Publications by authors named "Patricia L Lakin-Thomas"

7 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

The genetics of circadian rhythms in Neurospora.

Adv Genet 2011 ;74:55-103

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

This chapter describes our current understanding of the genetics of the Neurospora clock and summarizes the important findings in this area in the past decade. Neurospora is the most intensively studied clock system, and the reasons for this are listed. A discussion of the genetic interactions between clock mutants is included, highlighting the utility of dissecting complex mechanisms by genetic means. The molecular details of the Neurospora circadian clock mechanism are described, as well as the mutations that affect the key clock proteins, FRQ, WC-1, and WC-2, with an emphasis on the roles of protein phosphorylation. Studies on additional genes affecting clock properties are described and place these genes into two categories: those that affect the FRQ/WCC oscillator and those that do not. A discussion of temperature compensation and the mutants affecting this property is included. A section is devoted to the observations pertinent to the existence of other oscillators in this organism with respect to their properties, their effects, and their preliminary characterization. The output of the clock and the control of clock-controlled genes are discussed, emphasizing the phasing of these genes and the layers of control. In conclusion, the authors provide an outlook summarizing their suggestions for areas that would be fruitful for further exploration.
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http://dx.doi.org/10.1016/B978-0-12-387690-4.00003-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5027897PMC
December 2011

New models for circadian systems in microorganisms.

FEMS Microbiol Lett 2006 Jun;259(1):1-6

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

Microorganisms provide important model systems for studying circadian rhythms, and they are overturning established ideas about the molecular mechanisms of rhythmicity. The transcription/translation feedback model that has been accepted as the basis of circadian clock mechanisms in eukaryotes does not account for old data from the alga Acetabularia demonstrating that transcription is not required for rhythmicity. Moreover, new results showing in vitro rhythmicity of KaiC protein phosphorylation in the cyanobacterium Synechococcus, and rhythmicity in strains of the fungus Neurospora carrying clock gene null mutations, require new ways of looking at circadian systems.
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http://dx.doi.org/10.1111/j.1574-6968.2006.00211.xDOI Listing
June 2006

Transcriptional feedback oscillators: maybe, maybe not...

J Biol Rhythms 2006 Apr;21(2):83-92

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

The molecular mechanism of circadian rhythmicity is usually modeled by a transcription/translation feedback oscillator in which clock proteins negatively feed back on their own transcription to produce rhythmic levels of clock protein mRNAs, which in turn cause the production of rhythmic levels of clock proteins. This mechanism has been applied to all model organisms for which molecular data are available. This review summarizes the increasing number of anomalous observations that do not fit the standard molecular mechanism for the model organisms Acetabularia, Synechococcus, Drosophila, Neurospora, and mouse. The anomalies fall into 2 classes: observations of rhythmicity in the organism when transcription of clock genes is held constant, and rhythmicity in the organism when clock gene function is missing in knockout mutants. It is concluded that the weight of anomalies is now so large that the standard transcription/translation mechanism is no longer an adequate model for circadian oscillators. Rhythmic transcription may have other functions in the circadian system, such as participating in input and output pathways and providing robustness to the oscillations. It may be most useful to think in terms of a circadian system that uses a noncircadian oscillator consisting of metabolic feedback loops, which acquires its circadian properties from additional regulatory molecules such as the products of canonical clock genes.
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http://dx.doi.org/10.1177/0748730405286102DOI Listing
April 2006

Circadian clock genes frequency and white collar-1 are not essential for entrainment to temperature cycles in Neurospora crassa.

Proc Natl Acad Sci U S A 2006 Mar 14;103(12):4469-74. Epub 2006 Mar 14.

Department of Biology, York University, Toronto, Ontario, Canada M3J 1P3.

The fungus Neurospora crassa is a model system for investigating the mechanism of circadian rhythmicity, and the core of its circadian oscillator is thought to be a transcription/translation feedback loop involving the products of the frq (frequency), wc-1 (white-collar-1) and wc-2 (white-collar-2) genes. Several reports of rhythmicity in frq and wc null mutants have raised questions about how central the FRQ/WC loop is to the circadian system of Neurospora. Several research groups have attempted to answer this question by looking for entrainment of the conidiation banding rhythm in frq null mutants. Because the frq mutants are blind to light and cannot be entrained to light/dark cycles, these groups have used symmetric temperature cycles of equal-duration cool and warm phases to entrain the rhythm. Under these conditions, the direct effects of temperature on conidiation (masking effects) can compromise observations of the endogenous rhythm. I have reexamined this question by using short heat pulses to clearly separate masking from endogenous rhythms, and I have assayed entrainment in both frq and wc-1 null mutants. I found similar patterns of entrainment in the wild type and both mutant strains. Strong masking effects were found in the frq mutant but not in the wc-1 mutant. I conclude that a rapidly damping temperature-entrainable oscillator is present in the null mutants. A single temperature-entrainable oscillator may drive the conidiation rhythm in all strains, and additional properties such as light sensitivity and temperature compensation may be conferred by the intact FRQ/WC loop in the WT strain.
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http://dx.doi.org/10.1073/pnas.0510404103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1450195PMC
March 2006

Time-lapse analysis of the circadian rhythms of conidiation and growth rate in neurospora.

J Biol Rhythms 2004 Dec;19(6):493-503

Division of Science and Mathematics, University of Minnesota-Morris, Morris, Minnesota, USA.

The filamentous fungus Neurospora crassa has frequently served as a model organism for the study of circadian rhythms through its ability to form conidial spores on a daily basis. This phenomenon leaves a spatial pattern of conidiation bands along a solid surface of agar after several days of growth. Using time-lapse video, the authors have quantified the rate of conidiation. They have found that conidia do not form at a specified lag time after the growth front is laid down, but rather the band region tends to simultaneously develop over a short time frame. This produces a sharp peak when the conidiation rate is plotted against time. In addition, the authors used time-lapse video to assay growth rate with greater accuracy than previously reported. It is usually assumed that Neurospora's rate of growth is constant, and this assumption of linear growth has been used extensively to determine period and phase of the conidiation circadian rhythm. The authors have confirmed an earlier report of nonlinear growth rate and have shown that the growth rate varies by a factor of about 2 with each circadian cycle. They have demonstrated that the errors in calculating times of conidiation peaks are maximally 1 to 2 h if linearity is assumed. The conidiation rate and growth rate rhythms are not apparent under conditions (using mutants or high or low temperatures) where the spatial banding rhythm is not observed. In light/dark entraining conditions, the conidiation rate and growth rate rhythms maintain the same phase relationship in different T-cycles. These data are consistent with the hypothesis that the growth rate rhythm is a consequence of the conidiation rate rhythm.
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http://dx.doi.org/10.1177/0748730404270391DOI Listing
December 2004

Circadian rhythms in microorganisms: new complexities.

Annu Rev Microbiol 2004 ;58:489-519

Department of Biology, York University, Toronto, ON M3J 1P3, Canada.

Recent advances in understanding circadian (daily) rhythms in the genera Neurospora, Gonyaulax, and Synechococcus are reviewed and new complexities in their circadian systems are described. The previous model, consisting of a unidirectional flow of information from input to oscillator to output, has now expanded to include multiple input pathways, multiple oscillators, multiple outputs; and feedback from oscillator to input and output to oscillator. New posttranscriptional features of the frq/white-collar oscillator (FWC) of Neurospora are described, including protein phosphorylation and degradation, dimerization, and complex formation. Experimental evidence is presented for frq-less oscillator(s) (FLO) downstream of the FWC. Mathematical models of the Neurospora system are also discussed.
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http://dx.doi.org/10.1146/annurev.micro.58.030603.123744DOI Listing
December 2004