My research is aimed at answering this question: How do living things tell time? I am interested in circadian rhythms, the daily activity cycles driven by internal clocks in all eukaryotes and some prokaryotes. The goal of my research is to describe the mechanism of a circadian clock at the molecular and biochemical level. Because circadian rhythmicity is a fundamental property of all eukaryotic cells, an understanding of the mechanism of rhythmicity will give us important insights into how cells function.
I work with the filamentous fungus Neurospora crassa, a model organism that is at the forefront of circadian rhythm research. We use the rhythm of spore-formation (conidiation) as a visible marker for the state of the internal clock. Previous research has shown that the FRQ, WC-1 and WC-2 proteins are important for rhythmicity in Neurospora, but recent work has shown that conidiation rhythms can continue in their absence. I am interested in finding the oscillator that drives rhythmicity in the absence of FRQ (the FRQ-less oscillator, or FLO).
I have found that a mutation in lipid metabolism, chol-1, reveals rhythmicity in FRQ-less strains growing in constant conditions. I have also demonstrated that cycles of heat pulses reveal the existence of the FLO in FRQ-less strains without the chol-1 mutation. We are using these conditions as tools to visualize FRQ-less rhythms and assay the functioning of the FRQ-less oscillator. Our current goal is to identify the components of the FLO, determine how they interact to produce an oscillator mechanism, and determine how that oscillator interacts with the FRQ/WCC oscillator.
My lab’s strategy is to search for genes that affect the FLO, by using standard genetics to introduce known clock mutations into FRQ-less strains, and by mutagenesis to create new mutations affecting FLO. We have identified several mutations that disrupt FLO and we are characterizing these new FLO-affecting genes. We have mapped and identified two of these genes, and we are now carrying out functional analyses to determine where and when the products of these genes are expressed, and what proteins they interact with. We are also looking at the effects of our FLO-affecting mutations on the expression of clock-controlled genes and biochemical rhythms, and on the function of the FRQ/WC oscillator.
Graduate and undergraduate student projects are available. Some potential topics and methods include:
- Using standard genetics techniques to construct double-mutant fungal strains to assay the interactions between different clock-affecting mutations
- Using genetic engineering techniques to construct fungal strains carrying altered clock-affecting genes
- Using immunoblotting, qRT-PCR and Northern blotting to look at rhythmicity of clock-controlled proteins and their RNAs
- Using fluorescence microscopy of fluorescent protein-tagged strains to look at the subcellular localization of proteins
- Using co-immunoprecipitation to look at protein-protein interactions
- Using time-lapse photography to assay rhythms in spore formation