Evolution and our daily rhythms
Gen Kurosawa & Shingo Gibo (iTHEMS, RIKEN)
Consecu(ve*days*in*caveSleep>wake*cycle
awake
asleep

ShingoTime*of*day*(h)
Palmer*(2002)

Gibo and Kurosawa (2019) Biophys J
Gibo, Kunihiro, Hatsuda, Kurosawa in prep

The biggest challenge in the field of daily rhythms:
understanding sleep and sleep types
6pm

12am

6am

normaladvanceddelayedNon-24hnarcolepsy• Sleep types are related to daily rhythms.
• There are many people who are suffering from sleep disorder• Genes are important but, environments are also important.
• Sleep types change with ages.

The biggest mystery in the field of daily rhythms:“Why is our daily rhythms stable to temperature?”(Ex) Mammalian NIH3T3 cells

Daily rhythms
Cell division

Akin (2011)

Tsuchiya,..Nishida (2003) Genes Cells

Figure 2 Period and frequency estimates of circadian rhythm andcell cycle of NIH3T3 fibroblasts.The mean period and frequencyof circadian rhythm (!) and cell cycle (") were calculated for eachtreatment group. The value of each gene in the treatment groupis plotted as an open circle.

While enzymatic reactions usually accelerate with temperature,
the period of daily rhythms is stable to temperature.

To quantitatively analyze data of daily rhythms,
we focus on waveform distortion from sine-wave
sinusoidal Time-series, x4(t)⎡2 4⎤⎢∑ aj j ⎥NS = ⎢⎢ j=1∞ 2 ⎥⎥2⎢∑ aj j ⎥
⎣ j=1
⎦
∞

NS = 1.0

NS = 2.0

12

waveform of a variable∞x4 (t) = ∑ a j e

NS = 3.50Time

72i2πjtTj=−∞(oscillation can be expressed bysum of trigonometric function)

non-sinusoidal“We introduced the index, non-sinusoidal power (NS)”Gibo and Kurosawa (2019) Biophys J

To obtain theoretical basis of waveform,
we analyze the simple model
180 variable
Detailed model
Zhou et al. (2015)
Mol. Cell

Gibo model for circadian rhythms

Genedownregulation

PP

Protein(x3)
p2

RNA (x1)
124

p1

Protein(x2)

simplification
dx1= f (x3 ) − k1 x1 ,dt
dx2
= p1 x 1 − k 2 x 2 ,dt
dx3= p2 x 2 − k 3 x 3 ,dt
f(x3): any function

Fustin,..,Gibo,.., Kurosawa et al (2018) PNAS: decay

where x1 (t), x2 (t), Gibo
andandx3Kurosawa(t) are(2019)
concentrationof proteins, f (x3 ) iBiophys JResult 1/3

To obtain
theoreticalbasisof waveform,
we theoreticallyderived
the period
formulain the model as foll

mula, [

we derived the period of daily rhythms

2πPeriodτ == √k k + k k + k k
1 22 3
3 1"∞

2j4/|a|j=1 j"∞! "∞

2j4|a|
j=1 j"∞
2 2
j=1 |aj | j#12.

NS11222 si|a | j ] 2 . is waveform distortion fromjWaveform
Distortion (NS)j=1= 2πd as N SPeriod(Gibo andKurosawa 2019; Gibo and Kurosawa 2020).
Quadratic eq. of rates

orted waveform (larger N S) at higher temperature is required fo
1. Accelerated reactions tend to shorten period.
period. Indeed, a numerical simulation suggested that the wave
2. If accelerated reaction elongates period,
shouldbecome more
non-sinusoidal.
he period waveformis stable totemperaturechange(Fig. 2.2). Moreove
3. More non-sinusoidal at higher temp for stable period.
at entrainment region of the model became narrower when tem

Result 2/3

Period = 2π

Waveform Distortion (NS)12

Quadratic eq. of rates

Prediction: More non-sinusoidal waveform at higher temp generates
stable period to temperature in daily rhythms.

Low Temp

No waveform change

Waveform distortion

Period must shorten

Period can be stabilizedHigh Temp
Gibo and Kurosawa (2019) Biophys JResult 3/3

To obtain theoretical basis of waveform,
we solved the equation by the help of physics
Conventional perturbation approach: badx(t)= A1cos ωt + ε t A2 cos 2ωt + ..

Shingowhere Ai=fi(k1,k2,..), ω=g(k1,k2,..)Renormalization-group approachx(t)= A1cos ωt + ε A2 cos 2ωt + ..
“Now, we can understand waveform distortionby using the language of kinetic constants (ki)”Gibo, Kunihiro, Hatsuda, Kurosawa in prep

Other hypothesis:

Discussion 1/2

There should be a critical reaction for the period. If thatreaction is insensitive to temp, period can be stable to temp.

Isojima et al. (2009) PNAS, Shinohara et al. (2017) Mol Cell
(Prof Hiroki Ueda group (RIKEN/Univ Tokyo))Cf. Hong,.., Tyson (2007) PNAS, Terauchi et al. (2007) PNAS

Discussion 2/2
young (6mo)Daily rhythms in gene-activity
is known to change with age.
But we don’t know how.

aged (19month)Accosta-Rodriguez et al. (2022)
Science(Shingo’s idea)

From the analysis of waveform, cause of the change can be predicted.
Data of Young
Generation

Regulation of Aging (Simulation)

Mechanism
P

Data of Aged
Generationredi
ctionShingo(math)

Data analysis, ML

Quantificationof Aging(amp, waveform,..)Summary• Challenges about daily rhythms and sleep- sleep types- stability of daily rhythm to temperature• Waveform is an important indicator for period of daily rhythms• Dr. Singo Gibo is very smart (See poster #94 about hibernation)
Gibo and Kurosawa (2019) Biophys J
Gibo, Kunihiro, Hatsuda, Kurosawa in prep
JST CREST Biodynamics (2014-2020, PI: Prof Okamura (Kyoto U))
JST CREST Math and Info (2019-2025, PI: Prof Kawahara (Osaka U))
JSPS JP21K06105 and Continuous Support from RIKEN iTHEMS

SupplementarySuppose that you are in a cave..
Can you wake up tomorrow w/o clock?

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for
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also
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oursleeplog,
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andclintreated
with
CKIε(Fig. 4A).
the control
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gene foruse subjects
in fluorescencein
situ
wereunlinked(Fig.
1). Fouradditional
bjectsalso
supports
oursleeplog,
actigraphy
andclinmA
wasobtainedwith
wild-typhybridization
experiments(21).
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mappedto
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did
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ubjectsaddition,
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andfragments
clinment confirmed that this shi
we used a polymorphism in hPer2 tothey did show a strong tendency of early sleepTable
1Science;Phasemarkersof4). ov
Table
1Med,
Phase
markers
ofscoresovert
rhythms
Table
1
Phase
markersofovert
phosphorylation(Fig. NatuWh
genotype
K2174
and performed
two-pointlink-Tohwakepreference(Horne-Östberg
74.4rhythms" et(Jonesetal(1999)
Natetal.
(2001)
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Early-bird
familyintheworld
ment was repeated with a hig
age mapping with nine markers noted previous7.2; n # 4) (1).
markersovert
rhythms
Table1
1 PhasePhase
ofmutation
overt
rhythmstion of CKIε (6.5 ng/$l), mobily (13). A Table
recombination
demonstrated
theTo markers
establish whetherof
thiscauses

for both the wild-ty
FASPS, the S662G mutation was functionallyControlFASPS(nControlFASPS(n= 6)observed
DifferenhPER2Difference
fragments(Fig. 4B). TControl
FASPS(n= 6)S662
characterized (24).
To determine
whetherDr.
Fuof the phosphorylation status
is located within the CKIε binding site of(n=6)
Mean±pControl
FASPS(n=6)
Difference(n=6)
Mean±s.d(hours:minresidues
in thepeptide can be
CKIε, myc-epitope
taggedmPER2,FASPS(n= 6) (UCSF)
Differenc(nControl= 6) hPER2,
Mean±s.d(hours:minutewith excess kinase.
and the indicated truncation mutants of mPER2
Dr. PtacekCKIε
preferentiallyphospho(Fig.
3)
wereexpressed
inrabbit
reticulocyte
Mean±s.d.
(n=6)
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Mean±s.d.
6)
Mean ± s.d with (hours:minu
Mean(n±=s.d.
(UCSF)DD
acidic [for example,lysates and mPER2 peptides were immunoprephosphorylated residues [for excipitated with antibodies to myc (25). CKIε was
Mean
Mean± ±s.d.
s.d.
X-S] immediately upstream ofcoprecipitated with mPER2(1 to 763) but not
Sleep Onset
23:10±0:40
19:25±set
23:10±mPER2(10:40to 554),19:25
±1:44
3:45
23:10
± 0:4019:25±1:44
due (where D 3:45
is aspartate, S is sthus demonstrating that theX is any amino
CKIε binding site of mPER2 is located between
07:44
±1:44
1:13phosphoserine,
Sleep Offset
23:10
19:25
3:45
07:44
±0:40
1:13
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2:00
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seta
et
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underlined “S”04:18
is thetarget ±ofresidues
554 and04:18
763,
corresponding
to ±residues
phosphorylation) (29, 30). A
556 to 771 of hPER2 (Fig. 3).
a
1stSlow
Wave
Sleep
23:55
±
1:17
20:14
±a07:44
±
1:13
04:18
±2:00
3:26
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Sleep
23:55
±
1:17
20:14±2:35
3:41
reveals
fourStudies
ofdoubletime(dbt)
mutantsin
Dro07:44± 1:13 20:14
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3:26
et Sleepve
23:55± 1:17± 2:35
3:41
Human
Per2a gene
Fig.±2
1st REM
00:55
1:29
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ve Sleep
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structu
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tains
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abbnn==55for= 5 forDatacontrol
and bothDataofinclude
ASPS only.
forFASPS
controlonly.
andbFASPS.
includenightsstudy. botREM
hPer2 gene to be distal to marker D2S338. The
haplotype of the remaining eight markers wasfully linked to hPer2 in this family. The individuals in branch 3 were considered to represent phenocopies, and mutation analysis ofhPer2 was performed.
Human Per2 comprises 23 exons (Fig. 2A)(22). A sequencing error of the hPer2 cDNA
(GenBank accession number, NM 003894) wasidentified; the reported cDNA has a missing
base at position 3652 that shifts
a the reading
frame, predicting translation of 69 amino acids
that are not homologous to other PER proteins
before the stop codon. With the corrected se-occurs
S only. n = 5 for control and FASPS. Data include both nights of study. REM,
raThe “%Mutation
atthebinding
site
phosphorylation
enzymeb b DLMO,
dim-light
melatonin
onset;
Temp,temperature.
22 shom-lightmelatonin
onset;
Temp,temperature.
ght
melatoninTemp,temperature.
S only.
n =n 5forcontrolandFASPS.
Datainclude
bothnights
ofofstudy.
REM,SPSonly.
= onset;5 forcontrolandFASPS.
Data
includebothnights
study.
REMof theToh et al. (2001, 2007) Science(a 1 bght
melatonin
onset;
Temp,temperature.
-lightmelatonin
onset;
Temp,temperature.
tion)

cDNA
quencemutatCompetition experiment using
Evolution: Ouyang
et al.
an ancestorof plants(24h period type)(30hr environment)(24hr environment)

Ouyang et al. (1998) PNAS

“Resonance seems essential.”

P