Neurospora crassa: Circadian Rhythm

Temperature-Sensitive and Circadian Oscillatorsof Neurospora crassa Share Components

Introduction

Circadian rhythms are an endogenous part of plants, animals, and even bacteria. The rhythms are an innate part of an organism’s homeostasis that temporally organizes it for a 24-hour timescale. This biological clock responds to day-light and temperature cycles of a specific environment through a process known as entrainment (2). This allows an  organism to perform certain activities at a time that provides the best evolutionary value.  Neurospora crassa, a eukaryotic model organism, has played a major role in investigating the key molecular processes involved in maintaining a circadian clock.  It has been found that N. crassa keeps its clock through a series of autoregulatory feedback loops that involve four genes: frequency (frq), frq-interacting helicase (frh), white collar-1 (wc-1), and white collar-2 (wc-2). These proteins together create what is known as the frequency white collar oscillator (FWO). For N. crassa, the easiest way to measure the circadian rhythm is through asexual spore development, or conidiation. The purpose of conidiation is for N. crassa to disperse and protect its genome in adverse environmental conditions (3). In a wild-type colony, exposed to only dark and a constant temperature, go through conidiation with a 22-hour periodicity. Studies have shown that any disturbance in the FWO leads to a lapse in the circadian rhythm.  However, studies have also shown that mutants lacking only one of the core proteins in the FWO can still lead to a circadian dependent spore development under certain conditions.

Goal

The goal of this experiment was to determine the molecular factors behind a temperature-induced, frequency less oscillator (FLO). To do this, a complete FWO mutant strain was created  i.e.  all of the core genes of the FWO were knocked out (ΔFWO). Previous studies have only knocked out a single component, but this would leave the possibility that a temperature induced oscillator was the result of the remaining components. The creation of a ΔFWO would benefit future studies as a tool that could uncover novel oscillators independent of the FWO.

Results

Is the ΔFWO mutant a viable mutant for circadian rhythm studies??

The phenotype of the ΔFWO was tested to determine if it possessed any significant growth defects. Figure 1 shows that it did not possess and significant growth defects. It even grew similarly to the wt in the constant dark environment.

Well that is settled, but how does temperature affect its rhythm?

To test the temperature the dependence of the ΔFWO, its growth was compared alongside the wt, Δfrq, and Δwc mutants. The first tests were performed under constant dark at 22  with two hour perturbations of 30 . If the growth was independent of the temperature disturbances, meaning it is oscillator controlled, the peaks would also be independent of the temperature. The wt and Δfrq showed growth independent of the temperature changes. The Δwc-1 and ΔFWO were dependent on the temperature and growth decreases significantly at the onset of the 2-hour temperature increase.

Figure II: Conidiation rhythm in skeleton temperature cycles for wild-type, Δfrq, Δwc-1, and ΔFWO.

This result shows that the presence of the circadian like clock in the wt and the Δfrq was somehow dependent on the presence of a functioning wc-1 and wc-2 (white collar complex, WCC). This can be assumed because the Δwc and ΔFWO mutants both lacked functioning WCC where the wild-type and the Δfrq mutants possessed functioning WCC.

Who knew?! The WCC is required for creating frequency less oscillators rhythms!

Figure III: The temperature dependent experiments carried out in constant light conditions. It can be seen that the mutants lacking both WC-1 or WC-2 are dependent on temperature for rhythmic behavior (E and G).

The temperature dependence experiments were repeated in constant light conditions. Because only the wild type possessed a fully functional WCC, the conclusion was reached that the white collar genes are crucial for creating a circadian rhythm in cyclic temperature protocols. Obviously, the white collar and FWO mutants will not possess a functional WCC because they lack these genes all together. The frq mutant doesn’t possess a functional WCC because it contains low levels of WC-1. However, even this low level allowed for a slight rescue of the temperature independence and the FLO. In strains that possess no frq, but contain the WC-1 and WC-2 transcripts, a FLO still appears, but it is intermediate between a normal oscillator and one that is temperature dependent. Therefore, only wild type strains, possessing a full complement of frequency and white collar complex  possessed a circadian clock independent of the temperature pulses.

Major Conclusions!!

1) The ΔFWO did not show any significant growth defects. This makes it an ideal mutant for studying the novel molecular mechanisms underlying the circadian rhythms of Neurospora crassa.

2) A functioning white collar complex (WCC) is required for the development of a temperature inducedFLO.

3) Frq recruits the WCC in order to produce a robust wild-type circadian rhythm.

Strengths and Weaknesses

Strengths: The authors do a  good job in the introductions of laying out the specific genes involve in the circadian rhythm of N. crassa. They create a solid background required for the understanding of the results and their implications. Also, I appreciated how the authors combined the results and discussion. You encounter the results and their importance immediately after.

Weaknesses: Throughout the paper, the parenthetical citations the authors use break up the flow of the paper in a negative way. Some of the citations are overly long and interfere with the understanding of what is being said

Helpful Links

http://en.wikipedia.org/wiki/Circadian_rhythm

References

1. Hunt S, Elvin M, Heintzen C. Temperature-sensitive and circadian oscillators of Neurospora crassa share components. Genetics [serial online]. May 2012;191(1):119-131. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338254/

2. Ruoff P. Loros JJ. Dunlap JC. The relationship between FRQ-protein stability and temperature compensation in the Neurospora circadian clock. Proceedings of the National Academy of Sciences of the United States of America. 2005 Dec 6;102(49):17681-6. Epub 2005 Nov 28

3. Osherov N. May GS. The molecular mechanisms of conidial germination. FEMS Microbiology Letters. 2001 May 30;199(2):153-60.

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