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

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

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350000

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hrs after serum shock

Photon counts/min

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

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Cell 3 oscillation pattern

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Additional output parameters : • Cell size • Cell motility (distance and angle) • Cell division time • Easy correlation between cell

circadian phase, cell division time and cell motility

Cell 2 oscillation pattern

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Input

Output

Sage et al., Cell Division, 2010

Daniel SAGE

Per2luc insertion +/+

Primary mouse tail skin fibroblasts

photomultiplier tube

Reporter mouse expressing Per2::luc knock in

bioluminescence reporter

Individual cell oscillation analysis Population circadian profile analysis

Primary mouse tail fibroblasts from Per2::luciferase knockin mouse (63X objective)

Bioluminescence time lapse microscopy

DNA ligase I-RFP was kindly provided by C.Cordosa (Easwaran et al., Cell Cycle 4:3, 453-455, 2005)

DNA ligase I-RFP (S-phase marker) transient

expression in NIH3T3 cells

• Both methods have allowed us to perform in vivo non-invasive, high resolution and long term recording and analysis of individual circadian behavior at single cell level • Fluorescence microscopy allows higher spatial resolution and dual labeling, but is rather toxic and not suitable for sensitive/pre-treated cells

• In vivo bioluminescence monitoring of luciferase reporter driven from circadian promoters expressed in cultured cells is a powerful tool to study cellular clockwork

•Bioluminescence microscopy is absolutely non-toxic, allows the use of sensitive cells and more then 3 day recording. Spatial resolution and intensity limitations are yet to overcome

Summary-2

Single cell oscillation analysis using fluorescence or

bioluminescence time lapse microscopy

Circadian clock impacts critically on metabolic

regulation

The pancreas is composed of endocrine compartments

Islets of Langerhans

Insulin- β cells

Glucagon- α cells

Somatostatin- δ

cells Adapted from Cabrera et al, PNAS, 2006

L’horloge dans les îlots humains: cellules beta versus non-beta Organization of α and β cells in human islets Domenico Bosco et al., Diabetes 2010

Sections of human pancreata with islets of different sizes were either double-labeled for insulin

(red) and glucagon (green). Except for the 40- to 60-μm–diameter islets, all islets displayed one or

several unstained empty areas (vascular channels) at their core. Most glucagon-expressing cells

were located around vascular channels and at the mantle of islets, independent of their size.

Insulin-expressing cells seemed clustered into discrete ovoid areas surrounded by α-cells.

Obesity and metabolic syndrome in circadian Clock19Dmice

Bm

al1

Clo

ck

(Kohsaka et al., Cell Metab., 2007)

High-fat diet disrupts behavioral and molecular circadian rhythms

in mice

(Turek et al., Science, 2005)

Body w

eig

ht

mutant

WT

Regular High Fat

Metabolic parameter WT Clock P value

Triglyceride (g/dl) 136 ± 8 164 ± 8 < 0.05

Cholesterol (mg/ml) 141 ± 9 163 ± 6 < 0.05

Glucose (mg/dl) 130 ± 5 161 ± 7 < 0.05

Insulin (ng/ml 1.7 ± 0.3 1.1 ± 0.1 n.s.

Leptin (ng/ml) 3.4 ± 0.4 4.6 ± 0.3 < 0.05

Connection between obesity and the circadian clockwork

Disruption of the clock components CLOCK and BMAL1 leads

to hypoinsulinaemia and diabetes

Marcheva B. et al. 2010. Nature 466: 627-631

Bioluminescence

Time (days) Per2

-lu

cife

rase

Disruption of the circadian genes CLOCK and BMAL1 in mice

Diabetic phenotype of CLOCK and BMAL1 mutant mice

mutant

wt

Glucose Intolerance Insulin secretion Reduced b-cell growth

wt

mutant

The circadian activity of the pancreas

α-cell

β-cell

Pamela

PULIMENO Laurianne

GIOVANNONI

Impact of the human pancreatic islet clocks on islet gene

expression and function

Robust circadian clocks are ticking in human

pancreatic islets

Period length, h

23.6±0.4 hours N=20

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Bmal1-luc and CMV-GFP co-expression in intact human islets

Human islets-autonomous circadian oscillators

Time lapse bioluminescence microscopy in the individual human islets

Robust circadian clocks are ticking in dispersed

human islets cells

Period length, h

24.3±0.8 hours N=12

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Bmal1-luc and CMV-GFP co-expression in dispersed human islet cells

Rat Insuline2 promotor (RIP)-tomato expression in human islet cells

(collaboration with Patrick Salmon, CMU, University of Geneva)

Are different islet cell clocks coupled?

Visualizing clock in human β cells

Human islet cells oscillatory profile analysis (collaboration with Daniel Sage, EPFL)

Bioluminescence Fluorescence

Human islet cells: β versus non β-cell

oscillations

Bioluminescence

Fluorescence

Human islet cells: β versus non β-cell

oscillations

β non-β

26.28±2.26 h 26.01±1.37h

• Cell-autonomous high-amplitude

clocks are functional in human pancreatic islets: at islet population, single islet and single islet cell levels

• β-cells possess their own clocks, oscillating in synchrony with non-β-cells in primary human islet cell culture

•RIP-tomato labeling provides a valuable tool for studying human β- and non- β-cell function without FACS sorting, and is particularly useful for combined bioluminescence-fluorescence time lapse microscopy application

1. Is the circadian clock perturbed in obese and type 2 diabetic patients?

2. If so –how can we fix it?

3. Is it due to our lifestyle change?

Questions raised

• Proper clockwork is critical for the glucose homeostasis regulation

• Disruption of the circadian clockwork leads directly to the endocrine pancreas disfunction and diabetes development

Summary-3

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

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

• Proper clockwork is critical for the body metabolism and for hormone secretion.

• In vivo bioluminescence monitoring of luciferase reporter driven from circadian promoters expressed in cultured cells is a powerful tool to study cellular clockwork.

• Bioluminescence time-lapse microscopy opened new horizons in studies of the cellular clockwork and beyond.

Overall summary

Ueli SCHIBLER, UniGeneva

Christoph Bauer

Jerome Bosset

Michael Parkan

NCCR Bioimaging platform

University of Geneva

Daniel SAGE

Michael UNSER

BIG, EPFL

PROMEGA Joanna Stevenson Reka Nagy

Acknowledgments

Tiphaine Mannic

Laurent Perrin

Anne-Marie Makhlouf

Pamela Pulimeno

Dibner’s lab: