Publications by authors named "Lalanthi Ratnayake"

2 Publications

  • Page 1 of 1

Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in .

J Biol Rhythms 2021 Apr 7:748730421999948. Epub 2021 Apr 7.

Department of Biology, York University, Toronto, ON, Canada.

Molecular models for the endogenous oscillators that drive circadian rhythms in eukaryotes center on rhythmic transcription/translation of a small number of "clock genes." Although substantial evidence supports the concept that negative and positive transcription/translation feedback loops (TTFLs) are responsible for regulating the expression of these clock genes, certain rhythms in the filamentous fungus continue even when clock genes (, , and ) are not rhythmically expressed. Identification of the rhythmic processes operating outside of the TTFL has been a major unresolved area in circadian biology. Our lab previously identified a mutation () that abolishes FRQ-less rhythmicity of the conidiation rhythm and also affects rhythmicity when FRQ is functional. Further studies identified the gene product as a component of the TOR (Target of Rapamycin) nutrient-sensing pathway that is conserved in eukaryotes. We now report the discovery of TOR pathway components including GTR2 (homologous to the yeast protein Gtr2, and RAG C/D in mammals) as binding partners of VTA through co-immunoprecipitation (IP) and mass spectrometry analysis using a VTA-FLAG strain. Reciprocal IP with GTR2-FLAG found VTA as a binding partner. A Δ strain was deficient in growth responses to amino acids. Free-running conidiation rhythms in a FRQ-less strain were abolished in Δ. Entrainment of a FRQ-less strain to cycles of heat pulses demonstrated that Δ is defective in entrainment. In all of these assays, Δ is similar to Δ. In addition, expression of GTR2 protein was found to be rhythmic across two circadian cycles, and functional VTA was required for GTR2 rhythmicity. FRQ protein exhibited the expected rhythm in the presence of GTR2 but the rhythmic level of FRQ dampened in the absence of GTR2. These results establish association of VTA with GTR2, and their role in maintaining functional circadian rhythms through the TOR pathway.
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http://dx.doi.org/10.1177/0748730421999948DOI Listing
April 2021

A component of the TOR (Target Of Rapamycin) nutrient-sensing pathway plays a role in circadian rhythmicity in Neurospora crassa.

PLoS Genet 2018 06 20;14(6):e1007457. Epub 2018 Jun 20.

Department of Biology, York University, Toronto, ON, Canada.

The TOR (Target of Rapamycin) pathway is a highly-conserved signaling pathway in eukaryotes that regulates cellular growth and stress responses. The cellular response to amino acids or carbon sources such as glucose requires anchoring of the TOR kinase complex to the lysosomal/vacuolar membrane by the Ragulator (mammals) or EGO (yeast) protein complex. Here we report a connection between the TOR pathway and circadian (daily) rhythmicity. The molecular mechanism of circadian rhythmicity in all eukaryotes has long been thought to be transcription/translation feedback loops (TTFLs). In the model eukaryote Neurospora crassa, a TTFL including FRQ (frequency) and WCC (white collar complex) has been intensively studied. However, it is also well-known that rhythmicity can be seen in the absence of TTFL functioning. We previously isolated uv90 as a mutation that compromises FRQ-less rhythms and also damps the circadian oscillator when FRQ is present. We have now mapped the uv90 gene and identified it as NCU05950, homologous to the TOR pathway proteins EGO1 (yeast) and LAMTOR1 (mammals), and we have named the N. crassa protein VTA (vacuolar TOR-associated protein). The protein is anchored to the outer vacuolar membrane and deletion of putative acylation sites destroys this localization as well as the protein's function in rhythmicity. A deletion of VTA is compromised in its growth responses to amino acids and glucose. We conclude that a key protein in the complex that anchors TOR to the vacuole plays a role in maintaining circadian (daily) rhythmicity. Our results establish a connection between the TOR pathway and circadian rhythms and point towards a network integrating metabolism and the circadian system.
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http://dx.doi.org/10.1371/journal.pgen.1007457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028147PMC
June 2018