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mechanismandreduction - Mechanism and Reduc.on:...

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Unformatted text preview: 11/17/11 Mechanism and Reduc.on: Decomposing Circadian Clocks The Need to Decompose •  In order to explain the phenomenon in terms of a mechanism a researcher has to –  Locate the mechanism within the larger system that exhibits the phenomenon –  Decompose the mechanism into its parts and opera.ons •  Decomposing involves differen.a.ng the parts and opera.ons –  By physically detaching them from others –  By dis.nguishing them conceptually and securing evidence of their individual characteris.cs •  Not all ways of cuGng up a system differen.ate parts and opera.ons –  Need to find the working parts—the parts that work in coordina.on to produce the phenomenon Techniques for Decomposing Biological Mechanisms •  Structurally –  Physical dissec.on –  Microscopy and other imaging techniques (CAT and MRI scans) •  Func.onally –  Lesion or knockout a part •  To reveal what is lost without that part –  S.mulate a part •  To see what is behavior is increased –  Manipulate an input and record changes in the behavior of a part •  To see what parts are engaged in response to the input 1 11/17/11 Decomposi.on and Reduc.on •  Decomposing a mechanism into its parts and opera.ons is oQen characterized as reduc.onis.c •  For some philosophers, reduc.on has the connota.on of taking something all the way down to its simplest parts and opera.ons –  But that oQen fails to serve the goal of explana.on, which is to iden.fy the parts and opera.ons responsible for the phenomenon •  In going down further, one treats the opera.on performed by the part as the phenomenon of interest, and decomposes it into its parts and opera.ons –  But now one is asking a different ques.on: how does the part perform its opera.on? Localizing the Mammalian Circadian Mechanism •  Once it was accepted that circadian rhythms are maintained endogenously, the natural ques.on to ask is where the mechanism is located •  In the early 1970s Robert Moore localized the (?!) circadian clock in a small nucleus known as the suprachiasma.c nucleus (SCN). Located above the op.c chiasm in the anterior hypothalamus, the SCN consists of about 20,000 neurons in mice –  Pathways from the re.na project to the SCN –  Lesions to the SCN disrupt rhythms –  Much later: transplant of SCN into ventricle in lesioned rats restored circadian rhythms Localizing the Fly Circadian Rhythm •  Localizing fly circadian clock in the head –  Transplant head of flies with short cycle into abdomen of arrhythmic flies results in behavior with short cycle –  Since no neuronal projec.ons develop, must be a^ributed to diffusible factor •  Subsequently localized to specific neurons –  Dorsal neurons (DNs) oscillate when LNs destroyed, but are insufficient to maintain locomotor rhythms –  Oscilla.on in the lateral neurons (LNs) alone (achieved in transgenic flies) sufficient for locomotor rhythms 2 11/17/11 Localizing vs. Decomposing •  Localizing a phenomenon: Iden.fying where a phenomenon is controlled (locus of control) –  The occurrence of the phenomenon may require other en..es (your car requires the existence of oil fields), but typically there is a locus where the phenomenon is controlled) •  Localizing the phenomenon does not explain it –  That requires decomposing the locus of control into its parts and opera.ons and showing how they are organized to produce the phenomenon •  A second act of localiza.on occurs in iden.fying the part responsible for the opera.on The Task of Decomposing Circadian Rhythms •  Guiding metaphor: circadian rhythms are governed by a clock –  A dis.nct mechanism that keeps .me –  This clock is located in the SCN in mammals and the lateral neurons in fruit flies •  Two possibili.es: –  Clock required the interac.on of mul.ple neurons –  Clock was contained within individual neurons •  The fact that single ­cell organisms also exhibit circadian rhythms supported the la^er possibility •  So the challenge became to figure out what inside cells generates regular 24 ­hour oscilla.ons –  Presumably proteins which are synthesized from genes Fruit Flies as a Model Circadian Organism •  Eclosion occurs in the early morning hours before heat and dryness of the day •  In adults, many ac.vi.es, such as feeding behavior (including the physiological processes of diges.on) are restricted to day.me •  Since the work of Morgan, flies have been a highly produc.ve model system for gene.cs –  Rela.vely easy to induce muta.ons, iden.fy their phenotypic effects, and localize one or more genes involved in genera.ng the phenomenon—presumably via a protein for which it codes •  Strategy: induce muta.ons in fruit flies and screen for ones that exhibit unusual circadian rhythms 3 11/17/11 Seeking a Gene.c Mechanism •  For his disserta.on research, Ronald Konopka took up the challenge. In 1971 found muta.ons, all involving the same locus, that resulted in loss of rhythms or shortened or lengthened periods •  Called the gene period (per) –  In null muta.ons, normal rhythms could be restored by transplan.ng brain of WT into abdomen of fly with muta.on •  Note: this does not prove that per (and presumably its protein) is part of the clock mechanism –  It could be involved in something only tangen.ally related to the clock •  The more one can manipulate circadian rhythms by manipula.ng it, the more likely it seems that it is a part of the mechanism Figuring Out What the per Gene Does •  Only with the advent of cloning technology was it possible to iden.fy and measure concentra.ons of the protein PER and the mRNA transcript –  Both were shown to oscillate with an approximately 24 hour period •  Hardin, Hall, and Rosbash (1990) proposed that per, per mRNA and PER protein are organized into a delayed feedback loop capable of oscilla.on—a clock –  A mechanism sketch as there were several gaps or alterna.ve pathways Hardin et al., 1990 Next Challenge: How Does PER Inhibit per? •  PER lacks a domain (part of the protein) that is capable of binding to DNA and so serving to regulate transcrip.on –  Something else must be involved •  Seghal et al. (1994): PER has a partner TIMELESS (TIM) with which it formers a dimer –  The PER:TIM dimer was transported back into the nucleus •  Discovery of TIM s.ll leQ the mystery of how PER or TIM worked to inhibit transcrip.on of per or 4m –  Discovery of a promoter site (E ­box) on per and 4m pointed to a regulatory protein that controlled per and 4m transcrip.on –  Challenge: find this protein(s) 4 11/17/11 Mammals Provided the Clue •  Boldly trying to do in mice what Konopka had done in flies (since genera.on .mes are much longer, making it unlikely one would get a quick result), Vitaterna et al. (1995) discovered a gene whose mutants exhibited disrupted rhythms –  Called the gene Clock (Circadian Locomotor Output Cycles Kaput) –  Its dimeriza.on partner was soon iden.fied (BMAL1) •  Mouse CLOCK ­BMAL1 dimers can bind with Drosophila per and 4m –  Proposal: PER:TIM in some way interacts with CLOCK:BMAL1 to interfere with its ability to bind to the per and 4m promoters –  Soon Drosophila homologs of Clock and Bmal1 (cycle) were found: dCLOCK and CYCLE (which, ironically, doesn’t cycle) •  CLOCK ­BMAL1 completed the transcrip.on/transla.on feedback loop that cons.tutes the core oscillatory mechanism •  Basic Schema of Circadian Mechanism in Fruit Flies Keeping the Mechanism Entrained to our Planet •  Beyond endogenously maintaining approximately 24 hour oscilla.ons, the circadian clock is also entrainable to the external light/dark cycle •  Researchers soon discovered cryptochrome, a blue light photoreceptor, that acts to degrade TIM when acted upon by light •  When CRY is affected by light, it stops PER:TIM from interrup.ng the ability of CYC:CLK to bind to promoter on per and 4m –  If this is in the early morning, the effect is to keep the clock in day condi.on and delaying the clock –  If this is in the late night, the effect is to advance the clock to the condi.ons of the next day 5 11/17/11 More and More Clock Parts It is temp.ng to speculate that the Drosophila four ­component transcrip.onal feedback loop described here is sufficient to generate a rudimentary circadian rhythm. This oscilla.on would be amplified by other, unknown proteins that regulate RNA stability, protein stability, and phosphoryla.on of the oscillator components. »  Darlington et al., 1998 •  Other components were soon to be discovered: –  Kinases that phosphorylate PER and TIM, marking them for degrada.on •  Double.me (DBT) acts on PER •  Shaggy (SGG) acts on TIM –  Some of these involved a second feedback loop •  PDP1ε binds to the promoter of clock •  Vrille inhibits clock expression, and so per and 4m transcrip.on •  Diagramming the Whole Mechanism •  Yu and Hardin, 2006 •  Mid ­day: The Daily Cycle –  most PER and TIM is degraded –  CLK::CYC bind to E ­box on promoters of per, 4m, and vri •  AQernoon: –  VRI moves to the nucleus and suppresses clk expression –  PER is phosphorylated by DBT and CK2 and degraded •  Late aQernoon –  Accumula.on of TIM protects PER from degrada.on –  SGG phosphorylates TIM, making it able to enter nucleus •  Early evening –  PER and TIM heterodimerize and enter the nucleus –  PER in nucleus interferes with CLK::CYC ability to bind per and 4m promoter •  Late night –  PER and TIM peak, causing increased CLK expression –  PER::TIM dissociate, leading DBT to phosphorylate PER and subsequently its breakdown •  Dawn –  PER drops to low levels while CLK peaks 6 11/17/11 Conserved but Modified Mechanism in Mammals •  per is conserved in mammals with 3 variants: Per1, Per2, and Per3 –  Per1 and Per2 play much the same role in the clock as Drosophila per •  The mammalian homologs of CRY, CRY1 and CRY2, act like 4m in Drosophila –  Form dimers with PER1 and PER2 –  Inhibit their own transcrip.on by interac.ng with CLK:BMAL1 •  Also mammalian CLK doesn’t cycle, but BMAL1 does •  The discovery that CRY1 and CRY2 figure in the central oscillator in the mammalian clock raised the ques.on: what performs the entrainment role by responding to light? What Entrains the Mammalian Clock? •  Discovery of a new opsin compound, melanopsin, which is expressed in a dis.nct set of re.nal ganglion cells from the visual photopigment rhodopsin, –  intrinsically photosensi.ve Re.nal Ganglion Cells •  Through a signaling pathway, melanopsin ac.vates transcrip.on of Pers when light is present at night. Same Type of Mechanism in Other Orders •  Following the discovery of the core components of the fly and mouse clocks, researchers working on bacteria, fungi, and plants found similar transcrip.on/transla.on feedback loops in model species in each order •  The central components of the clocks in the different orders of life are different, but the overall organiza.on is very similar (interac.ng posi.ve and nega.ve feedback loops) 7 11/17/11 Across Orders: Conserved or Independently Evolved? •  The similar organiza.on with different components has suggested to many that the clock mechanism has evolved independently several .mes –  A case of convergent evolu.on •  But many of the ancillary components (e.g., kinases) are homologous (share a common descent) –  Sugges.ng that the mechanism may have been conserved •  Possibly the organiza.on has been conserved as well, with changes in some of the component parts (genes/proteins) –  Func.onal conserva.on A Clock Shock •  The transcrip.on/transla.on feedback loop seemed to provide a universal account of circadian clocks even if the parts were different across species •  In 2005 Takao Kondo’s laboratory demonstrated –  That circadian rhythms in cyanobacteria could be maintained without transcrip.on and transla.on –  That just combining the proteins Kai A, Kai B, and Kai C together with ATP they could generate circadian rhythms •  Kai C is both an autokinase and an autophosphorylase, with Kai A and Kai B helping to determining which process occurs The Clock Shock Comes to Mammals •  In January 2011 O’Neill et al. report circadian rhythms in mammalian red blood cells –  Which have no nucleus and hence no genes •  Mammals as well must have a mechanism that can work independently of the transcrip.on/transla.on feedback loop •  Has the work on transcrip.on/transla.on just been a mistake? –  Or does it play a role (e.g., in making the clock robust)? 8 ...
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