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

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 CLOCKS or THREE or MORE

DIFFERENT OSCILLATORS CONTROL GLOW & FLASHING Internnal Desynchronization

THREE RHYTHMS SIMULTANEOUSLY: PHASE-JUMPS ROENNEBERG & MORSE 1993 Glo FlsAgg

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.1984 Science 223 Western Blot

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

LBP mRNA DOES NOT CYCLE in Gonyaulax LBP 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, 1989 Naturwisenschaften 77

SYNTHESIS of PROTEINS in vitro is NOT CLOCK CONTROLLED MILOS ET AL, 1989 Naturwisenschaften 77

PATTERNS of CLOCK-CONTROLLED PROTEIN SYNTHESIS in Gony Markovic et al., 1996 J. Biol. Rhythms 11 p21 unknown p32 PCP p33 OEE1 p45 GAPDH p55 Rubisco II p75 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 RHYTHMS Fagan, 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 inhibitors Pulses 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

SPECIFIC INHIBITORS can REVEAL PATHWAYS of CLOCK BIOCHEMISTRY PROTEIN synthesis inhibitors pulses cause phase shifts PROTEIN phosphorylation inhibitors chronically cause period changes KINASES

6-DMAP (KINASE INHIBITOR) INCREASES Tau

6_DMAP (Kinase Inhibitor) INCREASES Tau

6_DMAP (KINASE INHIB) INCREASES Tau

NO AFTER-EFFECT of EXPOSURE to 6-DMAP COMOLLI and HASTINGS, 1995

STAUROSPORINE (kinase inhibitor) INCREASES Tau

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 inhibitors pulses 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 per o SHORT PERIOD per S LONG PERIOD per L Map location of gene Clone, sequence gene Measure mRNA Express encoded protein

A FEW CIRCADIAN CLOCK GENES 1) DROSOPHILA per ( PERIOD ) tim ( TIMELESS ) 2) NEUROSPORA frq ( FREQUENCY ) prd ( PERIOD ) 3) CYANOBACTERIA kai ( CYCLE IN JAPANESE ) 4) ARABIDOPSIS toc1 (TIMING OF CAB) lhy (LATE ELONG HYPOCOTYL) cca1 (CIRC CLOCK ASSOCIATED) 5) MOUSE clk ( CLOCK ) per1 ( PERIOD ) 6) HAMSTER tau ( PERIOD )

& COMPONENTS POSTULATED PATHWAYS & COMPONENTS in the REGULATION of CLOCK GENE EXPRESSION TTO TRANSCRIPTION 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 GENE CONIDIATION 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 NEUROSPORA Shinohara, Loros, &Dunlap J. Biol Chem 1998

MOLECULAR COMPONENTS of the NEUROSPORA CLOCK

MOLECULAR COMPONENTS of the MOUSE CLOCK

MOLECULAR COMPONENTS of the PLANT CLOCK

ACETABULARIA RHYTHMS:O 2 EVOL & CHLOROPLAST MOVEMENT SCHWEIGER 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:

BACTERIAL LUCIFERASE as a REPORTER of a TEMP COMPENSATED CIRCADIAN RHYTHM in a PROKARYOTE KONDO, STRAYER,KULKARNI, TAYLOR, ISHIURA, GOLDEN & JOHNSON PNAS 1993