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MCB 186 CIRCADIAN BIOLOGY Biochemistry of the Circadian Clock Lecture #3 October 3, 2007 J. W. Hastings. MODELING INPUT to and OUTPUT from THE CLOCK. - PowerPoint PPT Presentation
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MCB 186CIRCADIAN BIOLOGY
Biochemistry of the Circadian Clock
Lecture #3 October 3, 2007J. W. Hastings
MODELING INPUT to and OUTPUT from THE CLOCK
THIS IS ONLY A MODEL
DIFFERENT SYSTEMS MAY DIFFER: bacteria, plants, algae, fungi, animals
and, IT MAY BE INCORRECT
e.g., THERE MAY BE TWO CLOCKSor THREE or MORE
DIFFERENT OSCILLATORS CONTROL GLOW & FLASHING
Internnal Desynchronization
THREE RHYTHMS SIMULTANEOUSLY: PHASE-JUMPS ROENNEBERG & MORSE 1993
Glo
Fls Agg
INPUT to and OUTPUT from a TWO-CLOCK MODEL
CORE PACEMAKER OSCILLATOR
• BIOCHEMICAL ELEMENTS of the CLOCK
• AFFECTED by SIGNAL TRANSDUCTION
• CLOCK GENES vs CLOCK CONTROLLED
• CLOCK PROTEINS vs CLOCK CONTROLLED
INPUT PATHWAYS SIGNAL TRANSDUCTION
• MANY FACTORS AFFECT THE CLOCK
• EFFECTS on PHASE and PERIOD DISTINCT
• BIOCHEMICAL PATHWAYS UNKNOWN
OUTPUT PATHWAYS
HOW DOES the CLOCK TURN PROCESSES ON and OFF?
• TRANSCRIPTION: NEW mRNA, then protein
• TRANSLATION: REGULATE PROTEIN SYNTHEIS
• POST TRANSLATIONAL e.g. PHOSPHORYLATION
LUCIFERASE PROTEIN EXHIBITS A CIRCADIAN RHYTHM in LL
Johnson et al.1984Science 223
WesternBlot
WESTERN BLOTS LUCFERIN BINDING PROTEIN, LD & LL A CLOCK CONTROLLED GENE Morse et al., 1989 PNAS 86
GONYAULAX CELLS AT NIGHT (LEFT) AND DAY PHASES FLUORESCENCE OF LUCIFERIN IN SCINTILLONS
GONYAULAX CELLS AT NIGHT & DAY PHASES FLUORESCENCE OF LUCIFERIN IN SCINTILLONS
LBP mRNA DOES NOT CYCLE in GonyaulaxLBP SYNTHESIS & ABUNDANCE are STRONGLY CIRCADIAN
Morse et al., 1989 PNAS 86
LBP abundance
LBP synthesis
LBP mRNA
mRNA LEVELS ARE CONSTANT
SYNTHESIS of MANY PROTEINS is CIRCADIAN CONTROLLED In Vivo PULSE LABELING MILOS et al, 1989
MILOS ET AL, 1989Naturwisenschaften 77
SYNTHESIS of PROTEINS in vitro is NOT CLOCK CONTROLLED
MILOS ET AL, 1989Naturwisenschaften 77
PATTERNS of CLOCK-CONTROLLED PROTEIN SYNTHESIS in Gony
Markovic et al., 1996J. Biol. Rhythms 11
p21 unknown p32 PCPp33 OEE1p45 GAPDHp55 Rubisco IIp75 Luciferin binding
protein
ABUNDANCE vs SYNTHESIS
• SYNTHESIS RATE of a PROTEIN MAY EXHIBIT PRONOUNCED RHYTHM while RHYTHM in the ABUNDANCE of PROTEIN does NOT
• ABUNDANCE RHYTHM DEPENDS on STABILITY OF MOLECULE
GAPDH SYNTHESIS, ACTIVITY & ABUNDANCE RHYTHMSFagan, Morse & Hastings, 1999
HALF-LIFE of PROTEIN AFFECTS AMPLITUDE of ABUNDANCE RHYTHM
2 days
12 hr
IS THERE a CORE CIRCADIAN OSCILLATOR?
If so, HOW do we IDENTIFY the CELLULAR-BIOCHEMICAL
CLOCK COMPONENTS?
SPECIFIC INHIBITORS or MUTANTS AFFECTING CIRCADIAN RHYTHMS
SPECIFIC INHIBITORS can REVEAL PATHWAYS of CLOCK
BIOCHEMISTRY
PROTEIN synthesis inhibitorsPulses cause phase shifts
PROTEIN phosphorylation inhibitors Chronically cause period changes
PULSES of ANISOMYCIN (protein synthesis inhibitor)CAUSE PHASE SHIFTS in Gonyaulax
PHASE SHIFTS BY ANISOMYCIN 0.3 M, 1 HOUR
VERY BRIEF ANISOMYCIN PULSES CAUSE LARGE PHASE SHIFTS
TYPE 1 & 0 DRCs FOR BRIEF ANISOMYCIN PULSES
ARHYTHMICITY AT “CRITICAL” DOSE OF PHASE SHIFTING INHIBITOR
D-PRC for PHASE SHIFTS by an INHIBITOR of PROTEIN SYNTHESIS
D-PRC for PHASE SHIFTS by an INHIBITOR of PROTEIN SYNTHESIS
SPECIFIC INHIBITORS can REVEAL PATHWAYS of CLOCK
BIOCHEMISTRY
PROTEIN synthesis inhibitorspulses cause phase shifts
PROTEIN phosphorylation inhibitors chronically cause period changes
KINASES
6-DMAP (KINASE INHIBITOR) INCREASES Tau
Bioluminescence Experiment # 382
control
33µM
50 µM
75 µM
100 µM
120 µM
140 µM
160 µM
180 µM
200 µM
250 µM
275 µM
300 µM
350 µM
400 µM
1 2 3 4 5 6
day of experiment
21°C
time of 6-DMAP addition
Figure 1A
6-DMAP conc.
6_DMAP (Kinase Inhibitor) INCREASES Tau
Bioluminescence Exp.#381
90 180 270 360 450 540 630
control tau = 23.016
50µM 6-DMAP tau = 23.59
100µM 6-DMAP tau = 24.295
160µM 6-DMAP tau = 25.318
200µM 6-DMAP tau = 25.571
250µM 6-DMAP tau = 25.766
300µM 6-DMAP tau = 26.596
phase [°]
1
2
3
4
5
6
7
Figure 1C
6_DMAP (KINASE INHIB) INCREASES Tau
NO AFTER-EFFECT of EXPOSURE to 6-DMAP COMOLLI and HASTINGS, 1995
Bioluminescence Exp.# 393
90 180 270 360 450 540 630
control tau = 22.23
phase [°]
1
2
3
4
5
6
7
8
9
4 hour pul se t au = 2 2 .2 6
8 hour pulse tau = 22.26
12 hour pulse tau = 22.16
16 hour pulse tau = 22.06
Figure 2C
STAUROSPORINE (kinase inhibitor) INCREASES Tau
22
24
26
28
30
32
staurosporine (nM)
experiment #456
0 5 10 15 20 25 30 35 40 45
Figure 1B Comolli and Hastings
EFFECTS OF KINASE INHIBITORS ON PERIOD
6-DMAP (KINASE INHIB) BLOCKS LIGHT PHASE SHIFTING
STAUROSPORINE ENHANCES LIGHT PHASE SHIFTING
SPECIFIC INHIBITORS can REVEAL PATHWAYS of CLOCK
BIOCHEMISTRY
PROTEIN synthesis inhibitorspulses cause phase shifts
PROTEIN phosphorylation inhibitors chronically cause period changes
PROTEIN PHOSPHATASES
EFFECT of OKADAIC ACID (Protein phosphatase inhibitor) on CIRCADIAN BIOLUMINESCENCE RHYTHM
PERIOD EFFECTS of PROTEIN PHOSPHATASE INHIBITORS
EFFECTS OF OKADAIC ACID AND CALYCULIN ON THE LIGHT PRC
EFFECT OF CREATINE (FROM DIFFERENT SOURCES) ON PERIOD
PRCs: LIGHT-INDUCED DELAY-PHASE SHIFTS IN an LL BACKGROUND ARE EVOKED BY CREATINE
IS THERE a CORE CIRCADIAN OSCILLATOR?
If so, HOW do we IDENTIFY the CELLULAR-BIOCHEMICAL
COMPONENTS?
SPECIFIC INHIBITORS or MUTANTS AFFECTING CIRCADIAN RHYTHMS
DROSOPHILA PERIOD GENE CLOCK MUTANTS
WILD TYPE per+
ARHYHMIC pero
SHORT PERIOD perS
LONG PERIOD perL
Map location of geneClone, sequence geneMeasure mRNA Express encoded protein
A FEW CIRCADIAN CLOCK GENES
1) DROSOPHILAper (PERIOD)tim (TIMELESS)
2) NEUROSPORAfrq (FREQUENCY)prd (PERIOD)
3) CYANOBACTERIAkai (CYCLE IN JAPANESE)
4) ARABIDOPSIStoc1 (TIMING OF CAB)
lhy (LATE ELONG HYPOCOTYL)
cca1 (CIRC CLOCK ASSOCIATED)
5) MOUSEclk (CLOCK)per1 (PERIOD)
6) HAMSTERtau (PERIOD)
POSTULATED PATHWAYS & COMPONENTS& COMPONENTS in the REGULATION of CLOCK GENE EXPRESSION
ClockProtein
ClockProtein
| |P P
ClockGene
ClockmRNA
PositiveRegulators
Other ClockProteins
| |P P
ATP
Figure 1b
TTOTRANSCRIPTION TRANSLATION OSCILLATOR
COMMON ELEMENTS IN THE DESIGN OF CORE CIRCADIAN OSCILLATORS DUNLAP, 1999
MOLECULAR COMPONENTS of the DROSOPHILA CLOCK
NEUROSPORA CLOCK MUTANTS in the FREQUENCY GENECONIDIATION RHYTHM PERIOD Short long, & arhythmic
movie courtesy of Van Gooch
FRQ (frequency) GENE IN NEUROSPORA Dunlap et al
LIGHT CAUSES PHASE SHIFTS BY INDUCTION OF FRQ mRNA
CROSTHWAITE, LOROS & DUNLAP, 1995
INDUCED frq in NEUROSPORA BLOCKS RHYTHM & RESETS
Aronson, Johnson, Loros & Dunlap Science 1994
CLOCK CONTROLLED GENE & PROTEIN: GAPDH
NEUROSPORAShinohara, Loros, &DunlapJ. Biol Chem 1998
MOLECULAR COMPONENTS of the NEUROSPORA CLOCK
MOLECULAR COMPONENTS of the MOUSE CLOCK
MOLECULAR COMPONENTS of the PLANT CLOCK
ACETABULARIA RHYTHMS:O2 EVOL & CHLOROPLAST MOVEMENTSCHWEIGER ET AL, 1981
SINGLE CELL ACETABULARIA LIVES and EXHIBITS RHYTHM with NUCLEUS REMOVED
NUCLEUS IS IN ROOT- RHYTHM CONTINUES WHEN CUT OFF BUT A NEW NUCLEUS GRAFTED ON CONFERS ITS PHASE TO HOST
Schweiger 1964 Science 146: 658-659
BACTERIAL LUCIFERASE as a REPORTER of a TEMP COMPENSATED CIRCADIAN RHYTHM in a PROKARYOTE
KONDO, STRAYER,KULKARNI, TAYLOR, ISHIURA, GOLDEN & JOHNSON PNAS 1993