Combined Bioluminescence-Fluorescence Time Lapse ... · Combined Bioluminescence-Fluorescence Time Lapse Microscopy: Applications to Circadian Rhythm Studies Charna Dibner/Tiphaine
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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
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
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
0
50
100
150
200
250
300
0 4 7
11
14
18
21
25
28
32
35
39
42
46
49
53
56
60
63
67
70
Time, hours
Pix
els
Cell 3 oscillation pattern
0
50
100
150
200
250
300
0 4 7
11
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Time, hours
Pix
els
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
0
50
100
150
200
250
300
0 4 7
11
14
18
21
25
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Time, hours
Pix
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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
5000
7000
9000
11000
13000
15000
17000
19000
21000
23000
25000
0 9
19
28
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Time, hours
co
un
ts p
er
min
ute
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
5000
10000
15000
20000
25000
30000
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Time, hours
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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:
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