MODELING INPUT to and OUTPUT from THE CLOCK

1. MCB 186CIRCADIAN BIOLOGYBiochemistry of the Circadian ClockLecture #3 October 3, 2007J. W. Hastings

2. MODELING INPUTto and OUTPUT fromTHE CLOCK

3. THIS IS ONLY A MODELDIFFERENT SYSTEMS MAY DIFFER:bacteria, plants, algae, fungi, animalsand, IT MAY BE INCORRECTe.g., THERE MAY BE TWO CLOCKSor THREE or MORE

4. DIFFERENT OSCILLATORS CONTROL GLOW & FLASHING Internnal Desynchronization

5. THREE RHYTHMS SIMULTANEOUSLY: PHASE-JUMPS ROENNEBERG & MORSE 1993 Fls Agg Glo

6. INPUT to and OUTPUT from a TWO-CLOCK MODEL

7. CORE PACEMAKER OSCILLATOR • BIOCHEMICAL ELEMENTS of the CLOCK • AFFECTED by SIGNAL TRANSDUCTION • CLOCK GENES vs CLOCK CONTROLLED • CLOCK PROTEINS vs CLOCK CONTROLLED

8. INPUT PATHWAYSSIGNAL TRANSDUCTION • MANY FACTORS AFFECT THE CLOCK • EFFECTS on PHASE and PERIOD DISTINCT • BIOCHEMICAL PATHWAYS UNKNOWN

9. OUTPUT PATHWAYSHOW DOES the CLOCK TURN PROCESSES ON and OFF? • TRANSCRIPTION: NEW mRNA, then protein • TRANSLATION: REGULATE PROTEIN SYNTHEIS • POST TRANSLATIONAL e.g. PHOSPHORYLATION

10. LUCIFERASE PROTEIN EXHIBITS A CIRCADIAN RHYTHM in LL Johnson et al.1984 Science 223 Western Blot

11. WESTERN BLOTS LUCFERIN BINDING PROTEIN, LD & LL A CLOCK CONTROLLED GENE Morse et al., 1989 PNAS 86

12. GONYAULAX CELLS AT NIGHT (LEFT) AND DAY PHASES FLUORESCENCE OF LUCIFERIN IN SCINTILLONS

13. Morse et al., 1989 PNAS 86 LBP mRNA DOES NOT CYCLE in GonyaulaxLBP SYNTHESIS & ABUNDANCE are STRONGLY CIRCADIAN LBP mRNA LBP abundance LBP synthesis

14. mRNA LEVELS ARE CONSTANT

15. SYNTHESIS of MANY PROTEINS is CIRCADIAN CONTROLLEDIn Vivo PULSE LABELING MILOS et al, 1989 MILOS ET AL, 1989 Naturwisenschaften 77

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

17. Markovic et al., 1996 J. Biol. Rhythms 11 PATTERNS of CLOCK-CONTROLLED PROTEIN SYNTHESIS in Gony p21 unknown p32 PCP p33 OEE1 p45 GAPDH p55 Rubisco II p75 Luciferin binding protein

18. 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

19. GAPDH SYNTHESIS, ACTIVITY & ABUNDANCE RHYTHMSFagan, Morse & Hastings, 1999

20. HALF-LIFE of PROTEIN AFFECTS AMPLITUDE of ABUNDANCE RHYTHM 12 hr 2 days

22. IS THERE a CORE CIRCADIAN OSCILLATOR?If so, HOW do we IDENTIFY the CELLULAR-BIOCHEMICAL CLOCK COMPONENTS?SPECIFIC INHIBITORS or MUTANTS AFFECTING CIRCADIAN RHYTHMS

23. SPECIFIC INHIBITORS can REVEAL PATHWAYS of CLOCK BIOCHEMISTRYPROTEIN synthesis inhibitorsPulses cause phase shiftsPROTEIN phosphorylation inhibitors Chronically cause period changes

24. PULSES of ANISOMYCIN (protein synthesis inhibitor)CAUSE PHASE SHIFTS in Gonyaulax

25. PHASE SHIFTS BY ANISOMYCIN 0.3 M, 1 HOUR

26. VERY BRIEF ANISOMYCIN PULSES CAUSE LARGE PHASE SHIFTS

27. TYPE 1 & 0 DRCs FOR BRIEF ANISOMYCIN PULSES

28. ARHYTHMICITY AT “CRITICAL” DOSE OF PHASE SHIFTING INHIBITOR

29. D-PRC for PHASE SHIFTS by an INHIBITOR of PROTEIN SYNTHESIS

30. D-PRC for PHASE SHIFTS by an INHIBITOR of PROTEIN SYNTHESIS

31. SPECIFIC INHIBITORS can REVEAL PATHWAYS of CLOCK BIOCHEMISTRYPROTEIN synthesis inhibitorspulses cause phase shiftsPROTEIN phosphorylation inhibitors chronically cause period changesKINASES

32. 6-DMAP (KINASE INHIBITOR) INCREASES Tau

33. 6_DMAP (Kinase Inhibitor) INCREASES Tau

34. 6_DMAP (KINASE INHIB) INCREASES Tau

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

36. STAUROSPORINE (kinase inhibitor) INCREASES Tau

37. EFFECTS OF KINASE INHIBITORS ON PERIOD

38. 6-DMAP (KINASE INHIB) BLOCKS LIGHT PHASE SHIFTING

39. STAUROSPORINE ENHANCES LIGHT PHASE SHIFTING

40. SPECIFIC INHIBITORS can REVEAL PATHWAYS of CLOCK BIOCHEMISTRYPROTEIN synthesis inhibitorspulses cause phase shiftsPROTEIN phosphorylation inhibitors chronically cause period changesPROTEIN PHOSPHATASES

41. EFFECT of OKADAIC ACID (Protein phosphatase inhibitor) on CIRCADIAN BIOLUMINESCENCE RHYTHM

42. PERIOD EFFECTS of PROTEIN PHOSPHATASE INHIBITORS

43. EFFECTS OF OKADAIC ACID AND CALYCULIN ON THE LIGHT PRC

44. EFFECT OF CREATINE (FROM DIFFERENT SOURCES) ON PERIOD

45. PRCs: LIGHT-INDUCED DELAY-PHASE SHIFTS IN an LL BACKGROUND ARE EVOKED BY CREATINE

46. IS THERE a CORE CIRCADIAN OSCILLATOR?If so, HOW do we IDENTIFY the CELLULAR-BIOCHEMICAL COMPONENTS?SPECIFIC INHIBITORS or MUTANTS AFFECTING CIRCADIAN RHYTHMS

47. DROSOPHILA PERIOD GENE CLOCK MUTANTS WILD TYPE per+ ARHYHMIC pero SHORT PERIOD perS LONG PERIOD perL Map location of gene Clone, sequence gene Measure mRNA Express encoded protein

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

49. POSTULATED PATHWAYS & COMPONENTS in the REGULATION of CLOCK GENE EXPRESSION TTO TRANSCRIPTION TRANSLATION OSCILLATOR

50. COMMON ELEMENTS IN THE DESIGN OF CORE CIRCADIAN OSCILLATORS DUNLAP, 1999