Isolation of the flagellar swelling and identification of retinal in the phototactic flagellate, Ochromonas danica (Chrysophyceae)

Ochromonas danica, a freshwater, planktonic chrysophyte, is capable of sensing the light conditions of its environment. This biflagellate alga has a swelling near the base of the short flagellum and a chloroplastidic stigma in close association with it. A procedure is described for the isolation of this three dimensional flagellar swelling, the presumed photoreceptor. In contrast to an earlier method developed for the isolation of the paraflagellar swelling from Euglena gracilis, the protocol reported here for Ochromonas results in higher yields that should facilitate future biochemical investigations and could open avenues of investigation for the isolation and purification of the presumptive receptor protein. To verify the hypothesis that a rhodopsin-like protein might be present in this alga, we applied a standard extraction procedure successfully used in the identification of retinal. We here report the purification and identification of all-trans retinal in Ochromonas cells by column chromatography, HPLC and GC-MS. Since retinal is the chromophore of rhodopsin-like proteins, this finding may suggest that in these unicellular algae, too, a rhodopsin-like protein could be the photoreceptor pigment

Effects of hydroxylamine, digitonin and triton X-100 on photoreceptor (paraflagellarswelling) and photoreception of Euglena gracilis.

We present experiments that test the effects of agents commonly used in visual pigment investigations, namely hydroxylamine (NH 2OH), digitonin and triton X-100, on the photoreceptor and photoreception of Euglena. Hydroxylamine reacts with free and opsin-bound retinal, in aqueous solution, to form stable oximes, whereas digitonin and triton X-100 are the most common extractants of rhodopsin. Since previous data indicate that the chromophore present in Euglena photoreceptor is retinal, we investigated the influence of these chemicals on this organelle. The effects of these agents were studied by means of phase contrast, fluorescence and transmission electron microscopy and photobehaviour experiments. Hydroxylamine inhibited the formation of the Euglena photoreceptor. Photoaccumulation experiments on hydroxylamine-treated cells showed that they are unable to perceive light. Digitonin solubilized the crystalline structure of the photoreceptor, whereas the triton effect was limited to the membranous structures of the cell, leaving the photoreceptor unimpaired

TIN2 Stability Is Regulated by the E3 Ligase Siah2

Telomeres are coated by shelterin, a six-subunit complex that is required for protection and replication of chromosome ends. The central subunit TIN2, with binding sites to three subunits (TRF1, TRF2, and TPP1), is essential for stability and function of the complex. Here we show that TIN2 stability is regulated by the E3 ligase Siah2. We demonstrate that TIN2 binds to Siah2 and is ubiquitylated in vivo. We show using purified proteins that Siah2 acts as an E3 ligase to directly ubiquitylate TIN2 in vitro. Depletion of Siah2 led to stabilization of TIN2 protein, indicating that Siah2 regulates TIN2 protein levels in vivo. Overexpression of Siah2 in human cells led to loss of TIN2 at telomeres that was dependent on the presence of the catalytic RING domain of Siah2. In contrast to RNAi-mediated depletion of TIN2 that led to loss of TRF1 and TRF2 at telomeres, Siah2-mediated depletion of TIN2 allowed TRF1 and TRF2 to remain on telomeres, indicating a different fate for shelterin subunits when TIN2 is depleted posttranslationally. TPP1 was lost from telomeres, although its protein level was not reduced. We speculate that Siah2-mediated removal of TIN2 may allow dynamic remodeling of the shelterin complex and its associated factors during the cell cycle