PubMed · 4695819
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P A Sperber. 1973. The biologic time clock.. https://pubmed.ncbi.nlm.nih.gov/4695819/
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Using confocal laser scanning microscopy we investigated the Ca(2+) distribution in single corticotropin releasing factor- and urocortin-stimulated human skin cells. The models tested included melanoma cells, neonatal melanocytes and keratinocytes, and immortalized HaCaT keratinocytes. The changes in intracellular Ca(2+) signal intensities observed after stimulation of different cell types with corticotropin releasing factor and urocortin showed that: (1) the increase of intracellular Ca(2+) concentration was caused by a Ca(2+) influx (inhibition by EGTA); (2) this Ca(2+) influx took place through voltage-activated Ca(2+) ion channels (inhibition by d-cis-diltiazem, verapamil) and (3) cyclic nucleotide-gated ion channels were not involved in this process (no effect of Mg(2+)). The effects were also observed at very low peptide concentrations (10(-13) M) with no apparent linear correlation between peptide dosage and increase of fluorescence intensity, which implied co-expression of different corticotropin releasing factor receptor forms in the same cell. Immortalized (HaCaT) keratinocytes exhibited the strongest differential increases of a Ca(2+) fluorescence after peptide-stimulation. Corticotropin releasing factor induced Ca(2+) flux into the cytoplasm, while urocortin Ca(2+) flux into the nucleus with a remarkable oscillatory effect. The latter indicated the presence of an intracellular urocortin-induced signal transduction pathway that is unique to keratinocytes.
Cryptochromes are blue-light photoreceptors that regulate the circadian clock in animals and growth and development in plants. Cryptochromes have high sequence homology to DNA photolyase but appear to lack photorepair activity. All previous work on cryptochromes was performed with protein expressed in heterologous systems; hence, biochemical and photochemical studies performed with these proteins were subject to certain limitations. In this study, we purified cryptochrome 2 (hCRY2) from human cells and characterized it. We find that hCRY2 exhibits fluorescence properties consistent with the presence of folate and flavin cofactors. Cryptochrome 2 binds to double-stranded DNA weakly and to single-stranded DNA with higher affinity, and this binding is further stimulated by the presence of a (6-4) photoproduct. However, light has no effect on the cryptochrome 2-(6-4) photoproduct complex. These findings reveal new properties of this protein already known to function as a circadian photoreceptor and a light-independent negative transcriptional regulator of the clock genes.
Activation of sphingosine kinase (SPHK), thereby increasing cellular levels of sphingosine 1-phosphate (S1P), may be involved in a variety of intracellular responses including Ca(2+) signaling. This study uses mammalian SPHK1a, tagged with enhanced green fluorescent protein (eGFP), to examine whether translocation of this enzyme is linked with Ca(2+)-mobilizing responses. Real-time confocal imaging of SPHK1a-eGFP in human SH-SY5Y neuroblastoma cells visualized a relocation of the enzyme from the cytosol to the plasma membrane in response to Ca(2+)-mobilizing stimuli (muscarinic M(3)- or lysophosphatidic acid receptor activation, and thapsigargin-mediated store release). This redistribution was preceded by a transient increase in cytosolic SPHK1a-eGFP levels due to liberation of SPHK from localized higher intensity regions. Translocation was dependent on Ca(2+) mobilization from intracellular stores, and was prevented by pretreatment with the Ca(2+)/calmodulin inhibitor W-7, but not W-5 or KN-62. In functional studies, pretreatment with W-7 lowered basal and M(3)-receptor-mediated cellular S1P production. However, this pretreatment did not alter agonist-mediated Ca(2+) responses, and SPHK1a-eGFP activity itself appeared insensitive to Ca(2+)/calmodulin and W-7. These data suggest a role for Ca(2+)/calmodulin in controlling the subcellular distribution but not the activity of SPHK1a.