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

1
MCB 186CIRCADIAN BIOLOGY Lecture 4 Drugs as
probes of mechanism Phase shifts v.s. effects
on periodAnd some basic questions October 12,
2005J. 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
7
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 CLOCKCLOCK
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 PROCESSESPROTEIN 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
32
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 RHYTHMICITYSeveral 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
120 fc
380 fc
680 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
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.
Peterson and Saunders J. Theor Biol 1980
52
LOSS OF RHYTHMICITY BELOW 12O 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?

56
(No Transcript)
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
RHYTHMSROENNEBERG
62
ALTERNATE to RASTER PLOT- PEAK CIRCADIAN DAYS
63
GONYAULAX APPARENT PHASE JUMPSOTHERWISE 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
CELLHAAS, 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