Molecular Mechanisms of Circadian Photoreception in Neurospora crassa Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität München von Zdravko Dragovic aus Batajnica in Serbien Inaugural dissertation zur Erlangung des Doktorgrades der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Diplom Biologer Zdravko Dragovic aus Batajnica in Serbien Erstgutachter: Herr Prof. Reinhold Herrmann Zweitgutachter: Herr Prof. Eberhard Gwinner Sondergutachter: Herr Prof. Till Roenneberg Mündliche Prüfüng am 16. Februar 2004 2 1 Introduction.............................................................................................................7 1.1 Circadian rhythms............................................................................................7 1.2 Properties of circadian systems ........................................................................8 1.2.1 Free-running and self-sustainment............................................................8 1.2.2 Period length............................................................................................9 1.2.3 Circadian rhythms are entrainable ............................................................9 1.2.4 Circadian rhythms are compensated .........................................................9 1.2.5 Modules of circadian clock ......................................................................9 1.3 Entrainment of circadian rhythms by light......................................................10 1.3.1 Phase response curves (PRCs)................................................................10 1.3.2 Circadian photosensory system ..............................................................11 1.3.3 The Gonyaulax story ..............................................................................12 1.3.4 Circadian photoreception in Arabidopsis................................................12 1.3.4.1 Cryptochromes...................................................................................12 1.3.4.2 Phytochromes.....................................................................................13 1.3.4.3 Resetting of the Arabidopsis clock with light......................................14 1.3.5 Circadian Photoreception in mice...........................................................15 1.3.5.1 Specialized circadian photoreceptor cells in eye .................................15 1.3.5.2 Resetting of the clock within an SCN cell...........................................16 1.4 Neurospora crassa: A simple model system for clock and photobiology studies 17 1.4.1 The biology of Neurospora crassa .........................................................17 1.4.2 Asexual propagation...............................................................................17 1.4.3 Sexual cycle...........................................................................................18 1.5 Circadian Rhythms in Neurospora crassa ......................................................19 1.5.1 Conidiation is major output of circadian rhythm.....................................19 1.5.2 Genetic analysis of the clock ..................................................................20 1.5.3 Genetic analysis of the light input pathway ............................................21 1.5.4 The Neurospora genome suggests additional photoreceptors..................21 1.6 Action spectra analysis ..................................................................................22 1.7 Molecular studies of the photoreception in Neurospora..................................23 1.7.1 White Collars.........................................................................................23 1.7.2 FRQ protein ...........................................................................................24 1.7.3 Resetting of the Neurospora clock by light.............................................25 1.7.4 Photoadaptation and circadian photoreception........................................26 1.8 Open questions ..............................................................................................27 2 Results...................................................................................................................28 2.1 Molecular and genetic regulation of the light input pathway in Neurospora crassa 28 2.1.1 Rhythmicity in DD: FRQ and WC-1 ......................................................29 2.1.2 Regulation of frq and wc-1 is interdependent: WC-1 regulates frq and FRQ regulates wc-1 ...............................................................................................32 2.1.3 frq and the light signaling pathway.........................................................35 2.1.4 Circadian regulation of light signaling....................................................37 2.2 Posttranscriptional control of wc-1 expression ...............................................38 2.2.1 Identification of putative protein binding sites in wc-1 UTRs .................38 3 2.2.2 Proteins bind to 5’ and 3’ UTRs of wc-1 mRNA ....................................40 2.2.3 Examining specificity of protein-UTR complexes ..................................41 2.2.4 Circadian regulation of complex formation is not observable .................42 2.2.5 Light regulates UTR complex formation ................................................45 2.3 Light-regulated behavior in white collar mutants ...........................................46 2.3.1 white collar mutants synchronize conidiation in response to light...........47 2.3.2 Fluence titration of light induced conidiation in wc mutant strains..........53 2.3.3 A self–sustained circadian rhythm in the wc mutants..............................54 2.4 Molecular characterization of a series of white collar alleles ..........................55 2.4.1 Light induction of FRQ protein in different wc background....................55 2.4.2 Verification of the wc strains..................................................................56 2.4.3 Molecular dissection of light input mechanisms .....................................59 2.4.4 Presence of FRQ but not quantitative or qualitative changes are required for the regulation of conidiation by light................................................................63 2.4.5 Fluence titration of light induction of FRQ protein.................................64 2.5 Identification of light induced genes in wc-2 mutant.......................................65 2.6 Action spectrum for FRQ protein induction ...................................................68 2.6.1 Equal Intensity action spectrum..............................................................69 2.6.2 Range of reciprocity for FRQ protein light induction..............................71 2.6.3 Dose response curves .............................................................................73 2.6.4 Action spectrum for FRQ protein induction............................................75 2.7 Novel photoreceptor candidates .....................................................................77 3 DISCUSSION .......................................................................................................85 3.1 Regulation of the light input pathway in N. crassa .........................................85 3.1.1 Levels of WC-1 protein are circadianly regulated...................................85 3.1.2 Interdependent regulation of FRQ and WC-1 .........................................86 3.1.3 Circadian regulation of light responses...................................................86 3.2 Novel photoreceptors in Neurospora..............................................................87 3.2.1 wc mutants respond to light....................................................................87 3.2.2 What are the photoreceptors in Neurospora?..........................................89 3.2.3 Dual light input pathway in N. crassa.....................................................91 3.2.4 FRQ role in regulation of conidiation and carotenogenesis .....................92 3.2.5 A self-sustained circadian rhythm in the wc mutants ..............................93 3.2.6 Action spectrum of FRQ protein induction.............................................93 3.3 Posttranscriptional regulation of wc-1 expression...........................................94 3.3.1 Regulation of translation efficiency by binding to the 5’ UTR................95 3.3.2 Adaptation in constant light and regulation of mRNA stability by binding to the 3’ UTR ........................................................................................................96 4 Materials and Methods ..........................................................................................98 4.1 Physiological Methods...................................................................................98 4.1.1 Strain maintenance.................................................................................98 4.1.2 Media supplements ................................................................................98 4.1.3 Stock management .................................................................................98 4.1.4 Lab practice ...........................................................................................99 4.1.5 Conidial suspensions -inoculum .............................................................99 4.1.6 Liquid cultures .......................................................................................99 4 4.1.7 Sexual crossing ...................................................................................