1. MCB 186 CIRCADIAN BIOLOGY Lecture 4 Drugs as probes of mechanism: Phase shifts v.s. effects on period And some basic questions October 12, 2005 J. W. Hastings

2. LIMITS OF ENTRAINMENT HOW do you SPECIFY the LIMITS? ARE there EFFECTS OUTSIDE the LIMITS?

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

4. BACTERIAL COLONIES EXPRESSING BIOLUMINESCENCE Day phase Night phase Code numbers

5. MEASURING ALL OR ONLY SOME CULTURES

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

8. CLOCK MUTANTS REVEAL GENES REGULATING CIRCADIAN RHYTHMS Many but not all exhibit rhythms in expression of mRNA and protein Positive elements and negative feedback result in oscillation Not established how other systems are controlled (CCGs)

9. POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION

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

11. CORE CLOCK COMPONENTS IN FEEDBACK LOOPS OF 3 SYSTEMS

12. Cyanobacterial Clockworks Model -1998 Ishiura et al 1998 Science 281: 1519-1523

13. CCGs in Gonyaulax are CONTROLLED by RNA (translation not transcription) mRNA levels remain constant while protein levels exhibit rhythms Synthesis of many proteins is rhythmic

14. LUCIFERASE PROTEIN EXHIBITS A CIRCADIAN RHYTHM in LL

15. WESTERN BLOTS LUCFERIN BINDING PROTEIN, LD & LL

16. SYNTHESIS of MANY PROTEINS is CIRCADIAN CONTROLLED IN VIVO PULSE LABELING MILOS et al, 1989

17. GONYAULAX CIRCADIAN PULSED PROTEIN SYNTHESIS

18. LBP mRNA DOES NOT CYCLE IN GONYAULAX

19. A NOVEL SEQUENCE in the LBP 3’ UTR BINDS a PROTEIN

20. AN RNA-PROTEIN BASED FEEDBACK CLOCK CLOCK PROTEINS V.S. CLOCK CONTROLLED PROTEINS

21. MICROARRAY ANALYSIS of EXPRESSION of ~3000 DINOFLAGELLATE GENES at TWO CIRCADIAN TIMES

22. SPECIFIC INHIBITORS can REVEAL PATHWAYS of CELLULAR PROCESSES PROTEIN synthesis-phase shifts-as pulses PROTEIN phosphorylation- period changes-as continuous

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

24. EFFECT OF PROTEIN SYNTHESIS INHIBITORS ON RHYTHM KARAKASHIAN

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

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

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. DRUG PRCs in GONYAULAX are DOSE DEPENDENT

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

33. 6-DMAP (KINASE INHIBITOR) INCREASES Tau

34. 6_DMAP (KINASE INHIB) INCREASES Tau

35. 6_DMAP (Kinase Inhibitor) INCREASES Tau

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

37. STAUROSPORINE (kinase inhibitor) INCREASES Tau

38. EFFECTS OF KINASE INHIBITORS ON PERIOD

39. 6-DMAP (KINASE INHIB) BLOCKS LIGHT PHASE SHIFTING

40. STAUROSPORINE ENHANCES LIGHT PHASE SHIFTING

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. 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

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

48. EFFECT of WHITE LIGHT INTENSITYon PERIOD in Gonyaulax

49. 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.

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

51. 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.

52. LOSS OF RHYTHMICITY BELOW 12 O C

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

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

55. A SINGLE CLOCK or MANY CLOCKS? Can different rhythms have different periods?

57. DIFFERENT OSCILLATORS CONTROL GLOW & FLASHING

58. Gonyaulax NIGHT PHASE: LAWN ON BOTTOM OF DISH (LEFT) DAY PHASE: AGGREGATIONS (RIGHT)

59. GONYAULAX DAY PHASE AGGREGATIONS

60. GONYAULAX AGGREGATION RHYTHM

61. GONYAULAX INTERNAL DESYNCHRONIZATION OF TWO RHYTHMS ROENNEBERG

62. ALTERNATE to RASTER PLOT- PEAK # = CIRCADIAN DAYS

63. GONYAULAX APPARENT PHASE JUMPS OTHERWISE VERY PRECISE

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

65. MIXING TWO OUT-OF-PHASE CULTURES SEPARATE MIXED MIXED, FRESH MEDIUM

66. GLOW AND FLASHES FROM A SINGLE GONYAULAX CELL HAAS, DUNLAP & HASTINGS

67. INDIVIDUAL CELLS HAVE DIFFERENT TAUs; WIDTH INCREASES

68. BAND WIDTH OF GLOW IS LESS FROM A SINGLE THAN MANY CELLS

69. GONYAULAX EFFECT OF INTENSITY & COLOR ON TAU