Cryptochromes are blue-light photoreceptors sharing sequence similarity to photolyases, a class of flavoenzymes catalyzing repair of UV-damaged DNA via electron transfer mechanisms. Despite significant amino acid sequence similarity in both catalytic and cofactor-binding domains, cryptochromes lack DNA repair functions associated with photolyases, and the molecular mechanism involved in cryptochrome signaling remains obscure. Here, we report a novel ATP binding and autophosphorylation activity associated with Arabidopsis cry1 protein purified from a baculovirus expression system. Autophosphorylation occurs on serine residue(s) and is absent in preparations of cryptochrome depleted in flavin and/or misfolded. Autophosphorylation is stimulated by light in vitro and oxidizing agents that act as flavin antagonists prevent this stimulation. Human cry1 expressed in baculovirus likewise shows ATP binding and autophosphorylation activity, suggesting this novel enzymatic activity may be important to the mechanism of action of both plant and animal cryptochromes.

a b s t r a c tCryptochromes are widely distributed blue light photoreceptors involved in numerous signaling functions in plants and animals. Both plant and animal-type cryptochromes are found to bind ATP and display intrinsic autokinase activity; however the functional significance of this activity remains a matter of speculation. Here we show in purified preparations of Arabidopsis cry1 that ATP binding induces conformational change independently of light and increases the amount and stability of light-induced flavin radical formation. Nucleotide binding may thereby provide a mechanism whereby light responsivity in organisms can be regulated through modulation of cryptochrome photoreceptor conformation.

Vertebrates are able to visually identify moving objects and orient toward attractive ones or escape if the objects seem threatening. When there is more than one object in the visual field, they can attend to a particular object. The optic tectum (superior colliculus in mammals) (OT/SC) has long been known to mediate such functions (Schneider, 1969;Ingle, 1973a). Less well known is that the OT/SC is strongly affected by a smaller midbrain area called nucleus isthmi (parabigeminal nucleus in mammals) (NI/PB). We discuss how NI/PB influences OT/SC function and visual behavior.Anatomically, OT/SC makes reciprocal, topographic connections with ipsilateral NI/PB. Adjacent points in OT/SC project to adjacent points in NI/PB. The return projections from NI/PB terminate in many of the same superficial layers as retinotectal fibers, and their effects on tectal processing may facilitate selection of a single stimulus from an array of potential targets. In amphibians and mammals, NI/PB also project to the contralateral OT/SC (Fig. 1). Visual behavior and the frog NIWhen presented with a single prey stimulus anywhere in its visual field, a frog will approach and attack the stimulus. When presented with two prey stimuli, they will select one of the stimuli (Ingle, 1973b;Stull and Gruberg, 1998). After ablation of the optic tectum, frogs will not respond to prey stimuli (or to looming stimuli), although they retain other visual abilities, such as perceiving stationary objects (Ingle, 1973b).Other than the retina, the greatest input to the OT in frogs comes from NI. It can be divided into two functionally discrete regions: one region makes topographic reciprocal connections with the ipsilateral OT; the other region projects topographically to the contralateral tectal lobe. Unilateral ablation of NI results in a scotoma in the contralateral monocular visual field (Gruberg et al., 1991) that is similar to unilateral ablation of the OT. Partial ablation of NI results in a smaller scotoma that always includes the posteriormost part of the monocular field. Within the scotoma, the behavioral threshold to prey stimuli is considerably increased and resembles visual neglect.NI directly influences retinotectal transmission (King and Schmidt, 1991;Dudkin and Gruberg, 2003). Frog isthmotectal fibers are cholinergic (Desan et al., 1987;Wallace et al., 1990) and terminate in retino-recipient layers of the optic tectum. Retinal ganglion cell axons express nicotinic acetylcholine (ACh) receptors (Sargent et al., 1989). There do not appear to be conventional synapses between isthmotectal fibers and retinotectal axons (Gruberg et al., 1994). Nonetheless, by selectively filling retinotectal fibers with a fluorescent calcium-sensitive dye, NI influence on retinotectal fibers can be shown. Single-shock stimulation to the optic nerve causes a brief increase in fluorescence. Singleshock stimulation to NI causes no change in fluorescence. When single-shock stimulation of NI is paired with optic nerve stimulation, there is a greater than twofold incre...

Kinetoplastids are a group of parasites that includes several medically-important species. These human-infective species are transmitted by insect vectors in which the parasites undergo specific developmental transformations. For each species, this includes a stage in which parasites adhere to insect tissue via a hemidesmosome-like structure. Although this structure has been described morphologically, it has never been molecularly characterized. We are using Crithidia fasciculata , an insect parasite that produces large numbers of adherent parasites inside its mosquito host, as a model kinetoplastid to investigate both the mechanism of adherence and the signals required for differentiation to an adherent form. An advantage of C . fasciculata is that adherent parasites can be generated both in vitro , allowing a direct comparison to cultured swimming forms, as well as in vivo within the mosquito. Using RNAseq, we identify genes associated with adherence in C . fasciculata . As almost all of these genes have orthologs in other kinetoplastid species, our findings may reveal shared mechanisms of adherence, allowing investigation of a crucial step in parasite development and disease transmission. In addition, dual-RNAseq allowed us to explore the interaction between the parasites and the mosquito. Although the infection is well-tolerated, anti-microbial peptides and other components of the mosquito innate immune system are upregulated. Our findings indicate that C . fasciculata is a powerful model system for probing kinetoplastid-insect interactions.