1. MCB 186 CIRCADIAN BIOLOGY The cellular-molecular mechanism of the circadian clock DRUGS Lecture #3 October 18, 2006 J. W. Hastings

2. HOW DO YOU EXPERIMENTALLY PROBE FOR MECHANISM? ALTER CONDITIONS (temperature, light) APPLY INHIBITORS OR DRUGS (what kinds?) CHARACTERIZE MUTANTS (select for what?)

3. TEMPERATURE HAS ONLY SMALL EFFECT ON PERIOD

4. GONYAULAX TEMPERATURE COMPENSATION

5. TEMPERATURE-COMPENSATED PERIOD in VARIOUS ORGANISMS POSITIVE and NEGATIVE COEFFICIENTS-OPPOSING REACTIONS

6. POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION

7. light cycles LIMITS OF ENTRAINMENT light pulses PHASE SHIFTS HOW do you SPECIFY the LIMITS? ARE there EFFECTS OUTSIDE the LIMITS? CONSIDER HIGH FREQUENCY CYCLES

8. Turntable Screening Apparatus: 12 positions for petri dishes or titer plates

9. MEASURING ALL or ONLY SOME CULTURES MEASUREMENT REQUIRES a DARK PULSE

10. EFFECT OF NOT MEASURING (- - - -) ON PERIOD

11. LOSS OF RHYTHMICITY Several conditions, notably bright light and low temperature, lead to the loss of rhythm; has the clock stopped or is it simply not seen? Return to initial conditions results in a reappearance of rhythm at a fixed phase, CT12, independent of when the return occurs

12. EFFECT of WHITE LIGHT INTENSITY on PERIOD and AMPLITUDE in Gonyaulax 680 fc 380 fc 120 fc

13. EFFECT of WHITE LIGHT INTENSITYon PERIOD in Gonyaulax

14. JCCP 1957 Fig 3 After an extended period in bright LL, with no detectable bioluminescence rhythm, transfer to DD initiates a rhythm. The phase is determined by the time of transfer, as if the clock had stopped.

15. RHYTHM in Gonyaulax INITIATED by SHIFT from LL to DD is PHASED STARTING at CT 12

16. ANOTHER EXAMPLE of a CLOCK “STOPPED” in BRIGHT WHITE LIGHT Peterson and Saunders J. Theor Biol 1980 Eclosion rhythm of flesh-fly Sarcophaga argyrostoma. White triangle represents time of light exposure. Each point is the median eclosion time for the culture from the end of the light exposure. Note that the duration between end of light exposure and eclosion is constant (11.5 hrs, dotted line), as if the clock is stopped and restarts when the stimulus ends. Note the slight ~24 hr oscillation around the dotted line.

17. LOSS OF RHYTHMICITY BELOW 12 O C

18. LOW TEMPERATURE for 12 hr “ STOPS” the CLOCK for 12 hr

19. “STOPPED” Gonyaulax CLOCK RESTARTS with PHASE at CT12

20. Limit cycle depiction of light and temperature effects Phase shifts by light pulses Strong & weak: type 0 and 1 Bright light and low temperature Remote cycle

21. SPECIFIC INHIBITORS can REVEAL PATHWAYS of CLOCK BIOCHEMISTRY PROTEIN synthesis inhibitors- as pulses cause phase shifts PROTEIN phosphorylation-inhibit continuously cause period changes

22. EFFECT OF ACTINOMYCIN D (RNA synthesis) ON RHYTHM KARAKASHIAN

23. EFFECT OF PROTEIN SYNTHESIS INHIBITORS ON RHYTHM KARAKASHIAN

24. PROTEIN synthesis inhibitors as pulses cause phase shifts

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

26. PHASE SHIFTS BY ANISOMYCIN 0.3  M, 1 HOUR DRUG PRC

27. VERY BRIEF ANISOMYCIN PULSES CAUSE LARGE PHASE SHIFTS

28. TYPE 1 & 0 DRCs FOR BRIEF ANISOMYCIN PULSES

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

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

32. POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION

33. PROTEIN phosphorylation/dephos inhibitors chronically present cause period changes

34. 6-DMAP (KINASE INHIBITOR) INCREASES Tau

35. 6_DMAP (KINASE INHIB) INCREASES Tau

36. 6_DMAP (Kinase Inhibitor) INCREASES Tau

37. NO AFTER-EFFECT of EXPOSURE to 6-DMAP COMOLLI

38. STAUROSPORINE (kinase inhibitor) INCREASES Tau

39. EFFECTS OF KINASE INHIBITORS ON PERIOD

40. 6-DMAP (KINASE INHIB) BLOCKS LIGHT PHASE SHIFTING

41. STAUROSPORINE ENHANCES LIGHT PHASE SHIFTING

42. EFFECT of OKADAIC ACID (Protein phosphatase inhibitor) on CIRCADIAN BIOLUMINESCENCE RHYTHM- Tau INCREASE

43. PERIOD EFFECTS of PROTEIN PHOSPHATASE INHIBITORS

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

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

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

47. DRUG PRCs in GONYAULAX are DOSE DEPENDENT