truly circadian (see Dunlap and Loros 2017 for
a recent review). While there is much more to
learn about these cellular oscillations, it is
interesting that studies continue to find previously unknown links between these oscillations
and the circadian clock, such as a widely conserved daily oscillation in Mg
2+
, which can now
be explained by clock regulation of genes
involved in both active ion transport (via
pumps) and passive ion channels (Feeney
et al. 2016; Dunlap and Loros 2016).
In addition, the clock plays a role in the
regulation of the cell cycle. Hong et al. (2014)
found that there was an overall circadian
rhythm in mitosis, with most cells dividing in
the evening, while morning is the peak time for
cells to be in interphase. The circadian clock
rhythmically regulates the kinase STK-29 and
the G1 and G2 cyclins, CLN-1 and CLB-1, which
in turn regulate entry into mitosis (Hong et al.
2014). The clock and cell cycle are also
interconnected by Chk2 (PRD-4 in N. crassa),
a key kinase in both processes, and it is through
this kinase that the cell cycle can impact the
clock (Pregueiro et al. 2006). Chk2 binds to
FRQ and phosphorylates it in a DNA damagedependent manner, triggering destabilization
and leading to FRQ degradation, shifting the
phase of the clock and enabling circadian
responsiveness to DNA damage (Gamsby et al.
2009; Pregueiro et al. 2006). This research in N.
crassa demonstrates that the cross-talk between
the circadian clock and cellular systems contributes to the optimal timing of cellular growth
and development, important relationships that
are conserved and are now being elucidated in
higher eukaryotes (e.g., Gaucher et al. 2018;
Reinke and Asher 2019).
V. Methods for Detecting Circadian
Rhythms
As noted earlier (see Sect. I), for something to
be defined as a circadian rhythm, the period
must be free running and at or near 24 h. Therefore, to identify circadian rhythms in Neurospora crassa, it is standard procedure to collect a
time series of regularly recorded observations
under constant conditions, after a strong resetting cue is given. From this time series, the
period, amplitude, and phase can be calculated,
as it can be for any oscillatory wave. To look at
rhythms in vivo in N. crassa, two common
methods are utilized: the “band” or bd mutant
strain background and a luciferase reporter
system. To track the period of the clock using
the bd mutant background, the strain of interest with a bd background is grown in constant
conditions on a race-tube, a long glass tube
with agar media in the bottom (Fig. 4.1).
Bands of conidia and aerial hyphae form at
regular intervals as the strain grows and the
periodicity of this banding is linked to the circadian clock (Brandt 1953; Pittendrigh et al.
1959). This banding is used as a proxy for the
clock but in reality is a measure of the periodicity of the clock’s output.
To look more directly at the core clock, a
luciferase reporter system was developed using
an N. crassa codon-optimized luciferase gene
from fireflies fused to a promoter region or
gene of interest. When fused to the core clock
gene frq or its promoter, this system allows for
the tracking of changes in steady-state protein
levels or transcriptional activity, respectively,
over the course of multiple days (Gooch et al.
2008; Larrondo et al. 2012; Morgan et al. 2003).
With the advent of RNA microarrays, RNAsequencing and mass spectrometry, ex vivo
methods of tracking rhythms in mRNA or protein levels have allowed further investigation of
circadian regulation within the cell. Samples are
extracted from tissue grown under constant
conditions, which has been shown to preserve
the oscillations in clock genes and proteins in
N. crassa (Loros et al. 1989). From the extracted
samples, a variety of assays and computational
approaches can be performed to measure
rhythms in the core clock and identify clock
output, including Western blots, qPCR, RNAseq, and proteomics (e.g., Hurley et al. 2014,
2018). Novel techniques and technologies are
developed in N. crassa on a regular basis to
allow circadian biologists to learn more about
the core clock and its regulation of cellular
output (e.g., De los Santos et al. 2017; Pelham
et al. 2018).
94
M. S. Jankowski et al.
a recent review). While there is much more to
learn about these cellular oscillations, it is
interesting that studies continue to find previously unknown links between these oscillations
and the circadian clock, such as a widely conserved daily oscillation in Mg
2+
, which can now
be explained by clock regulation of genes
involved in both active ion transport (via
pumps) and passive ion channels (Feeney
et al. 2016; Dunlap and Loros 2016).
In addition, the clock plays a role in the
regulation of the cell cycle. Hong et al. (2014)
found that there was an overall circadian
rhythm in mitosis, with most cells dividing in
the evening, while morning is the peak time for
cells to be in interphase. The circadian clock
rhythmically regulates the kinase STK-29 and
the G1 and G2 cyclins, CLN-1 and CLB-1, which
in turn regulate entry into mitosis (Hong et al.
2014). The clock and cell cycle are also
interconnected by Chk2 (PRD-4 in N. crassa),
a key kinase in both processes, and it is through
this kinase that the cell cycle can impact the
clock (Pregueiro et al. 2006). Chk2 binds to
FRQ and phosphorylates it in a DNA damagedependent manner, triggering destabilization
and leading to FRQ degradation, shifting the
phase of the clock and enabling circadian
responsiveness to DNA damage (Gamsby et al.
2009; Pregueiro et al. 2006). This research in N.
crassa demonstrates that the cross-talk between
the circadian clock and cellular systems contributes to the optimal timing of cellular growth
and development, important relationships that
are conserved and are now being elucidated in
higher eukaryotes (e.g., Gaucher et al. 2018;
Reinke and Asher 2019).
V. Methods for Detecting Circadian
Rhythms
As noted earlier (see Sect. I), for something to
be defined as a circadian rhythm, the period
must be free running and at or near 24 h. Therefore, to identify circadian rhythms in Neurospora crassa, it is standard procedure to collect a
time series of regularly recorded observations
under constant conditions, after a strong resetting cue is given. From this time series, the
period, amplitude, and phase can be calculated,
as it can be for any oscillatory wave. To look at
rhythms in vivo in N. crassa, two common
methods are utilized: the “band” or bd mutant
strain background and a luciferase reporter
system. To track the period of the clock using
the bd mutant background, the strain of interest with a bd background is grown in constant
conditions on a race-tube, a long glass tube
with agar media in the bottom (Fig. 4.1).
Bands of conidia and aerial hyphae form at
regular intervals as the strain grows and the
periodicity of this banding is linked to the circadian clock (Brandt 1953; Pittendrigh et al.
1959). This banding is used as a proxy for the
clock but in reality is a measure of the periodicity of the clock’s output.
To look more directly at the core clock, a
luciferase reporter system was developed using
an N. crassa codon-optimized luciferase gene
from fireflies fused to a promoter region or
gene of interest. When fused to the core clock
gene frq or its promoter, this system allows for
the tracking of changes in steady-state protein
levels or transcriptional activity, respectively,
over the course of multiple days (Gooch et al.
2008; Larrondo et al. 2012; Morgan et al. 2003).
With the advent of RNA microarrays, RNAsequencing and mass spectrometry, ex vivo
methods of tracking rhythms in mRNA or protein levels have allowed further investigation of
circadian regulation within the cell. Samples are
extracted from tissue grown under constant
conditions, which has been shown to preserve
the oscillations in clock genes and proteins in
N. crassa (Loros et al. 1989). From the extracted
samples, a variety of assays and computational
approaches can be performed to measure
rhythms in the core clock and identify clock
output, including Western blots, qPCR, RNAseq, and proteomics (e.g., Hurley et al. 2014,
2018). Novel techniques and technologies are
developed in N. crassa on a regular basis to
allow circadian biologists to learn more about
the core clock and its regulation of cellular
output (e.g., De los Santos et al. 2017; Pelham
et al. 2018).
94
M. S. Jankowski et al.