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