mcb 186 circadian biology lecture 4 drugs as probes of mechanism: phase shifts v.s. effects on...
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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
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