closely associated with circadian clocks. They have been shown to function as photoreceptors but also to fulfill light-independent roles as transcriptional repressors within the negative feedback loop of the circadian clock. In addition, there is evidence for Crys being involved in light-dependent magneto-sensing, and regulation of neuronal activity in insects, adding to the functional diversity of this cryptic protein class. In mammals, Crys are essential components of the circadian clock, but their role in other vertebrates is less clear. In invertebrates, Crys can function as circadian photoreceptors, or as components of the circadian clock, while in some species, both light-receptive and clock factor roles coexist. In the current study, we investigate the function of Cry proteins in zebrafish (Danio rerio), a freshwater teleost expressing 6 cry genes. Zebrafish peripheral circadian clocks are intrinsically light-sensitive, suggesting the involvement of Cry in light-resetting. Echinoderms (Strongylocentrotus purpuratus) represent the only class of deuterostomes that possess an orthologue (SpuCry) of the light-sensitive Drosophila melanogaster Cry, which is an important component of the light-resetting pathway, but also works as transcriptional repressor in peripheral clocks of fruit flies. We therefore investigated the potential of different zebrafish cry genes and SpuCry to replace the light-resetting and repressor functions of Drosophila Cry by expressing them in fruit flies lacking endogenous cry function. Using various behavioral and molecular approaches, we show that most Cry proteins analyzed are able to fulfill circadian repressor functions in flies, except for one of the zebrafish Crys, encoded by cry4a. Cry4a also shows a tendency to support light-dependent Cry functions, indicating that it might act in the light-input pathway of zebrafish.

Keywords: Danio rerio; Strongylocentrotus purpuratus; circadian clock; luciferase; period; timeless.