keynote lecture instrumentation challenges for systems biology john wikswo vanderbilt institute for...
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Keynote Lecture
Instrumentation Challenges for Systems Biology
John Wikswo
Vanderbilt Institute for Integrative Biosystems Research and EducationVanderbilt University, Nashville, TN, USA
Third IEEE Sensors Conference, Vienna, Austria, October 25, 2004
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AbstractBurgeoning genomic and proteomic data are motivating the development of numerical models for systems biology. However, specification of the almost innumerable dynamic model parameters will require new measurement techniques. The problem is that cellular metabolic reactions and the early steps of intracellular signaling can occur in ms to s, but the 100 to 100k s temporal resolution of measurements on milliliter culture dishes and well plates is often limited by diffusion times set by the experimental chamber volume. Hence the instruments themselves must be of cellular dimension to achieve response times commensurate with key intracellular biochemical events, as is done with microelectrode recording of ion-channel conductance fluctuations and fluorescence detection of protein binding. The engineering challenge is to develop BioMEMS and molecular-scale sensors and actuators to study the breadth of mechanisms involved in intracellular signaling, metabolism, and cell-cell communication.
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Acknowledgements• Mike Ackerman – Nanophysiometer fabrication• Franz Baudenbacher, Ph.D. – Nanophysiometer and dynamic profiling• Darryl Bornhop, Ph.D. – Optical detection of protein binding• Richard Caprioli, Ph.D. – MALDI-TOF and mass spectrometry• Eric Chancellor -- picocalorimetry• David Cliffel, Ph.D. – Cytosensor/electrochemical electrodes• Elizabeth Dworska – Cell culture• Sven Eklund -- Microphysiometry• Shannon Faley – T-cell activation and signaling• Todd Giorgio, Ph.D. – messenger recognition • Igor Ges, Ph.D. – Nanophysiometer fabrication• Frederick Haselton, Ph.D. – cell culture and protein capture• Jacek Hawiger, M.D., Ph.D. – T cell activation/intracellular targeting• Borislav Ivanov – pH sensors• Duco Jansen, Ph.D. – T-cell activation• Amanda Kussrow – Optical determination of protein binding• Eduardo Andrade Lima – Multichannel potentiostats• Jeremy Norris – MALDI-TOF• Phil Samson – Microscopy, microfluidics, and cell lysing• David Piston, Ph.D. – Spectroscopy and fluorescent detection• Sandra Rosenthal, Ph.D. – Q-Dots• David Schaffer – Nanophysiometer fabrication• Ian Thomlinson, Ph.D., – Q-Dots• Roy Thompson, ECBC/Aberdeen – Class A toxin studies• Momchil Velkovsky, Ph.D. – Statistical Analysis• Mike Warnement – Glow in the dark• Andreas Werdich – Cardiac nanophysiometer• DARPA, AFOSR, NIH, Vanderbilt
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Definition
Systems Biology is … quantitative, postgenomic, postproteomic, dynamic, multiscale physiology
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Theme I
The complexity of postreductionist biology
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Step 1 in Science:Reductionism
Thermodynamics Statistical mechanics
Molecular/atomic dynamics
Electrodynamics
Quantum Chromodynamics
Bulk solids
Devices
Continuum models
Microscopic models
Atomic physics
Anatomy
Physiology
Organ
Cell
Protein
Genome
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Spatial Resolution in Physiology
Unaided eye
10-9 10-6 10-3 100
Resolution, Meters
-3000
-2000
-1000
0
1000
2000
3000
His
toric
al T
ime,
Yea
rs
Animal
TissueMagnifying glass
X-Ray / SEM / STMCell
Optical microscope
PhysiologyCell
Molecular Biology
Molecule Systems BiologyComputer
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The Problems• Our understanding of biological phenomena is
often based upon – experiments that measure the ensemble averages of
populations of 106 – 107 cells, or– measurements of a single variable while all other
variables are hopefully held constant, or– recordings of one variable on one cell, or– averages over minutes to hours, or– combinations of some of the above, as with a 10 liter
bioreactor that measures 50 variables after a one-week reactor equilibration to steady state.
• Genomics is providing an exponential growth in biological information
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The rate at which DNA sequences began The rate at which DNA sequences began accumulating was exponentialaccumulating was exponential
0
2,000,000
4,000,000
6,000,000
8,000,000
10,000,000
12,000,000
14,000,000
1965 1970 1975 1980 1985 1990 1995 2001
Human GenomeProject begun
National Library of Medicine
Rapid DNA sequencing invented
~13 million sequence entries in GenBank
Nearly 13 billion bases from ~50,000 species
Year
GB
Courtesy of Mark Boguski
2002: 22,318,883
http://www.ncbi.nlm.nih.gov/Genbank/genbankstats.html
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1.00
10.00
100.00
1,000.00
10,000.00
100,000.00
1,000,000.00
10,000,000.00
100,000,000.00
Transistors/chip
DNA Sequences
Moore’s Law vs.Growth of GenBank
Courtesy of Mark Boguski
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X-Ray and NMRS
Pore conductance
Motility & ECM
Step 2 in Science:Post-Reductionism
Thermodynamics Statistical mechanics
Molecular/atomic dynamics
Electrodynamics
Quantum Chromodynamics
Bulk solids
Devices
Continuum models
Microscopic models
Atomic physics
Behavior
Physiology
Organ
Cell
Protein
Genome
P-P Cross-Section at low Pt Structural Biology
Systems BiologySi Step Edge Diffusion
Systems Biology
Systems Biology
Systems Biology
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Key Questions in Systems Biology
• Given the shockwave of genetic and proteomic data that is hitting us, what are the possible limitations of computer models being developed for systems biology?
• What are promising approaches?– Multiphasic, dynamic cellular instrumentation– Exhaustively realistic versus minimal models– Dynamic network analysis
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‘Postgenomic’ Integrative/Systems Physiology/Biology
• Suppose you wanted to calculate how the cell responds to a toxin…
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The complexity of eukaryotic gene transcription control mechanisms
Courtesy of Tony Weil, MPB, Vanderbilt
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Molecular Interaction Map: Cell Cycle
KW Kohn, “Molecular Interaction Map of the Mammalian Cell Cycle Control and DNA Repair Systems,” Mol. Biol. of the Cell, 10: 2703-2734 (1999)
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Molecular Interaction Map: DNA Repair
KW Kohn, “Molecular Interaction Map of the Mammalian Cell Cycle Control and DNA Repair Systems,” Mol. Biol. of the Cell, 10: 2703-2734 (1999)
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Proteins as Intracellular Signals
A cell expresses between 10,000 to 15,000proteins at any one time for four types of activities:• Metabolic• Maintaining integrity of subcellular structures• Intracellular signaling• Producing signals for other cells
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4300 5940 7580 9220 10860 12500
Inte
nsi
ty
4300 4500 4700 4900 5100 5300m/z
Inte
nsi
ty
Courtesy of Richard Caprioli, Mass Spectrometry Research CenterVanderbilt University
MALDI-TOF: Cells express a lot of proteins…
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G-Protein Coupled Receptors
Courtesy of Heidi HammPharmacology, Vanderbilt
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s04114
The Time Scales of Systems Biology
• 109 s Aging• 108 s Survival with CHF• 107 s Bone healing• 106 s Small wound healing• 105 s Atrial remodeling with AF• 104 s • 103 s Cell proliferation; DNA replication• 102 s Protein synthesis • 101 s Allosteric enzyme control; life with VF• 100 s Heartbeat• 10-1 s Glycolosis• 10-2 s Oxidative phosphorylation in mitochondria• 10-3 s • 10-4 s Intracellular diffusion, enzymatic reactions• 10-5 s • 10-6 s Receptor-ligand, enzyme-substrate reactions• 10-7 s • 10-8 s Ion channel gating• 10-9 s
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“A cell is a well-stirred
bioreactor enclosed by a
lipid envelope”….
Sure….
• ER, yellow; • Membrane-bound
ribosomes, blue;• free ribosomes, orange; • Microtubules, bright green;• dense core vesicles, bright
blue; • Clathrin-negative vesicles,
white;• Clathrin-positive
compartments and vesicles, bright red;
• Clathrin-negative compartments and vesicles, purple;
• Mitochondria, dark green. .
3.1 x 3.2 µm3
Marsh et al., Organellar relationships in the Golgi region of the pancreatic beta cell line, HIT-T15, visualized by high resolution electron tomography. PNAS 98 (5):2399-2406, 2001.
6319movie6.mov
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“A cell is a well-stirred
bioreactor enclosed by a
lipid envelope”….
ODEs become PDEs …
Lots and lots and lots of PDEs
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‘Postgenomic’ Integrative/Systems Physiology/Biology
• Specify concentrations and• Rate constants• Add gene expression,• ProteinN interactions, and• Signaling pathways• Time dependencies• Include intracellular spatial
distributions, diffusion, and transport: ODE → PDE(t)
• … and then you can calculate how the cell behaves in response to a toxin
• Suppose you wanted to calculate how the cell responds to a toxin…
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The Catch• Modeling of a single mammalian cell may require
>100,000 dynamic variables and equations
• Cell-cell interactions are critical to system function
• 109 interacting cells in some organs
• Cell signaling is a highly DYNAMIC, multi-pathway process
• Many of the interactions are non-linear
• The data don’t yet exist to drive the models
• Hence we need to experiment…
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The Grand ChallengeA cell expresses between 10,000 to 15,000 proteins at any one time for four types of activities:• Metabolic• Maintaining integrity of subcellular structures• Intracellular signaling• Producing signals for other cells.
There are no technologies that allow the measurement of a hundred, time dependent, intracellular variables in a single cell (and their correlation with cellular signaling and metabolic dynamics), or between groups of different cells.
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Theme II
Instrumenting the Single Cell
Goal: Develop devices, algorithms, and measurement techniques that will allow us to instrument single cells and small populations of cells and thereby explore the complexities of quantitative, experimental systems biology
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Sizes, Volumes, DiffusionTime Constants
X V, m3 V TauDiff Example N
1 m 1 1000 L 109 s Animal, bioreactor 100
10 cm 10-3 1 L 107 s Organ, bioreactor 100
1 cm 10-6 1 mL 105 s = 1 day Tissue, cell culture 10
1 mm 10-9 1 uL 103 s μenviron, well plate 10
100 um 10-12 1 nL 10 s Cell-cell signaling 5
10 um 10-15 1 pL 0.1 s Cell 10
1 um 10-18 1 fL 1 ms Subspace 2
100 nm 10-21 1 aL 10 us Organelle 2
10 nm 10-24 1 zL 100 ns Protein 1
1 nm 10-27 1 npL 1 ns Ion channel 1
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High-Content Toxicology Screening Using Massively Parallel, Multi-Phasic Cellular Biological Activity Detectors
MP2-CBAD
F Baudenbacher, R Balcarcel, D Cliffel, S Eklund, I Ges, O McGuinness, A Prokop, R
Reiserer, D Schaffer, M Stremler, R Thompson, A Werdich, and JP Wikswo
Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE)
Edgewood Chemical and Biological Center (SBCCOM / ECBC)
DARPADARPADARPADARPA
ctivity etection
echnologies
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MP2-CBAD Discrimination
NBHep
G2
HeLa
Cell T
ypes
Toxin
Sensor Array
Output
pH DO Glc Lac CO2 NADH
Discrimination Matrix
NanoPhysiometer
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Simplified Metabolic Network
OxidativePhosphorylation
Heat
Glycogen
Lactate
CO2
NADHNAD+
NADH
Glycolysis
TCA Cycle
NADPHOxidase
NAD+
Acidification
OxidativePhosphorylation
Glucose
Glycogen
Oxygen
Lactate
CO2
NADHNAD+
NADH
Glycolysis
TCA Cycle
O2
NADPHOxidase
NAD+
Acidification
Reaction Set for Metabolic Network: 5 reactions + 1 transport
Glucose + 2 ADP + 2 NAD+ 2 Pyruvate + 2 ATP + 2 NADH
Pyruvate + NADH Lactate + NAD+
Pyruvate + CoA + FAD
+ GDP + 3 NAD+ + NAD(P)+
3 CO2 + FADH2 + GTP
+ 3 NADH + NAD(P)H
0.5 O2 + 3 ADP + NADH 3 ATP + NAD+
0.5 O2 + 2 ADP + FADH2 2 ATP + FAD
O2 (extracellular) O2
•Robert Balcarcel
•Franz Baudenbacher
•David Cliffel
•Ales Prokop
•Owen McGuinness
•John Wikswo
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The well size determines the bandwidth
• Microliter – 10-100 secondsModified Cytosensor MicroPhysiometer
• SubNanoliter – 10-100 millisecondsVanderbilt NanoPhysiometer
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Multianalyte Microphysiometry
• The Multianalyate MicroPhysiometer (MMP) serves as a platform for studying large numbers of cells simultaneously
• Upon activation, we can measure acidification rate, O2, lactate, glucose with ~1 minute resolution
MicroPhysiometer:
Modified sensor head
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Multianalyte Microphysiometry for Biotoxin Discrimination
Pyruvate
O 2
Oxidative phosphorylation
ADP ATP
NAD+
TCA
OxamateFluoride
Hydrazine
Antimycin
H+
Pyruvate
NADHOxidative
phosphorylation
ADP ATP
NAD+
TCA
Hydrazine
Antimycin
Lactate
CO2
Glucose
DNPO2
H+
H+
CHO cells with 720 s of 20 mM fluoride.
Figure 8.
0.0
0.1
0.2
0.3
0.4
0.5
0 1000 2000 3000
Time (s)
Lactate C
on
cen
tratio
n (
mM
)
Peak height
Peak area
0.21
0.22
0.23
0.24
0 1000 2000 3000
Time (s)
Oxyg
en
Co
ncen
tratio
n (
mM
)
0
50
100
150
200
250
0 1000 2000 3000
Time (s)
Acid
ific
atio
n R
ate (
-u
V/s
)9.0
9.2
9.4
9.6
9.8
10.0
0 1000 2000 3000
Time (s)
Glu
co
se C
on
cen
tratio
n (
mM
)
Oxygen
Lactate
Acidification
Glucose
S.E. Eklund, D.E. Cliffel, et al., – Anal.Chim.Acta 496 (1-2):93-101, 2003; – Anal.Chem. 76 (3):519-527, 2004; – Nanobiotechnology, Humana Press, In Press.
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Automated Data Acquisition and Analysis
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The well size determines the bandwidth
• Microliter – 10-100 secondsModified Cytosensor MicroPhysiometer
• SubNanoliter – 10-100 millisecondsVanderbilt NanoPhysiometer
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Lactate Diffusion Times
Volume, liters
Linear Dimension, micronsD
iffus
ion
Tim
e, s
econ
ds
10-15 10-5 1 105
10-4
10-2
1
102
104
106
108
1010
10-6
10-10
1 104102 106
mL = 3 x 104 sec
L = 300 sec
nL = 3 sec
pL = 30 msec
Smaller = m
uch faster
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PDMS Soft Lithography
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Nanophysiometer for Rapid Activation Dynamics (Baudenbacher)
• The Multianalyte NanoPhysiometer (MNP) will serve as a platform for studying, one at a time, large numbers of single cells
• Upon activation, we will measure pH, O, Vm, [Ca], lactate, glucose, Q-Dot binding
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Microelectrodesto measureextracellular potentialsand stimulate cells
Optical fiber array to measure propagating calcium waves in a single cardiomyocyte
101 102 103 104 105 106 107 108 109 110 111 112-50
-40
-30
-20
-10
0
10
20
pote
ntial (u
V)
time (s)
A
E
C
D
B
101 102 103 104 105 106 107 108 109 110 111 112-50
-40
-30
-20
-10
0
10
20
pote
ntial (u
V)
time (s)
A
E
C
D
B
EF
D
Cardiomyocyte in the NanophysiometerF Baudenbacher and A Werdich
A. Werdich, et al Lab on a Chip 4 (4):357-362, 2004
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Microfabricated pH ElectrodesI. Ges, B. Ivanov, F Baudenbacher
A) pH electrodesB) pH calibrationC) Reference
electrodeD) Calibration deviceE) Temporal
response to a 1 pH step change.
F) and G) Stop-flow acidification for A9L HD2 fibroblasts and M3 WT4 CHO cells
Ges et al., Submitted for publication
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Goal: Instrumented bioreactors with individually addressable traps
First Generation Autoloading NanoPhysiometer
A.Prokop, et al., • Biological and
Bioinspired Materials and Devices, MRS, 2004,
• Biomedical Microdevices 6 (4):In Press, 2004.
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Viability of Activated Jurkat cells in NanoPhysiometer Using CO2-free media
Time in trap = 9.5 hrs
Red = non-viable cells (Yopro-1 fluorescence)
Round, Smooth, No Fluorescence = Viable Cells
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Nanophysiometer ModelingMark Stremler
• Possible device flow and sensing scenarios:
• 3D computational model:
Sensor: • 10 m wide, 100 m long• Zero concentration at surface• Sensor flux proportional to current
Flow Sensing
Continuous Continuous
Intermittent Intermittent
Single Cell: • 10 m diameter• Specified membrane fluxes
Symmetryplane
Channel Walls: • No transport• Zero velocity condition
Inlet Flow: • Specified flowrate, velocity profile• Specified concentrations• Upstream diffusion allowed
25 m
50 m100 m
Outlet
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• Model diffusion and reactions within the polymer matrix of the sensor.
• Enzyme concentration within the sensor assumed uniform. • Production of H2O2 within sensor modeled with Michaelis-
Menten kinetics.• Sensor signal given by gradient of H2O2 at the surface.• Model implemented analytically and with CFD-ACE
Inverse Sensor Model
S+E1 H202+E2
Michaelis-Menten kinetics
Substrate: (∂Cs/ ∂n)=0 on surface, CH2O2=0 in bulk
S
S
Fluid: analyte S carried to sensor by diffusion
H202 H202
Sensor matrix
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The Next Steps
• Inverse sensor model• Inverse metabolic network model• Additional metabolic parameters• Apply experiments, models and analysis to examine the
blocking or enhancing of metabolic pathways
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Theme III
Instrumenting and Controlling The Single Cell
Goal: Develop devices, algorithms, and measurement techniques that will allow us to instrument and control single cells and small populations of cells and thereby explore the complexities of quantitative, experimental systems biology
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How do we study cellular-level responses to stimuli in both normal and patho-
physiologic conditions?
Hypothesis: Great advances in physiology have been made by opening the feedback loop and taking control of the biological system
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Negative versus Positive Feedback
Negative Feedback Positive Feedback
Metcalf, Harold J.; Topics in Classical Physics, 1981, Prentice-Hall, Inc., p.108
Sense Sense
Control Control
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Hypoxia-Red Blood Cell Concentration
Variables– Erythropoietin E– Hypoxia A– RBC
Guyton, Arthur C.; Textbook of Medical Physiology, 6rd ed.; 1981, W.B. Saunders, p.59
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Glucose-Insulin Control
Guyton, Arthur C.; Textbook of Medical Physiology, 6rd ed.; 1981, W.B. Saunders, p.9
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Opening the Feedback Loop
Hypothesis: Great advances in physiology have been made by opening the feedback loop
– Starling cardiac pressure/volume control
– Kao neuromuscular/humeral feedback
– Voltage clamp of the nerve axon
Khoo,Michael C.K.; Physiological Control Systems; 2000, IEEE Press, p.183
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Opening the Feedback Loop
Hypothesis: Great advances in physiology have been made by opening the feedback loop
– Starling cardiac pressure/volume control
– Kao neuromuscular/humeral feedback
– Voltage clamp of the nerve axon Khoo,Michael C.K.; Physiological Control Systems;
2000, IEEE Press, p.184
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Opening the Feedback Loop
Hypothesis: Great advances in physiology have been made by opening the feedback loop
– Starling cardiac pressure/volume control
– Kao neuromuscular/humeral feedback
– Voltage clamp of the nerve axon
Guyton, Arthur C.; Textbook of Medical Physiology, 6rd ed.; 1981, W.B. Saunders, p.110
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Simplified Hodgkin-Huxley
• For the resting cell, ENa, RNa and inward INa depolarize the cell with positive feedback
• EK, RK and outward IK repolarize the cell and serve as negative feedback
• Ignore Cl
Khoo,Michael C.K.; Physiological Control Systems; 2000, IEEE Press, p.187
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Hodgkin-Huxley: Closed-loop with positive and negative feedback
Adapted from Khoo,Michael C.K.; Physiological Control Systems; 2000, IEEE Press, p.259
Sodium Conductance
Potassium Conductance
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Overriding Internal Control: Voltage Clamp
Adapted from Khoo,Michael C.K.; Physiological Control Systems; 2000, IEEE Press, p.259
Current Source
Cla
mp
Cur
rent
Vol
tage
S
ense
Control Voltage
Sodium Conductance
Potassium Conductance
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Potassium Conductance
Sodium Conductance
Opening the Loop During External Control
Adapted from Khoo,Michael C.K.; Physiological Control Systems; 2000, IEEE Press, p.259
Current Source
Cla
mp
Cur
rent V
oltage S
ense
TTX Control Voltage
TEA
Specific ions
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Voltage clamp of the nerve
axon
Guyton, Arthur C.; Textbook of Medical Physiology, 6rd ed.; 1981, W.B. Saunders, p.110
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How do we study cellular-level responses to stimuli in both normal and patho-
physiologic conditions?
Required: New devices to sieze control of subsecond, submicron cellular processes.
Hypothesis: Great advances in physiology have been made by opening the feedback loop and taking control of the biological system
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A Key to the Future of Systems Biology: External Control of Cellular
Feedback
Electrical
• Mechanical
• Chemical
• Cell-to-cell…
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Signatures of Control
• Stability in the presence of variable input (DT= 50o F)
• Oscillations when excessive delay or too much gain
• Divergent behavior when internal range is exceeded or controls damaged Guyton, Arthur C.; Textbook of Medical Physiology,
6rd ed.; 1981, W.B. Saunders, p.9
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Control Stability
• Proportional control
• Proportional control with finite time delay
• Higher gain, same delay
• Same gain, longer delay Metcalf, Harold J.; Topics in Classical Physics,
1981, Prentice-Hall, Inc., p.111, p.113
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Intracellular Metabolic and Chemical Oscillations
• We know that oscillations and bursts exist– Voltage– Calcium– Glucose/insulin– Neurotransmitter– Repair enzymes
http://www.intracellular.com/app05.html
•Prediction: At higher bandwidths than provided by present instrumentation, we will see in biosystems other chemical bursts, oscillations, and chaotic behavior. FIND THEM, USE THEM!
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Ok, we’re convinced about feedback and control….
What do we need to study cellular dynamics?
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What Do We Need to Study Cellular Dynamics?
• Multiple, fast sensors
• Intra- and extracellular actuatorsfor controlledperturbations
• Openers (Mutations,siRNA, drugs) for the internal feedback loops
• System algorithms and models that allow you to close and stabilize the external feedback loop
• …
Actuator
Actuator
Integration and Feedback
Integration and Feedback
Actuator
Integration and Feedback
Sensor
Sensor
Sensor
Cell
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Lck
chain
P
Zap-70
P
P
Intracellular Ca2+ stores
IL-2Rα
MHCCD4/CD8
Adaptor protein
PIP2
DAG
PKCIP3
IL-2
IL-2
Nucleus
IB
RelA RelB
Ionomycin target site
PMA target site
T cell membrane
APC membrane
IL-2
IL-2Rα
GFP-LUCCalcineurin
Inactive NFATP
IBkinase
Active NFB
transcribe
Ras-GTP
ERK
ELK
P
Rac-GTP
P
P
c-Jun
P
JNK
c-Fos
Active NFAT
Ca2+
Ag peptide
Vm
O2pHGlu Lac T
Short-term goal: Measure ~ 10 dynamic variables from a single cell with sub-second response!
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Quantum Dots to Report Protein
Presence
• Quantum dots can be congugated to an antibody that then binds to a membrane protein
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QD Detection of Gene Upregulation
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Activated Jurkat Cells Labeled with IL-2Ab Conjugated QDots
Red: Anti-IL2 QDots
Green: Yopro-1 nucleic acid stain (i.e. non-viable cells)
Activated using PMA & Ionomycin for 72 hrs
QDots label 50-70% of viable activated cells
Unactivated Cells Activated Cells
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Quantum Dot Quenching for Detection of Protein Binding and Enzyme Activity
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Metal Nanoshells as Substrates for Surface-Enhanced Raman Spectroscopy
• 1012 Raman enhancement– optically-addressable intracellular nanothermometer?
• Molecular (vibrational) spectroscopy for protein identification and nanoparticle labeling, (Cullum at U. Maryland, Baltimore)
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We need more cellular nanosensors!!
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What about the cellular nanocontrollers/nanoactuators?
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Lck
chain
P
Zap-70
P
P
Intracellular Ca2+ stores
IL-2Rα
MHCCD4/CD8
Adaptor protein
PIP2
DAG
PKCIP3
IL-2
IL-2
Nucleus
IB
RelA RelB
Ionomycin target site
PMA target site
T cell membrane
APC membrane
IL-2
IL-2Rα
GFP-LUCCalcineurin
Inactive NFATP
IBkinase
Active NFB
transcribe
Ras-GTP
ERK
ELK
P
Rac-GTP
P
P
c-Jun
P
JNK
c-Fos
Active NFAT
Ca2+
Ag peptide
Vm
O2pHGlu Lac T
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What should our cellular controllers look like?
They should be very, very small..
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Targeted Optical Delivery of Heat or Charge
• Metallic NanoShells (Halas at Rice, Cliffel at Vanderbilt, Tomchek at UES, ….)
• Infrared heating by bioconjugate nanoshells– Local control of enzymatic reactions– Selected destruction of tagged organalles
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Magnetic Nanoparticles
• Translational and rotational forces– Viscosity -- Nanorheometry– Molecular motor characterization
• Magnetic separation
• Magnetic identification – Tagged cells– Tagged molecules
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We need more cellular nanoactuators!!
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What is the cellular sensor/actuator competition?
Proteins, proteins, proteins…
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Plasma Membrane
Biochemistry, 2nd ed. Voet, D.; Voet, J.G.; NY, John Wiley & Sons, 1995, p. 292
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Bacterial Photosynthetic Reaction Center
Biochemistry, 2nd ed. Voet, D.; Voet, J.G.; NY, John Wiley & Sons, 1995, p. 296
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Calcium Control of Conductance
Molecular Cell Biology, 2nd ed. Darnell, J.; Lodish, H.; Baltimore, W.H Freeman & Co. 1990, p.525
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Gap Junctions
Biochemistry, 2nd ed. Voet, D.; Voet, J.G.; NY, John Wiley & Sons, 1995, p. 304
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84s04138s02740
The Ultimate NanoMachine: The 1 nm pore in a gated ion channel
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Cells have LOTS of
different ion channels
that serve as sensors and actuators!
Clancy, C. E. and Y. Rudy. Linking a genetic defect to its cellularphenotype in a cardiac arrhythmia. Nature 400 (6744) 566-569, 1999.
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The Ultimate Instrumentation Question for Systems Biology
Can we develop nanodevices that allow sensing and control of cellular functions more effectively than natural or bioengineered proteins, but also provide readout and external control?
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Sizes, Volumes, Time Constants
X V, m3 V TauDiff Example N
1 m 1 1000 L 109 s Animal, bioreactor 100
10 cm 10-3 1 L 107 s Organ, bioreactor 100
1 cm 10-6 1 mL 105 s = 1 day Tissue, cell culture 10
1 mm 10-9 1 uL 103 s μenviron, well plate 10
100 um 10-12 1 nL 10 s Cell-cell signaling 5
10 um 10-15 1 pL 0.1 s Cell 100
1 um 10-18 1 fL 1 ms Subspace 2 - ?
100 nm 10-21 1 aL 10 us Organelle 2 - ?
10 nm 10-24 1 zL 100 ns Protein 1
1 nm 10-27 1 npL 1 ns Ion channel 1
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Then…. Statistical Analysis of Activation Responses
• Correlations of protein expression and dynamical state
• Effective metabolic and signaling model– Metabolic Flux Analysis is primarily steady state– Dynamic measurements require dynamic
network models• Accumulation and depletion of intracellular stores
in short times• Enzyme concentrations fixed in the intermediate
time period– Inverse analysis of exact models is intractable,
so effective models are required
Problem to be solved
Simple Eqs. or rules
Mathematical construct
Decomposition
Numerical method
Executableprogram
Direct mapping of Bi-layered Generic Net
Abstraction
Problem to be solved
Simple Eqs. or rules
Mathematical construct
Decomposition
Numerical method
Executableprogram
Direct mapping of Bi-layered Generic Net
Abstraction
1g3
1g23g41g1
2g3
2b11b1
3b22b2
2b3
4b3
p3 p4p2p1
2b
a2a1a3
2g4
1g
g4
b32b3
1g3
1g23g41g1
2g3
2b11b1
3b22b2
2b3
4b3
p3 p4p2p1p1
2b
a2a1a3
2g4
1g
g4
b32b3
OxidativePhosphorylation
Heat
Glycogen
Lactate
CO2
NADHNAD+
NADH
Glycolysis
TCA Cycle
NADPHOxidase
NAD+
OxidativePhosphorylation
Glucose
Glycogen
Oxygen
Lactate
CO2
NADHNAD+
NADH
Glycolysis
TCA Cycle
O-2
NADPHOxidase
NAD+
Acidification
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The Payoff
• The simultaneous measurement of the dynamics of a hundred intracellular variables will allow an unprecedented advance in our understanding of the response of living cells to pharmaceuticals, cellular or environmental toxins, CBW agents, and the drugs that are used for toxin prophylaxis and treatment.
• The general application of this technology will support the development of new drugs, the screening for unwanted drug side effects, and the assessment of yet-unknown effects of environmental toxins
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– Systems Biology –
The Ultimate Sensor Challenge for the 21st Century