Engineering Interactive BiologicalMaterial Interfaces with Nanotechnology

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Cell Membrane Physical Chemistry
Phys. Rev. Lett. 96 118101 (2006) Phys. Rev.
Lett. 95 048101 (2005) J. Am. Chem. Soc. 127 36
(2005) PNAS 101 12798 (2004)
Science 310 1191 (2005) Angew. Chem. Int. Ed.
44 3524 (2005) Sci. STKE 2005, pe45
(2005) Nature Chem. Biol. 1 283 (2005)
Nature 427 139 (2004) Adv. Mater. 17 1477
(2005) Anal. Chem. 77 6985 (2006)
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T Cell Recognition

• Front line of adaptive immune system
• Detect foreign (e.g. viral) antigen
• peptides displayed on antigen presenting cell
  (APC) surface by major histocompatibility complex
  (MHC)
• T cell receptor (TCR)
• Diverse combinatorial array
• Individual proteins well understood
• How it works remains unclear

Quade Paul EchoMedicalMedia.com
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Synapses of the Immune System

• Central supramolecular activation cluster
  (c-SMAC) containing MHC, Peripheral ICAM
• Signal triggered cytotoxicity

Dustin et al. Science, 285 221 (1999)

• Interspersed MHC and ICAM
• Signal triggered thymocyte apoptosis

Davis et al. Immunity, 16. 839 (2002)

• Central ICAM, peripheral MHC
• Signal triggered inhibition of cytotoxicity

Strominger et al. PNAS, 96 15062 (1999)
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TCR Signaling Reaction Network
How does spatial patterning impact signal
transduction?
Friedl et al., Nat. Rev. Immunol. 5 532 (2005)
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Hybrid Live Cell - Supported Membrane Synapse

• Supported membrane replaces APC in synapse with
  live cell
• Proteins in supported membrane must be mobile
• GPI-linked cell surface receptors functional in
  this configuration

Mature T cell
Thymocyte
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Groves, Angew. Chem. Int. Ed. 44 3524 (2005)
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Repatterning the T Cell Immune Synapse
Experimental configuration living T cell on
supported membrane displaying active proteins.
Phase Contrast
Spatial mutations of the signaling apparatus.
Mossman et al. Science 310 p. 1191 (2005)
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Tracking Synapse Assembly

• Total internal reflection fluorescence (TIRF)
• Single molecule resolution
• Flow tracking algorithm
• Automated statistical analysis of protein movement

DeMond et al. in preparation
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Tracking Synapse Assembly
DeMond et al. in preparation
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Probing Membrane Structures

• Extended clusters of proteins and/or lipids
  needed for signaling
• Sizes likely to be below optical resolution

?
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Fluorescence Correlation Spectroscopy

• Whole T cell membranes probed in hybrid
  live-cell format

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Mapping Intracellular Signaling
Friedl et al., Nat. Rev. Immunol. 5 532 (2005)
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Phosphotyrosine Signaling
Friedl et al., Nat. Rev. Immunol. 5 532 (2005)
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Phosphotyrosine Signaling
PY (purple)
TCR (green)

• Fixed at 2 min
• Strong PY co-localized with alll TCR clusters

Unrestricted
2 µm grid
Mossman et al. Science 310 p. 1191 (2005)
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Phosphotyrosine Signaling
PY (purple)
TCR (green)

• Fixed at 5 min
• No TCR signaling from c-SMAC
• Sustained TCR signaling in periphery

Unrestricted
2 µm grid
Mossman et al. Science 310 p. 1191 (2005)
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Calcium Signaling
Friedl et al., Nat. Rev. Immunol. 5 532 (2005)
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Calcium Signaling Waves
Winslow Crabtree, Science 307 56 (2005)
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Calcium Signaling
Mossman et al. Science 310 p. 1191 (2005)
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Summary Synaptic Pattern Formation
Biological Conclusions

• Distinctive patterns correlate with different
  types of signaling activity.
• Can causality be established?
• Cytoskeleton is a key regulator of pattern
• Mechanism of cytoskeletal signal regulation
  spatial translocation
• Patterned supported membrane substrates enable
  molecular-scale dissection of live signaling
  processes
• Spatial mutations of chemical signaling apparatus

Technological Conclusions
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Membrane Forces
What are the physical mechanisms of spatial
pattern formation? Membrane curvature and
bending generate important driving forces.
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Topography of Membrane Rafts
Phase separated membrane domains create
topography

• Fluorescence

• DOPCCholSM
• Coexisting fluid phases

• Reflection Interference Contrast Microscopy

Kaizuka Groves in preparation
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Bending-Mediated Superstructure
Fluorescence
RICM
Time
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Substrate-Imposed Curvature
AFM

• Fused quartz wafer
• Anisotropic oxide etch (CF4 and CHF3)
• Isotropic wet etch (HF)

• Upper membrane exhibits normal miscibility phase
  transitions

Parthasarathy, Yu, Groves Langmuir 22 5095,
2006
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Curvature-Mediated Domain Organization

• Liquid ordered domain positions governed by
  substrate curvature.
• Strong preference for low curvature region.
• Substrate exhibits mild curvature variation.

Parthasarathy, Yu, Groves Langmuir 22 5095,
2006
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Curvature-Mediated Domain Organization
Cell
GUV

• Threshold curvature differential for ordering
  between 0.04 and 0.4 µm-1
• Combination of kinetic and thermodynamic
  influences

Parthasarathy, Yu, Groves Langmuir 22 5095,
2006
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Synapse repatterning by curvature
TCR
Brightfield
ICAM

• Substrate curvature does NOT influence
  diffusion
• Synapse repatterning is driven by a different
  process

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Summary Membrane Forces

• Mechanical bending leads to long-range forces
• Protein sorting at membrane interfaces
• Repulsive forces between membrane rafts
• Bending mediated phase organization

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Outlook
Dynamical manipulation of living chemical networks

• In vivo photolithography
• Electrically addressable substrate
• Stochastic noise in T cell signaling

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The Group
Graduate Students Michael Baksh Kaspar
Mossman Nathan Clack Cheng-Han Yu Bill
Galush Bryan Jackson Jeff Nye Esther Winter Andy
Demond Amber Wise Joe Hickey Boryana
Rossenova Pradeep Nair Nina Caculitan Dr. Yoshi
Kaizuka
Postdocs Dr. Raghu Parthasarathy Dr. Sharon
Rozovsky Dr. Martin Forstner Collaborators Prof.
Carolyn Bertozzi Prof. Matt Francis Prof. John
Kuriyan Prof. Ehud Isacoff Prof. Randy
Sheckman Prof. Joe Gray (LBNL) Prof. Mike Dustin
(NYU) Prof. Arup Chakraborty (MIT) Prof. Atul
Parikh (UCD) Prof. Mark Davis (Stanford)
Financial Support Burroughs Wellcome Career Award
in the Biomedical Sciences Beckman Young
Investigator Searle Scholars Award Hellman
Faculty Award NIH NSF CAREER DOE NSET DOE
Biomolecular Materials NuvoMetrix Inc. Center
Participation CBST NSF (UC Davis) CPIMA NSF
(Stanford)
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Thermal Fluctuation Imaging
Fluorescence Interference Contrast
Microscopy (FLIC)
Kaizuka Groves Phys. Rev. Lett., 96 118101,
2006
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Spatial Correlation Functions
Membrane Bending Energy
Kaizuka Groves Phys. Rev. Lett., 96 118101,
2006
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Temporal Correlation Functions
Hydrodynamic Drag
Kaizuka Groves Phys. Rev. Lett., 96 118101,
2006
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Photoreleasable Agonist Peptide
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Synapse Inhibition
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Uncaging Induces Synapse Formation
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Schematic