Human and Drosophila Cryptochromes Are Light Activated by Flavin Photoreduction in Living Cells

Abstract

Cryptochromes are a class of flavoprotein blue-light signaling receptors found in plants, animals, and humans that control plant development and the entrainment of circadian rhythms. In plant cryptochromes, light activation is proposed to result from photoreduction of a protein-bound flavin chromophore through intramolecular electron transfer. However, although similar in structure to plant cryptochromes, the light-response mechanism of animal cryptochromes remains entirely unknown. To complicate matters further, there is currently a debate on whether mammalian cryptochromes respond to light at all or are instead activated by non–light-dependent mechanisms. To resolve these questions, we have expressed both human and Drosophila cryptochrome proteins to high levels in living Sf21 insect cells using a baculovirus-derived expression system. Intact cells are irradiated with blue light, and the resulting cryptochrome photoconversion is monitored by fluorescence and electron paramagnetic resonance spectroscopic techniques. We demonstrate that light induces a change in the redox state of flavin bound to the receptor in both human and Drosophila cryptochromes. Photoreduction from oxidized flavin and subsequent accumulation of a semiquinone intermediate signaling state occurs by a conserved mechanism that has been previously identified for plant cryptochromes. These results provide the first evidence of how animal-type cryptochromes are activated by light in living cells. Furthermore, human cryptochrome is also shown to undergo this light response. Therefore, human cryptochromes in exposed peripheral and/or visual tissues may have novel light-sensing roles that remain to be elucidated.

Chemical amplification of magnetic field effects relevant to avian magnetoreception

“…In vivo photoexcitation of cryptochromes leads to accumulation of states of the protein that contain reduced forms of FAD 60 implying efficient reduction of the donor radical and hence substantial enhancement of the magnetic field effect (i.e. k D >> k F and hence E >> 1).…”

Resolving cryptic aspects of cryptochrome signaling

“…Spectroscopic studies of CRY proteins in a cellular system confirmed an oxidized FAD ground state under cellular conditions (18). These proteins, including Trp triad mutants, rapidly photoreduce in the presence of blue light to generate semiquinone intermediates (18).…”

“…Spectroscopic studies of CRY proteins in a cellular system confirmed an oxidized FAD ground state under cellular conditions (18). These proteins, including Trp triad mutants, rapidly photoreduce in the presence of blue light to generate semiquinone intermediates (18). The ability to rescue photoreduction efficiency in a cellular system led to a hypothesis that cellular metabolites, such as ATP and NADH, may contribute to photoreduction in CRY proteins.…”

A metal left spinning

“…The data almost defy imagination -on the order of a million cells in every cubic centimetre of sediment buried half a kilometre below the sea floor, or more than 50% of all microbial cells on Earth 12 10,11,13 , and a hung jury has been declared 14 over the question of the predominance of Bacteria or Archaea. But why the protracted debate?…”

“…These include approaches that detect ribosomal RNA (rRNA) or intact polar lipids (IPLs) of cell membranes (both are proxies for live cells), or that quantify rRNA gene copies from extracted DNA. Of these techniques, in situ fluorescent tagging of ribosomes in methods known as FISH or CARD-FISH produce the most disparate (indeed, opposite) results 10,11,13 . Although potentially an ideal …”