Drosophila Clock Flashcards
Konopka and Benzer, 1971?
Konopka and Benzer (1971) screened for flies with abnormal circadian behaviour (abnormal eclosion rhythms/periods) after chemical mutagenesis. Thye found a strain of flies with a longer period (28h), another strain that had a shorter period (19h) and a third strain that didn’t show rhythmic eclosion at all. They then found that all three fly strains had a mutation in the same gene locus, located on the X-chromosome. This locus was called period with mutant sdesignated periodLong, periodShort or period0 and the first so called ‘clock gene’ was revealed.
What is the molecular mechanism of per and time?
The finding that per RNA and protein are expressed cyclically and rising levels of protein are associated with declining levels of the mRNA led to the postulate that the PER protein negatively regulates its own transcription to generate an autoregulatory circadian loop (Siwicki et al., 1988; Hardin et al.,1990)
Subsequent studies with tim supported this idea, showing that the two mRNAS cycle in phase and the PER and TIM proteins interact directly and affect their own transcription (Gekakis et al. 1995; Sehgal et al. 1995). The negative feedback loop thus generated constitutes the basis of overt rhythms in Drosophila.
What is the current model of the drosophila clock?
(in dark)
The current model predicts that the helix-loop-helix transcription factors dclock (Clk) and cycle (Cyc) bind as heterodimers to E-Box sequences (CACGTG) at midday in the genome of the fly (Allada et al., 1998, Rutila et al., 1998). E-Boxes are found in the promoter region of many circadian regulated genes like period (per), timeless (tim).
The subsequent rise of the per and tim mRNA in the evening/night leads to accumulation of Per and Tim in the cytoplasm. This only occurs after dark, because of the light sensitivity of Tim and the fact that Tim stabilizes Per. Without Tim’s protection Per is phosphorylated by the Double-Time (Dbt) kinase (Price et al., 1998) and afterward ubiquitinated by the F-Box protein Slimb and then degraded in the proteasome (Grima et al.,2002).
If PER is not degraded, PER binds to TIM and the dimer enters the nucleus, where it inhibits the Dclock:Cycle function on e-box.
Rutila et al., 1998?
- homozygous Cyc flies = completely arrhythmic, no transcription of Per and Tim
- heterozygous Cyc/+ flies are rhythmic but have altered periods – suggests that Cyc has dosage effect on the clock period
How does Cry fit into the molecular clock of flies?
Cry is a blue-light photopigment (Emery et al., 1998, Stanewsky et al., 1998) that is expressed in specific subsets of the clock neurons and in the compound eyes. This protein is activated in the light. Light activated Cryptochrome can bind to Tim and thus triggers Tim degradation. As a consequence to the lack of Tim, Period is now vulnerable to Dbt phosphorylation and is subsequently degraded (and the clock reset).
What is the molecular response of circadian clock to light?
Light dcereases TIM protein levels. CRY is a circadian photopigment which absorbs blue light and is able to bind to TIM. This degrades TIM through the ubiquitin ligase component JET. As a result the accumulation of TIM and therefore PER:TIM dimers is slowed. Thus, it takes longer for PER:TIM dimers to inhibit Dclock:Cycle function on e-boxes, leading to a phase shift of the circadian clock.
How does Cry affect the amplitude of the phase shift to light?
in CryB mutant Drosophila, amplitude of phase response curve was reduced àClock does not respond to light as well (Stanewsky et al 1998)
in Cry overexpressed Drosophila, phase shift size increased, more sensitive to low intensity light, no effect on period or strength of locomotor activity rhythms (Emery et al 1998)
Is Cry essential for synchronisation of circadian clock to normal L:D cycles?
No, as drosophila have a visual system that can recieve light information for entrainment, although this is poorly characterised
Why are there no expression of behavioural rhythms in constant light (LL) conditions?
Tim is presumably continously degraded by constantly activated Cry.
How does Cry and Tim contribute to the phase responses of light pulses in different times of day?
Light pulses during the early night allows Cry to bind to Tim, which is rapidly degraded so delays internal clock
Throughout the night, there is no light so Cry activity is reduced, allowing shorter cycles through the loop, therefore the phase shifting begins to advance so advances internal clock
Light pulse in late night/early morning causes phase advance.
A light pulse applied early at night will generate a delay in the rhythm, whereas one given late at night produces an advance. These dynamic changes are collectively termed the phase–response curve (PRC), and are very similar in all organisms.
TIM rapidly degrades in response to light due to activation of CRY, so a light pulse given early at night depletes TIM during its rising phase when there is an available pool of tim mRNA. The time it takes to reconstitute the previous TIM levels, generates the delay in the molecular (and behavioral) cycle. The same pulse given late at night, when TIM levels are falling, again depletes TIM, but at a time when there is little tim mRNA. TIM levels are prematurely reduced to a level that corresponds to that several hours in the future, thereby generating a phase advance. This simple molecular model provides a compelling explanation for the apparently complex PRC.
Differences between drosophila and mammal clocks
Several differences in the structure or function of these mammalian clock components are notable. mPER-mCRY functions to repress CLOCK-BMAL1 transcription, but mCRY is the major repressor as opposed to PER in flies (Reppert and Weaver, 2002).
Although CRY functions as a circadian photoreceptor in flies, its role as a transcriptional repressor has been retained in at least some fly peripheral tissues (Collins et al., 2006).
Their entrainment to light differs markedly. In flies, light entrains the circadian oscillator by inducing TIM degradation, whereas light entrains the mammalian oscillator by inducing Per1 transcription (Reppert and Weaver, 2002). Since light can directly entrain Drosophila oscillators (Ashmore and Sehgal, 2003), but indirectly entrains mammalian oscillators (Reppert and Weaver, 2002), it is not surprising that different mechanisms have evolved in these animals.
Anatomy of drosophila clock
The drosophila master clock is believed to be made up of dorsal and ventral lateral neurons. The traditional view is that lateral neurons are the key pacemaker clock neurons which play critical roles in imposing circadian structure on the daily pattern of rest and activity, and which demonstrate cell-(or cluster-)autonomous oscillator function. In contrast, dorsal neurons have been thought to play more subtle roles in modulating circadian rhythmicity.
These neurons have direct contact with ocelli and eyes, which are both involved in detecting sunlight.
Differences between drosphila and mammal clock?
Anatomical difference between drosophila and mammal clock
In mammals the central `master’ clock is located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus ( Klein et al., 1991). The SCN is entrained to light-dark (LD) cycles by a distinct set of photosensitive retinal ganglion cells that project to the SCN through the retinohypothalamic tract ( Berson et al., 2002; Hattar et al., 2002; Moore et al., 1995; Provencio et al., 2002). The SCN in turn activates rhythms in behavior (e.g. locomotor activity), by secreting factors (e.g. TGFα and prokineticin) that act locally within the hypothalamus ( Cheng et al., 2002; Kramer et al., 2001), and entrains subservient circadian oscillators in peripheral tissues (e.g. liver and kidney) via humoral signals (e.g. glucocorticoids) (Balsalobre et al., 2000a; Balsalobre et al., 2000b; Oishi et al., 1998; Ueyama et al., 1999). Such peripheral oscillators can, however, become uncoupled from the SCN if their specific needs dictate — as occurs in liver, lung and skeletal muscle after entrainment by food ( Yamazaki et al., 2000). The SCN maintains robust (>2 week) rhythms when entrained to LD cycles in vivo and cultured in vitro, whereas peripheral oscillators lose rhythmicity after just 4-5 days under the same conditions ( Yamazaki et al., 2000).
In flies the central clock is located in a group of 5-6 bilaterally symmetric small ventral lateral neurons (sLNvs) situated in the lateral brain close to the optic lobes (Helfrich-Forster, 1996). As in the SCN, sLNvs receive light input from retinal photoreceptors in the compound eyes and extra-retinal photoreceptors within the brain; however, they can also be entrained directly by light that penetrates the cuticle ( Helfrich-Forster et al., 2001; Stanewsky et al., 1998). In constant dark (DD) conditions, sLNvs maintain robust rhythms in gene expression and locomotor activity ( Ewer et al., 1992; Frisch et al., 1994;Helfrich-Forster, 1998; Zerr et al., 1990). Peripheral oscillators in flies (e.g. antennal clock cells and Malpighian tubules) can also maintain robust (>7 day) rhythms in cell culture, which suggests that fly peripheral oscillators depend less on the sLNvs than do their mammalian counterparts on the SCN ( Emery et al., 1997;Giebultowicz and Hege, 1997; Plautz et al., 1997). Indeed, precisely how much influence the sLNvs have over fly peripheral oscillators is not known, given that light penetrating through the cuticle appears to entrain peripheral clocks, thus negating the requirement for a `master’ clock to synchronize other oscillators in the fly.
Despite this difference, the sLNvs can be regarded as a central oscillator because, like the SCN, they drive behavioral rhythms in locomotor activity.
Can blind drosophila still entrain to L-D cycles?
Although CRY can entrain peripheral oscillators in the fly tissue-autonomously ( Emery et al., 2000b), light input to the sLNvs can also occur through retinal and extra-retinal photoreceptors ( Helfrich-Forster et al., 2001). As such, the sLNvs can still entrain in flies that contain a single amino acid mutation that disrupts the flavin-binding domain of CRY (cryb). Entrainment in these flies to pulses of light is, however, impaired compared with the wildtype (Stanewsky et al., 1998).
