MCB 186CIRCADIAN BIOLOGY

Lecture 4 Drugs as probes of mechanism: Phase shifts v.s. effects on period

And some basic questions

October 12, 2005J. 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

Dayphase

Nightphase

Codenumbers

MEASURING ALL OR ONLY SOME CULTURES

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

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

ClockProtein

ClockProtein

| |P P

ClockGene

ClockmRNA

PositiveRegulators

Other ClockProteins

| |P P

ATP

Figure 1b

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

CORE CLOCK COMPONENTS IN FEEDBACK LOOPS OF 3 SYSTEMS

Cyanobacterial Clockworks Model -1998

Circadian rhythms

C

CA

ABB

Protein Interactions

A B C

C

KaiC

B

KaiB

A

KaiA

rhythmicmRNAs

Kai proteins

kaiBCpkaiAp

Ishiura et al 1998 Science 281: 1519-1523

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

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

6-DMAP (KINASE INHIBITOR) INCREASES TauBioluminescence Experiment # 382

control

33µM

50 µM

75 µM

100 µM

120 µM

140 µM

160 µM

180 µM

200 µM

250 µM

275 µM

300 µM

350 µM

400 µM

1 2 3 4 5 6

day of experiment

21°C

time of 6-DMAP addition

Figure 1A

6-DMAP conc.

6_DMAP (KINASE INHIB) INCREASES Tau

6_DMAP (Kinase Inhibitor) INCREASES Tau

Bioluminescence Exp.#381

90 180 270 360 450 540 630

control tau = 23.016

50µM 6-DMAP tau = 23.59

100µM 6-DMAP tau = 24.295

160µM 6-DMAP tau = 25.318

200µM 6-DMAP tau = 25.571

250µM 6-DMAP tau = 25.766

300µM 6-DMAP tau = 26.596

phase [°]

1

2

3

4

5

6

7

Figure 1C

NO AFTER-EFFECT of EXPOSURE to 6-DMAP COMOLLI

Bioluminescence Exp.# 393

90 180 270 360 450 540 630

control tau = 22.23

phase [°]

1

2

3

4

5

6

7

8

9

4 hour pul se t au = 2 2 .2 6

8 hour pulse tau = 22.26

12 hour pulse tau = 22.16

16 hour pulse tau = 22.06

Figure 2C

STAUROSPORINE (kinase inhibitor) INCREASES Tau

22

24

26

28

30

32

staurosporine (nM)

experiment #456

0 5 10 15 20 25 30 35 40 45

Figure 1B Comolli and Hastings

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 12O 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 RHYTHMSROENNEBERG

ALTERNATE to RASTER PLOT- PEAK # = CIRCADIAN DAYS

GONYAULAX APPARENT PHASE JUMPSOTHERWISE 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 CELLHAAS, 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