Biomedical subjects

Trey K Sato

Publications and source records attributed to Trey K Sato.

8 recordsLinked to original sources

PubMed2006

Feedback repression is required for mammalian circadian clock function.

Direct evidence for the requirement of transcriptional feedback repression in circadian clock function has been elusive. Here, we developed a molecular genetic screen in mammalian cells to identify mutants of the circadian transcriptional activators CLOCK and BMAL1, which were uncoupled from CRYPTOCHROME (CRY)-mediated transcriptional repression. Notably, mutations in the PER-ARNT-SIM domain of CLOCK and the C terminus of BMAL1 resulted in synergistic insensitivity through reduced physical interactions with CRY. Coexpression of these mutant proteins in cultured fibroblasts caused arrhythmic phenotypes in population and single-cell assays. These data demonstrate that CRY-mediated repression of the CLOCK/BMAL1 complex activity is required for maintenance of circadian rhythmicity and provide formal proof that transcriptional feedback is required for mammalian clock function.

3T3 Cells↗

PubMed2004

A functional genomics strategy reveals Rora as a component of the mammalian circadian clock.

The mammalian circadian clock plays an integral role in timing rhythmic physiology and behavior, such as locomotor activity, with anticipated daily environmental changes. The master oscillator resides within the suprachiasmatic nucleus (SCN), which can maintain circadian rhythms in the absence of synchronizing light input. Here, we describe a genomics-based approach to identify circadian activators of Bmal1, itself a key transcriptional activator that is necessary for core oscillator function. Using cell-based functional assays, as well as behavioral and molecular analyses, we identified Rora as an activator of Bmal1 transcription within the SCN. Rora is required for normal Bmal1 expression and consolidation of daily locomotor activity and is regulated by the core clock in the SCN. These results suggest that opposing activities of the orphan nuclear receptors Rora and Rev-erb alpha, which represses Bmal1 expression, are important in the maintenance of circadian clock function.

ARNTL Transcription Factors↗

PubMed2003

Melanopsin is required for non-image-forming photic responses in blind mice.

Although mice lacking rod and cone photoreceptors are blind, they retain many eye-mediated responses to light, possibly through photosensitive retinal ganglion cells. These cells express melanopsin, a photopigment that confers this photosensitivity. Mice lacking melanopsin still retain nonvisual photoreception, suggesting that rods and cones could operate in this capacity. We observed that mice with both outer-retinal degeneration and a deficiency in melanopsin exhibited complete loss of photoentrainment of the circadian oscillator, pupillary light responses, photic suppression of arylalkylamine-N-acetyltransferase transcript, and acute suppression of locomotor activity by light. This indicates the importance of both nonvisual and classical visual photoreceptor systems for nonvisual photic responses in mammals.

Animals↗

PubMed2003

An array of insights: application of DNA chip technology in the study of cell biology.

The advent of DNA microarray technology has ushered in an era of systems biology whereby researchers can study the transcriptional behavior of thousands of genes in parallel. Advances in manufacturing techniques and informatics, and the availability of several genome sequences have furthered these capabilities to the point where whole-transcriptome studies can be accomplished in yeast, flies and plants, and soon will be possible in mammals. Concomitant with the expanding ability of the technology has been the development of novel techniques and their application towards the study of cellular biology.

Animals↗

PubMed2003

DNA arrays: applications and implications for circadian biology.

DNA arrays can be powerful tools to investigate transcriptional regulation of biological systems in a highly parallel and genome-wide manner. Their use has furthered the investigation of basic cell and developmental biology and is now being applied toward the understanding and diagnosis of human disease. DNA arrays are of particular use in the study of circadian biology because the clock is, at its heart, a transcriptional/translational feedback loop. Here, we review the underlying technology behind DNA arrays, discuss general applications, and focus on the use of the arrays in the study of circadian biology in plants, flies, and mice.

Animals↗

PubMed2002

Melanopsin (Opn4) requirement for normal light-induced circadian phase shifting.

The master circadian oscillator in the hypothalamic suprachiasmatic nucleus is entrained to the day/night cycle by retinal photoreceptors. Melanopsin (Opn4), an opsin-based photopigment, is a primary candidate for photoreceptor-mediated entrainment. To investigate the functional role of melanopsin in light resetting of the oscillator, we generated melanopsin-null mice (Opn4-/-). These mice entrain to a light/dark cycle and do not exhibit any overt defect in circadian activity rhythms under constant darkness. However, they display severely attenuated phase resetting in response to brief pulses of monochromatic light, highlighting the critical role of melanopsin in circadian photoentrainment in mammals.

Animals↗

PubMed2002

Cooperative binding of the cytoplasm to vacuole targeting pathway proteins, Cvt13 and Cvt20, to phosphatidylinositol 3-phosphate at the pre-autophagosomal structure is required for selective autophagy.

Autophagy is a catabolic membrane-trafficking mechanism involved in cell maintenance and development. Most components of autophagy also function in the cytoplasm to vacuole targeting (Cvt) pathway, a constitutive biosynthetic pathway required for the transport of aminopeptidase I (Ape1). The protein components of autophagy and the Cvt pathway include a putative complex composed of Apg1 kinase and several interacting proteins that are specific for either the Cvt pathway or autophagy. A second required complex includes a phosphatidylinositol (PtdIns) 3-kinase and associated proteins that are involved in its activation and localization. The majority of proteins required for the Cvt and autophagy pathways localize to a perivacuolar pre-autophagosomal structure. We show that the Cvt13 and Cvt20 proteins are required for transport of precursor Ape1 through the Cvt pathway. Both proteins contain phox homology domains that bind PtdIns(3)P and are necessary for membrane localization to the pre-autophagosomal structure. Functional phox homology domains are required for Cvt pathway function. Cvt13 and Cvt20 interact with each other and with an autophagy-specific protein, Apg17, that interacts with Apg1 kinase. These results provide the first functional connection between the Apg1 and PtdIns 3-kinase complexes. The data suggest a role for PtdIns(3)P in the Cvt pathway and demonstrate that this lipid is required at the pre-autophagosomal structure.

Amino Acid Sequence↗

PubMed2002

Regulation of Fab1 phosphatidylinositol 3-phosphate 5-kinase pathway by Vac7 protein and Fig4, a polyphosphoinositide phosphatase family member.

The Saccharomyces cerevisiae FAB1 gene encodes the sole phosphatidylinositol 3-phosphate [PtdIns(3)P] 5-kinase responsible for synthesis of the polyphosphoinositide PtdIns(3,5)P(2). VAC7 encodes a 128-kDa transmembrane protein that localizes to vacuolar membranes. Both vac7 and fab1 null mutants have dramatically enlarged vacuoles and cannot grow at elevated temperatures. Additionally, vac7Delta mutants have nearly undetectable levels of PtdIns(3,5)P(2), suggesting that Vac7 functions to regulate Fab1 kinase activity. To test this hypothesis, we isolated a fab1 mutant allele that bypasses the requirement for Vac7 in PtdIns(3,5)P(2) production. Expression of this fab1 allele in vac7Delta mutant cells suppresses the temperature sensitivity, vacuolar morphology, and PtdIns(3,5)P(2) defects normally exhibited by vac7Delta mutants. We also identified a mutant allele of FIG4, whose gene product contains a Sac1 polyphosphoinositide phosphatase domain, which suppresses vac7Delta mutant phenotypes. Deletion of FIG4 in vac7Delta mutant cells suppresses the temperature sensitivity and vacuolar morphology defects, and dramatically restores PtdIns(3,5)P(2) levels. These results suggest that generation of PtdIns(3,5)P(2) by the Fab1 lipid kinase is regulated by Vac7, whereas turnover of PtdIns(3,5)P(2) is mediated in part by the Sac1 polyphosphoinositide phosphatase family member Fig4.

Alleles↗