Single Cell Variability
The contribution of noise to
biological systems
Outline
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Background
Why single cells?
Noise in biological systems
Cool studies
Conclusions
Background – Microscale Life
Sciences Center
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Funded by NIH
CEGS
To develop technologies for single cell
research
Lab-on-a-chip modality
Collaborative approach
Why Single Cells?
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Variable of interest
Bulk data represents
averages
Averages may not
represent behavior of
subpopulations
Potential Resonse Profiles for a Population
10
9
8
7
6
5
4
3
2
1
0
1
2
3
4
Intensity of Response
5
Range of Response
50% response
Singular Resonse
6
7
Why Single Cells? – One
Example
=?
=?
Why Single Cells? – One
Example
9
8
7
6
5
4
3
=?
2
1
0
1
2
3
4
5
6
7
8
9
Gaussian
=?
7
6
5
4
3
2
1
0
1
2
3
4
5
6
Bimodal
7
8
9
Why Single Cells? – One
Example
9
8
7
6
5
4
3
=?
2
1
0
1
2
3
4
5
6
7
8
9
Gaussian
=?
=
7
6
5
4
3
2
1
0
1
2
3
4
5
6
Bimodal
7
8
9
Variability in populations –
What we know so far
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Population response is governed by:
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Variability at the single cell level
Subpopulations
Noise inherent to any complex system
Noise in biological systems
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“Chemical analysis are affected by two types
of noise: chemical noise and instrumental
noise”*
What is chemical noise?
What is instrument noise?
In general: Noise = σ/mean
*Principals of Instrumental Analysis. 1998. Skoog, Holler, and Nieman.
Noise in biological systems
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“Chemical analysis are affected by two types
of noise: chemical noise and instrumental
noise”*
What is chemical noise?
 Fluctuations in Temp, concentration,
vibrations, light, gradients, etc
What is instrument noise?
 Composite of noise from individual
components of a system
*Principals of Instrumental Analysis. 1998. Skoog, Holler, and Nieman.
Noise in biological systems
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Noise in a nutshell
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Chemical noise = intrinsic (inherent) variability
Instrument noise = extrinsic (global) variability
Will show examples from literature and my
research
Noise in biological systems
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Intrinsic noise:
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Inherent
Order of events
Entropy
Binding of substrate to enzyme
Noise in biological systems
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Extrinsic noise:
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Concentrations of system components
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Chemical flux through components
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Regulatory proteins, polymerase
Enzyme activities
Substrate to product conversion
Global effects of all components
Extrinsic Noise – cell growth
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Global variability that is a composite of
intrinsic noise from each component of a
system.
First observed by Kelly and Rahn in 1932*
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Measured 2-3 fold variation in the division times of
single E. coli cells
No correlation between division time of mother
cell and division time of either of the two daughter
cells
*Kelly & Rahn, J. Bacteriol., 1932
Extrinsic Noise – cell growth
200
180
160
Frequency
140
120
100
80
60
40
Cells imbedded in soft agar
20
Minutes
*Kelly & Rahn, J. Bacteriol., 1932
70-75
65-70
60-65
55-60
50-55
45-50
40-45
35-40
30-35
25-30
20-25
15-20
10-15
0
Extrinsic Noise – cell growth
Air tank
Light Source
vent
Pump
Reservoir
Lung (50ft tubing)
hv
Environmental
Chamber
Objective
Waste
Extrinsic Noise
LSM Data
Extrinsic Noise
Single Cell Growth over Time
5.5
5
Cell Length (mm)
4.5
4
3.5
3
2.5
2
1.5
1
0
5
10
Time (hrs)
Strovas et al. In preparation.
15
20
Extrinsic Noise
Single Cell Growth over Time
5.5
5
Cell Length (mm)
4.5
4
3.5
3
2.5
2
1.5
0.73 mm/hr
0.55mm/hr
1
0
5
10
Time (hrs)
Strovas et al. In preparation.
15
20
Extrinsic Noise
Methanol
25
20
20
Time (hrs)
3.12 +/- 0.55 hrs (N = 115)
3.73 +/- 0.63 hrs (N = 195)
6.5
6.2
5.9
Time (hrs)
•Over 2 fold range in division rates
•Extrinsic noise differs based on carbon source
Strovas et al. In preparation.
5.6
5.3
5
4.7
4.4
4.1
2.6
4.5
4.3
4.1
3.9
3.7
3.5
3.3
3.1
2.9
2.7
0
2.5
0
2.3
5
2.1
5
3.8
10
3.5
10
15
3.2
15
2.9
Frequency
25
1.9
Frequency
Succinate
Intrinsic Noise - Transcription
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The noise inherent to a system component
What are components of a biological system?
Focus on noise in transcription
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How does one measure transcription rates?
Intrinsic Noise - Transcription
Promoter Activities via Transcriptional Fusions
light
Plac
Intrinsic Noise - Transcription
http://meds.queensu.ca/~mbio318/EXTRA_MATERIAL.html
Intrinsic Noise - Transcription
http://meds.queensu.ca/~mbio318/EXTRA_MATERIAL.html
Intrinsic Noise
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Elowitz et al, 2002
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Elegant experiment to show intrinsic noise
Made two transcriptional fusions in E. coli:
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Plac-YFP
Plac-CFP
Observed YFP and CFP fluorescence w/ and
w/out IPTG present
Intrinsic Noise
Elowitz et al, Science, 297, 1183-1186, 2002
Intrinsic Noise
Fluorescence vs. Growth rate
Methanol
2
2800
2600
2400
2200
2000
1800
1600
1400
1200
1000
RFU/mm
RFU/mm
2
Succinate
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
2800
2600
2400
2200
2000
1800
1600
1400
1200
1000
0
0.2
0.4
0.6
0.8
1
Growth Rate (mm/hr)
Growth Rate (mm/hr)
R2 = 0.0257
R2 = 0.0049
Strovas et al. In preparation.
1.2
1.4
1.6
Intrinsic Noise
Single Cell RFU/mm2
Succinate -> Methanol Carbon Shift
3000
2800
2600
2400
2200
2000
1800
1600
1400
1200
1000
0
5
10
15
20
25
30
35
40
Time (hrs)
Succinate: 1993.15 +/- 468.14 RFU/mm^2 (N = ~1000)
Methanol: 3075.30 +/- 243.35 RFU/mm^2 (N = ~1000)
Strovas et al. In preparation.
Noise in biological systems Summary
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Variability in biological systems at the
population and single cell level is governed
by intrinsic and extrinsic noise.
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Extrinsic noise dominates variability as a whole
Intrinsic noise dominates the variability observed
from individual components of a system
Intrinsic noise can be independent of extrinsic
noise
Now what?
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Since noise in biological systems can govern
biological variability, can’t we cure cancer and
move on?
No! Like all complex systems we must
characterize them!
What we know is just the tip of the iceberg!
Nifty stuff – Balaban et al.
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Bacterial persistence as a phenotypic switch
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Balaban et al. 2004. Science. 305: 1622-1625
Demonstrated the ability of single cells from
an E. coli clonal population to survive
treatment with antibiotics.
Nifty stuff – Balaban et al.
Nifty stuff – Balaban et al.
Nifty stuff – Balaban et al.
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Persister cells were susceptible to
subsequent antibiotic treatment
Heterogeneity (variance) within the
population attributed to presence of
persisters
Why can persisters survive and how is it
useful?
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What type of noise governs this response?
Nifty stuff – Raser and Shea
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Control of stochasticity in eukaryotic gene
expression
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Raser and Shea. 2004. Science. 304: 1811-1814
Used similar methods to Elowitz et al. only
using yeast.
Suggests that noise is an evolvable trait that
can help balance fidelity and diversity
Nifty stuff – Raser and Shea
Time course during phosphate starvation
Nifty stuff – Raser and Shea
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Showed extrinsic noise dominates total noise
in yeast
Intrinsic noise only contributed 2-20%
Transcription in eukaryotes has been
described as pulsative
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Results in variable mRNA levels from cell to cell
Causes phenotypic diversity in clonal yeast
populations
Conclusions
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Population averages skew the underlying
contributions of subpopulations
Subpopulations are the result of variable
cellular response within a clonal population
Cellular variability arises from intrinsic noise,
but governed by extrinsic noise
Cellular variability allows for adaptation to
environmental perturbations