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

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

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

BACTERIAL COLONIES EXPRESSING BIOLUMINESCENCE Day phase Night phase Code numbers

MEASURING ALL OR ONLY SOME CULTURES

EFFECT OF NOT MEASURING ( ) ON PERIOD

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)

POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION

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

CORE CLOCK COMPONENTS IN FEEDBACK LOOPS OF 3 SYSTEMS

Cyanobacterial Clockworks Model Ishiura et al 1998 Science 281:

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

LUCIFERASE PROTEIN EXHIBITS A CIRCADIAN RHYTHM in LL

WESTERN BLOTS LUCFERIN BINDING PROTEIN, LD & LL

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

GONYAULAX CIRCADIAN PULSED PROTEIN SYNTHESIS

LBP mRNA DOES NOT CYCLE IN GONYAULAX

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

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

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

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

EFFECT OF ACTINOMYCIN D (RNA synthesis) ON RHYTHM KARAKASHIAN

EFFECT OF PROTEIN SYNTHESIS INHIBITORS ON RHYTHM KARAKASHIAN

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

DRUG PRCs in GONYAULAX are DOSE DEPENDENT

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

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

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

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

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

DIFFERENT OSCILLATORS CONTROL GLOW & FLASHING

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

GONYAULAX DAY PHASE AGGREGATIONS

GONYAULAX AGGREGATION RHYTHM

GONYAULAX INTERNAL DESYNCHRONIZATION OF TWO RHYTHMS ROENNEBERG

ALTERNATE to RASTER PLOT- PEAK # = CIRCADIAN DAYS

GONYAULAX APPARENT PHASE JUMPS OTHERWISE VERY PRECISE

INPUT to and OUTPUT from a TWO-CLOCK MODEL

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

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

INDIVIDUAL CELLS HAVE DIFFERENT TAUs; WIDTH INCREASES

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

GONYAULAX EFFECT OF INTENSITY & COLOR ON TAU