Decisions, Decisions!
How Cells Sense Their Environment
and
Make Decisions to Adapt and Respond
Prof. Thomas E. Kuhlman
Department of Physics
Center for the Physics of Living Cells
University of Illinois at Urbana-Champaign
!
Saturday Physics for Everyone
September 13th, 2014
http://cdn.physorg.com/newman/gfx/news/hires/2013/minusenviron.jpg
Introduc7on
CELLS, DNA, AND GENETIC REGULATION
Your Body is Made of CELLS
http://www.turbosquid.com/3d-models/einstein-cartoon-human-3d-model/286031
Neuronal Cells
http://www.turbosquid.com/3d-models/einstein-cartoon-human-3d-model/286031
Neuronal Cells
Re7nal Cells
http://www.turbosquid.com/3d-models/einstein-cartoon-human-3d-model/286031
Neuronal Cells
Re7nal Cells
Cardiac Muscle Cells
http://www.turbosquid.com/3d-models/einstein-cartoon-human-3d-model/286031
Neuronal Cells
Re7nal Cells
Intes7nal Cells
Cardiac Muscle Cells
http://www.turbosquid.com/3d-models/einstein-cartoon-human-3d-model/286031
Neuronal Cells
40,000,000,000,000 Cells! Re7nal Cells
Of Different Kinds, Shapes, and Sizes!
Intes7nal Cells
Cardiac Muscle Cells
http://www.turbosquid.com/3d-models/einstein-cartoon-human-3d-model/286031
Neuronal Cells
40,000,000,000,000 Cells! Re7nal Cells
Of Different Kinds, Shapes, and Sizes!
The Cell’s Gene7c Informa7on (DNA) Contains All the Informa7on Required to Produce Materials (Proteins) for Different Cell Types
Intes7nal Cells
Cardiac Muscle Cells
http://www.turbosquid.com/3d-models/einstein-cartoon-human-3d-model/286031
Neuronal Cells
40,000,000,000,000 Cells! Re7nal Cells
Of Different Kinds, Shapes, and Sizes!
The Cell’s Gene7c Informa7on (DNA) Contains All the Informa7on Required to Produce Materials (Proteins) for Different Cell Types
What Is It That’s Different About the Gene7c Informa7on Intes7nal Cellsof Different Cell Types?
Cardiac Muscle Cells
http://www.turbosquid.com/3d-models/einstein-cartoon-human-3d-model/286031
(Most) Cells Types Have the Same DNA Information
♂
♀
[Gurdon, Proc R Soc (Lond) B Biol Sci 1977]
(Most) Cells Types Have the Same DNA Information
♂
♀
DNA
♀ Egg Cell
[Gurdon, Proc R Soc (Lond) B Biol Sci 1977]
(Most) Cells Types Have the Same DNA Information
♂
♀
DNA
♂ Intes7nal Cell
♀ Egg Cell
[Gurdon, Proc R Soc (Lond) B Biol Sci 1977]
(Most) Cells Types Have the Same DNA Information
♂
♀
♂ Intes7nal Cell
♀ Egg Cell
[Gurdon, Proc R Soc (Lond) B Biol Sci 1977]
(Most) Cells Types Have the Same DNA Information
♂
♀
♂ Intes7nal Cell
♀ Egg Cell
[Gurdon, Proc R Soc (Lond) B Biol Sci 1977]
(Most) Cells Types Have the Same DNA Information
♂
♀
♂ Intes7nal Cell
♀ Egg Cell
[Gurdon, Proc R Soc (Lond) B Biol Sci 1977]
(Most) Cells Types Have the Same DNA Information
♂
♀
♂ Intes7nal Cell
♀ Egg Cell
[Gurdon, Proc R Soc (Lond) B Biol Sci 1977]
(Most) Cells Types Have the Same DNA Information
♂
USE of DNA is Different in Different Cells! “GENETIC REGULATION”
♀
♂ Intes7nal Cell
♀ Egg Cell
[Gurdon, Proc R Soc (Lond) B Biol Sci 1977]
Introduc7on
GENE REGULATION AND DECISION MAKING
We <3 Escherichia coli
t=0
N=1
t=0
We <3 Escherichia coli
t=0
5 cm
N=1
1 μm
t=0
We <3 Escherichia coli
t=0
5 cm
N=2
N=1
1 μm
t=0
We <3 Escherichia coli
t=0
t = 25 years
5 cm
N=2
N=1
1 μm
t=0
We <3 Escherichia coli
t=0
t = 25 years
5 cm
N=2
N=1
1 μm
t=0
t = 20 minutes
We <3 Escherichia coli
t=0
t = 25 years
5 cm
N=2
N=1
1 μm
t=0
t = 20 minutes
N=
1,500,000,000
We <3 Escherichia coli
t=0
t = 25 years
t = 760 years
N=2
N=
1,500,000,000
5 cm
N=1
1 μm
t=0
t = 20 minutes
We <3 Escherichia coli
t=0
t = 25 years
t = 760 years
N=2
N=
1,500,000,000
t = 20 minutes
t = 10 hours
5 cm
N=1
1 μm
t=0
We <3 Escherichia coli
t=0
t = 25 years
t = 760 years
5 cm
With E. coli we can manipulate and study MANY CELLS N=
N = 2TIME
N=1
in a SHORT 1,500,000,000
1 μm
t=0
t = 20 minutes
t = 10 hours
Anatomy of E. coli
Anatomy of E. coli
Anatomy of E. coli
DNA Genome
Encodes Information
Anatomy of E. coli
A
T
G
C
G
A
DNA Genome
Encodes Information
T
A
C
G
C
T
Basepair Sequence
Anatomy of E. coli
Proteins
Machines That Build
and Run the Cell
A
T
G
C
G
A
DNA Genome
Encodes Information
T
A
C
G
C
T
Basepair Sequence
Anatomy of E. coli
Proteins
Machines That Build
and Run the Cell
A
T
G
C
G
A
DNA Genome
Encodes Information
T
A
C
G
C
T
Basepair Sequence
Gene Regulation - A Crash Course in Molecular Microbiology
1 μm
Escherichia coli (400x)
Gene Regulation - A Crash Course in Molecular Microbiology
1 μm
Escherichia coli (400x)
DNA
Gene Regulation - A Crash Course in Molecular Microbiology
1 μm
Escherichia coli (400x)
DNA
Gene Regulation - A Crash Course in Molecular Microbiology
E. coli Genome
4.6 Million Base Pairs
1 μm
Escherichia coli (400x)
DNA
Gene Regulation - A Crash Course in Molecular Microbiology
E. coli Genome
4.6 Million Base Pairs
gene 1
gene 2
gene 3
gene 4
Gene Regulation - A Crash Course in Molecular Microbiology
“Gene”
- Sequence of bases (A, T, G, C) that encodes information to make a protein
gene 1
gene 2
gene 3
gene 4
Gene Regulation - A Crash Course in Molecular Microbiology
“Gene”
- Sequence of bases (A, T, G, C) that encodes information to make a protein
gene 1
“Promoter”
-Required for genes to be expressed
gene 2
gene 3
gene 4
Gene Regulation - A Crash Course in Molecular Microbiology
“Gene”
- Sequence of bases (A, T, G, C) that encodes information to make a protein
gene 1
“Promoter”
-Required for genes to be expressed
gene 2
“Operator”
- Protein Binding Site
gene 3
gene 4
Genome
gene 1
gene 2
gene 3
gene 4
Genome
Gene
gene 1
gene 2
gene 3
gene 4
Genome
Gene
gene 1
gene 2
gene 3
gene 4
Genome
Gene
gene 1
“RNA polymerase” Protein
gene 2
gene 3
gene 4
Genome
Gene
gene 1
messenger RNA (”mRNA”)
“RNA polymerase” Protein
“Transcription”
gene 2
gene 3
gene 4
Genome
Gene
gene 1
messenger RNA (”mRNA”)
“RNA polymerase” Protein
“Transcription”
gene 2
gene 3
gene 4
Genome
messenger RNA (”mRNA”)
Ribosome
Gene
gene 1
“RNA polymerase” Protein
“Transcription”
gene 2
gene 3
gene 4
Genome
messenger RNA (”mRNA”)
“Translation”
Ribosome
Gene
gene 1
“RNA polymerase” Protein
Protein
“Transcription”
gene 2
gene 3
gene 4
Gene is “ON”
Genes Are Regulated!
1 μm
Escherichia coli (400x)
[Monod, Thesis 1941]
Genes Are Regulated!
1 μm
Number of Bacteria
Escherichia coli (400x)
Glucose Lactose
Time
[Monod, Thesis 1941]
Genes Are Regulated!
1 μm
Number of Bacteria
Glucose
Lactose
Glucose Lactose
Time
[Monod, Thesis 1941]
Genes Are Regulated!
1 μm
Number of Bacteria
Glucose
Lactose
Glucose Lactose
Time
[Monod, Thesis 1941]
Genes Are Regulated!
1 μm
Glucose Lactose
Lactose
Concentra7on
Number of Bacteria
Glucose
Time
[Monod, Thesis 1941]
Genes Are Regulated!
1 μm
Lactose
Glucose Lactose
Time required to turn on genes to eat lactose!
Concentra7on
Number of Bacteria
Glucose
Time
[Monod, Thesis 1941]
Gene Regulation by Protein Transcription Factors
RNA polymerase
gene 1
gene 2
gene 3
gene 4
Gene is “ON”
gene 1
gene 2
gene 3
gene 4
gene 1
gene 2
gene 3
gene 4
“Transcription Factor”
Lac Repressor Protein
gene 1
gene 2
gene 3
gene 4
gene 1
gene 2
gene 3
gene 4
gene 1
gene 2
gene 3
gene 4
Gene is “OFF”
Genome
gene 1
Gene
gene 2
gene 3
gene 4
Gene is “OFF”
Genome
gene 1
Protein Transcription Factors
Gene
gene 2
gene 3
gene 4
Gene is “OFF”
Genome
Protein Transcription Factors
Gene
LACTOSE
gene 1
gene 2
gene 3
gene 4
Genome
Protein Transcription Factors
Gene
LACTOSE
gene 1
gene 2
gene 3
gene 4
Genome
Protein Transcription Factors
Gene
LACTOSE
gene 1
gene 2
gene 3
gene 4
Genome
Protein Transcription Factors
Gene
LACTOSE
gene 1
gene 2
gene 3
gene 4
Physics!
GENE REGULATION AND DECISION MAKING
Where’s the Physics?
gene 1
gene 2
gene 3
gene 4
Where’s the Physics?
[Golding, Paulsson, Zawilski, and Cox, Cell 2005]
Transcription and Expression
gene 1
gene 2
gene 3
gene 4
Where’s the Physics?
gene 1
gene 2
gene 3
gene 4
Where’s the Physics?
KI
KO
gene 1
gene 2
Transcriptional Regulation
[TF] + [Operator] ←⎯
→[TF − Operator]
KO
[TF] + [Inducer] ←⎯
→[TF − Inducer]
KI
gene 3
gene 4
Where’s the Physics?
[Kuhlman et al, PNAS 2007]
Theoretical
Experimental
KI
KO
gene 1
gene 2
Transcriptional Regulation
[TF] + [Operator] ←⎯
→[TF − Operator]
KO
[TF] + [Inducer] ←⎯
→[TF − Inducer]
KI
gene 3
gene 4
Where’s the Physics?
gene 1
gene 2
gene 3
gene 4
Where’s the Physics?
Intracellular Diffusion
gene 1
[Kuhlman and Cox, MSB 2012,
Kuhlman and Cox, PRE 2013]
gene 2
gene 3
gene 4
Our Experimental Toolbox
Chemical Reactions
Lactose
Genes
Our Experimental Toolbox
Chemical Reactions
Lactose
Genes
Colorless
Our Experimental Toolbox
Chemical Reactions
Lactose
Genes
Colorless
Our Experimental Toolbox
Microfluidics
Our Experimental Toolbox
Microfluidics
Our Experimental Toolbox
Microfluidics
Microscopy and Fluorescence
An Example
PROTEIN TRANSCRIPTION FACTOR DIFFUSION AND GENOME ORGANIZATION
Example: Transcription Factor Diffusion in E. coli
gene 1
[Elf et al, Science 2007]
gene 2
gene 3
gene 4
Example: Transcription Factor Diffusion in E. coli
“Transcription Factor”
Lac Repressor
gene 1
[Elf et al, Science 2007]
Venus
Fluorescent Reporter
gene 2
gene 3
gene 4
Example: Transcription Factor Diffusion in E. coli
gene 1
[Elf et al, Science 2007]
gene 2
gene 3
gene 4
Example: Transcription Factor Diffusion in E. coli
gene 1
gene 2
gene 3
gene 4
“IPTG” prevents lac repressor from binding to operator
[Elf et al, Science 2007]
Genome
Gene
Genome
Gene
Can imagine different ways to organize:
Genome
Gene
Can imagine different ways to organize:
Highly Ordered
Genome
Gene
Can imagine different ways to organize:
Highly Ordered
vs.
Random Pile
Highly Ordered
vs.
Random Pile
Highly Ordered
CON: Takes a lot of time
and energy to keep
organized!
PRO: If you are a slow
searcher, it makes things
easier to find
vs.
Random Pile
Highly Ordered
CON: Takes a lot of time
and energy to keep
organized!
PRO: If you are a slow
searcher, it makes things
easier to find
vs.
Random Pile
PRO: Things naturally
tend towards random
pile; doesn’t require
energy to maintain
CON: Extremely hard to
find what you want
unless you are extremely
fast!
Highly Ordered
GOOD FOR:
vs.
Random Pile
GOOD FOR:
Highly Ordered
GOOD FOR:
[Elf et al, Science 2007]
vs.
Random Pile
GOOD FOR:
But The E. coli Genome is Highly Organized!
lacI
lacZ
lacY
lacA
But The E. coli Genome is Highly Organized!
lacZ
lacI
d ~100 bp
lacY
lacA
But The E. coli Genome is Highly Organized!
lacZ
lacI
lacY
lacA
d ~100 bp
Blue:
d < 10 kbp
Green: 10 kbp < d < 500 kbp
Yellow:
d > 500 kbp
[Warren, ten Wolde JMB 2004]
E. coli
Random
But The E. coli Genome is Highly Organized!
lacZ
lacI
lacY
lacA
d ~100 bp
Blue:
d < 10 kbp
Green: 10 kbp < d < 500 kbp
Yellow:
d > 500 kbp
[Warren, ten Wolde JMB 2004]
E. coli
Random
But The E. coli Genome is Highly Organized!
lacZ
lacI
lacY
lacA
d ~100 bp
Blue:
d < 10 kbp
Green: 10 kbp < d < 500 kbp
Yellow:
d > 500 kbp
[Warren, ten Wolde JMB 2004]
Random
E. coli
WHY?
D
120x magnification
488 nm laser excitation
D
Rate of Diffusion Depends Upon Cell Growth
Happy, Growing Cells
Starving Cells
400x magnification
514 nm laser excitation
D
120x magnification
488 nm laser excitation
D
Rate of Diffusion Depends Upon Cell Growth
Happy, Growing Cells
Starving Cells
400x magnification
514 nm laser excitation
Another Example
MRNA DIFFUSION
Example: mRNA tracking in E. coli
Gene
[Golding and Cox, PNAS 2004;
Golding and Cox, PRL 2006]
rfp
96x MS2 bs
Example: mRNA tracking in E. coli
...
mRNA
Transcription
Gene
[Golding and Cox, PNAS 2004;
Golding and Cox, PRL 2006]
rfp
96x MS2 bs
Example: mRNA tracking in E. coli
MCP
...
mRNA
GFP “Green Fluorescent
Protein”
“MS2 Coat Protein”
Transcription
Gene
[Golding and Cox, PNAS 2004;
Golding and Cox, PRL 2006]
rfp
96x MS2 bs
Example: mRNA tracking in E. coli
GFP GFPGFP GFP GFP
MCPMCPMCPMCPMCP
mRNA
...
Transcription
Gene
[Golding and Cox, PNAS 2004;
Golding and Cox, PRL 2006]
rfp
96x MS2 bs
Example: mRNA tracking in E. coli
GFP GFPGFP GFP GFP
MCPMCPMCPMCPMCP
mRNA
...
Transcription
Gene
[Golding and Cox, PNAS 2004;
Golding and Cox, PRL 2006]
rfp
96x MS2 bs
Intracellular mRNA motion is subdiffusive!
=> Cytoplasm is highly crowded
D ~ 10-2 um2/s
Acknowledgements
Center for Biophysics and
Computational Biology
Cac Nguyen
Neil Kim
Nigel Goldenfeld
Huseyin Tas
Nick Sherer
Michael Martini