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

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

TEMPERATURE HAS ONLY SMALL EFFECT ON PERIOD

GONYAULAX TEMPERATURE COMPENSATION

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

POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION

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

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

MEASURING ALL or ONLY SOME CULTURES MEASUREMENT REQUIRES a DARK PULSE

EFFECT OF NOT MEASURING ( ) ON PERIOD

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

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

EFFECT of WHITE LIGHT INTENSITYon PERIOD in Gonyaulax

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.

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

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.

LOSS OF RHYTHMICITY BELOW 12 O C

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

“STOPPED” Gonyaulax CLOCK RESTARTS with PHASE at CT12

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

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

EFFECT OF ACTINOMYCIN D (RNA synthesis) ON RHYTHM KARAKASHIAN

EFFECT OF PROTEIN SYNTHESIS INHIBITORS ON RHYTHM KARAKASHIAN

PROTEIN synthesis inhibitors as pulses cause phase shifts

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

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

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

POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION

PROTEIN phosphorylation/dephos inhibitors chronically present cause period changes

6-DMAP (KINASE INHIBITOR) INCREASES Tau

6_DMAP (KINASE INHIB) INCREASES Tau

6_DMAP (Kinase Inhibitor) INCREASES Tau

NO AFTER-EFFECT of EXPOSURE to 6-DMAP COMOLLI

STAUROSPORINE (kinase inhibitor) INCREASES Tau

EFFECTS OF KINASE INHIBITORS ON PERIOD

6-DMAP (KINASE INHIB) BLOCKS LIGHT PHASE SHIFTING

STAUROSPORINE ENHANCES LIGHT PHASE SHIFTING

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

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

DRUG PRCs in GONYAULAX are DOSE DEPENDENT