Combined Bioluminescence-Fluorescence Time Lapse ... Combined Bioluminescence-Fluorescence Time Lapse

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  • Combined Bioluminescence-Fluorescence Time Lapse

    Microscopy: Applications to Circadian Rhythm Studies

    Charna Dibner/Tiphaine Mannic

    Division of Endocrinology, Diabetes,

    Nutrition and Hypertension

    University Hospital of Geneva

    PROMEGA

    Bioluminescent Cell-Based Assay

    Seminar Tour

    13-14th March 2013

  • 1. A body-wide web of circadian

    oscillators

    2. Fluorescence and

    bioluminescence time-lapse

    microscopy: new era in circadian

    clock studies

    3. Application of combined

    bioluminescence-fluorescence

    time lapse microscopy for human

    pancreatic islet clock studies

    Overview

    Central

    Clock

    Peripheral

    Clocks

  • Digital composite image of the day/night terminator passing over Europe and Northern Africa.

    This picture gives a good impression about the enormous environmental changes organisms

    have to cope with during the course of each day (source: www.nasa.gov; adapted from the site

    of Dr. Henric Oster, Max-Planck-Institute, Circadian Rhythms Group)

    Geophysical Time Circadian Clocks: Time-Measuring Devices Allowing Synchronization to Geophysical Time

    http://circadianrhythms.mpibpc.mpg.de/ http://www.nasa.gov/

  • Body temperature Cardiovascular system: heartbeat, blood pressure

    Renal activity

    Endocrine system

    Activity of the digestive tract

    Visual acuity

    Rest- activity cycles

    Circadian rhythms in mammals

  • INPUT photoperiod

    OUTPUTS

    overt rhythms in physiology

    SCN neuron SCN

    OSCILLATOR

    cellular oscillators of coupled SCN neurons

    The central clock resides in the suprachiasmatic nuclei

    of the hypothalamus (SCN)

  • Visualization of the neuronal cells of suprachiasmatic

    nuclei (SCN)

    Courtesy of Prof. Mick Hastings (MRC, Cambridge, UK)

    luciferase mPer1

    promoter

    Circadian oscillation of mPer1-luciferase expression in neurons of SCN kept in

    organotypic tissue culture

  • Rhythmic physiology

    Central pacemaker (SCN) Peripheral clocks

    Central and peripheral circadian clocks

  • Peripheral clock entrainment pathways

    Dibner et al., Ann.Rev.Physiol. 2010

  • Activator

    Repressor

    Circadian oscillator underlying principle: negative

    feeback loops of gene expression

  • Rev-Erba

    Cry1, Cry2

    Per1, Per2

    Bmal1

    Clock

    The mammalian circadian oscillator molecular

    makeup

  • Working model for the mammalian circadian

    clockwork circuitry

  • SCN

    Circadian clocks ticking everywhere?..

    Per1 mRNA

    …but clock (e.g. Per1) gene expression is circadian in most body cells

    Universal character of the circadian clock

  • Cell cycle arrested

    Balsalobre et al., Science, 1998

    ..yes, even in cultured fibroblast cells

  • 1. Acute phase shift (jet lag)

    2. Chronic phase shift (shift work)

    3. Drug effectiveness on various parameters that are under

    endogenous rhythm control (eg: urinary excretion rates, BP,

    heart rate, etc)

    4. Sensitivity to anesthetic agents

    5. Delayed Sleep Phase Syndrome (DSPS); Advanced Sleep

    Phase Syndrome (FASPS)

    6. Delayed sleep phase insomnia

    7. Psychiatric illnesses

    8. Epilepsy

    What if the clock does not tick properly?

    Common circadian rhythm disorders

  • Jet lag and shift work: a circadian phase shifting

    • When we travel east, the sun rises and sets earlier. The natural light

    is advanced with respect to that at home. This results in an

    apparently shorter night followed by a new cycle. When we travel

    west, sunrise and sunset is later than at home, giving an apparently

    longer day, followed by a new cycle. In both these cases, we

    undergo a phase shift of the Zeitgebers in our environment.

    • In humans, the period of readaptation during travel is called jet

    lag. However phase shifting does not exclusively occur with travel.

    The increased need for 24 hour service, or the constant use of

    expensive machines has resulted in the use of shift work to provide

    a constant work force. Such schedules often require the frequent

    resynchronisation of individuals to new time cues as a result

    of working shifts.

  • Shiftwork: health effects

    • Increased likelihood of obesity

    • Increased risk of cardiovascular disease

    • Higher risk of mood changes

    • Increased risk of gastrointestinal problems, such as constipation and stomach discomfort

    • Higher risk of motor vehicle accidents and work- related accidents

    • Increased likelihood of family problems, including divorce

  • • Physiology and behavior of light sensitive organisms oscillate with a period length of ~24h.

    • These “circadian rhythms” are driven by a self-sustaining clock-like mechanism.

    • A master pacemaker in the brain, the SCN, synchronizes the internal clock with external time (light, temperature) and transmits the signal to the periphery.

    • The core clock ticking in cells in the whole body have similar molecular make up.

    • Fibroblasts in culture could be induced by different stimuli and exhibit strong oscillations. In vivo bioluminescence monitoring of luciferase reporter driven from circadian promoters expressed in these cells has been extensively used to study cell clock work.

    Summary-1

  • WHY?

    • Signal analysis from entire dish does not allow single cell and spatial analysis

    • Cell desynchronization: dephased oscillators or non cycling cells? • Dividing cells: does cell division change oscillation pattern?

    Single cell oscillation analysis using fluorescence or

    bioluminescence time-lapse microscopy

  • Nagoshi et al., Cell (2004)

    Rev-erb a

    ex1 2 3 4 5 6 7 8 ATG TGA

    Venus PolyA KmR

    NLS PEST1

    TGA ATG

    Rev-Venus

    Circadian Yellow Fluorescent Protein (Venus)

    expression in individual NIH3T3 fibroblasts

    • In 2008, Osamu Shimomura, Martin Chalfie and Roger Y. Tsien have received the Nobel Price of chemistry for the discovery and development of green fluorescent protein, GFP

  • 0.5%

    Serum

    50% Serum

    3.5 t=0 2.0 2.5 80hrs 3.0

    0.5% Serum

    Circadian Yellow Fluorescent Protein (Venus)

    expression in individual NIH3T3 fibroblasts

  • Circadian Reverb alpha Venus NLS PEST1 expression

    in individual NIH3T3 fibroblasts

  • Luciferase reporters are extensively used to monitor

    circadian rhythms

    Firefly Luciferase

    Luciferin + ATP + O2 Oxyluciferin + AMP + CO2 + light

    Luciferase catalyzes the oxidation of luciferin photon emitter

    (photon emitter)

    Chemical reaction in luminescence

    Firefly eggs

  • Circadian rhythm analysis in the individual cells:

    bioluminescent reporters

    • Bioluminescent reporter is a biological construct where firefly luciferase synthesis is driven by the regulatory sequence of the gene of interest

    •Expression of the bioluminescent reporter in the cell allows quantification of the emitted light, reflecting the level of expression of the gene of interest

    luciferase

    Bmal1 promoter and 5’UTR

    Bmal1 3’UTR and polyA site

  • luciferase

    Bmal1 promoter and 5’UTR

    Bmal1 3’UTR and polyA site

    0

    50000

    100000

    150000

    200000

    250000

    300000

    350000

    0 20 40 60 80 100

    hrs after serum shock

    P h o to n c o u n ts / m in

    photomultiplier tube

    Period length=26.2 hrs

    Real time recording of bioluminescence generated by a transgenic

    NIH3T3 cell line expressing firefly luciferase from the Bmal1 promoter

    Nagoshi et al., Cell, 2004

  • Olympus Luminoview LV-200

    Bioluminescence work station

    Bioluminescence time lapse microscopy

    Advantages Non-toxic (no problem with drug pre-treatment)

    Limitations Low signal intensity (luciferase-expressing cell lines often give extremely dim signals)

    To overcome this we use Ultrasensitive camera cooled to -80°C Measurement in total darkness

  • Robust image processing software for cell tracking

    and analysis (collaboration with Daniel Sage, BIG, EPFL)

    Preprocessing

    Tracking

    Cell 1 oscillation pattern

    0

    50

    100

    150

    200

    250

    300

    0 4 7

    1 1

    1 4

    1 8

    2 1

    2 5

    2 8

    3 2

    3 5

    3 9

    4 2

    4 6

    4 9

    5 3

    5 6

    6 0

    6 3

    6 7

    7 0

    Time, hours