core 1: technology core 1 project 1 – measure a. network and pathway data integration. b. virtual...
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CORE 1: TECHNOLOGYCore 1 Project 1 – MEASURE
a. Network and Pathway Data Integration. b. Virtual Experiment. c. Optical Probe Development. d. Fluorescence Correlation Spectroscopy.
Core 1 Project 2 - MODELa. Modularity and Multistate Complexes. b. Molecular Flux in Crowded Spaces. c. Stochastic Modeling and Discrete Particles. d. Moving Boundaries.
Core 1 Project 3 – MANIPULATEa. Signaling Platforms.b. In Vivo Nanofabrication.c. Holographic Optical Tweezers.
CORE 2: DRIVING BIOLOGICAL PROJECTSDBP 1 “Study of Neutrophil Polarity” Dianqing WuDBP 2 “Cell Polarity and Cell Fusion” William MohlerDBP 3 “Polarity in A2 RNA Trafficking Pathway in Neurons John H. CarsonDBP 4 “Regulation of Cell Locomotion by Microtubules” Vladimir I. RodionovDBP 5 “Spatial Asymmetry, Nuclear Transport, and Signaling” Ian G. MacaraDBP 6 “Simulation of Cytoskeletal Structure and Mobility” Thomas D. Pollard
CORE 3: FACILITIESCORE 4: TRAININGCORE 5: DISSEMINATIONCORE 6: ADMINISTRATION
Measure
Model Manipulate
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Quantitative Cell BiologyQuantitative Cell Biology
Predictions
Dynamics of Cellular Structures and
Molecules
Simulation
Hypothesis (Model)
• What are the initial concentrations, diffusion coefficients and locations of all the implicated molecules?
• What are the rate laws and rate constants for all the biochemical transformations?
• What are the membrane fluxes and how are they regulated?
• How are the forces controlling cytoskeletal mechanics regulated?
ExperimentExperiment
Trends in Cell Biology 13:570-576 (2003)
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Today’s Talks
• Bill Mohler – Contrast Mechanisms in Non-linear Optics (SHG of muscle)
• Paul Campagnola - New Photoactivators for Nano/Microfabrication
• Les Loew – Novel Environmentally-Sensitive Chromophores for Fluorescence and SHG Imaging
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MeasureMeasure - - Fluorescent ProbesLes Loew
N
N+
N
O
O
ONH Br
Membrane Domains
Membrane Association and Fusion
Environmentally-Sensitive Labels
TPEF SHG Overlay
500 550 600 650 7000
2x106
4x106
>550 nm Emission
Q = 0.04
Q = 0.08
4039
= 1.47ns (2500 M) = 1.60ns (7.7 m) = 1.60ns (1.5 M) = 1.65ns (0.25 m) = 1.75ns (0 M)
0
5x106
1x107
>590 nm Emission
Q = 0.03
4042
= 1.83ns (2500 M) = 1.80ns (7.7 M)
nsns = 2.09ns (0 M)
Fluorescence
0
2x106
4x106
>590 nm Emission
Q = 0.12
Q = 0.03
Q = 0.15
= 1.96ns (24.0 M) = 1.93ns (2500 M)
= 2.05ns (7.6 M) = 2.13ns (1.6 M)ns = 2.23ns (0 M) 4045
500 550 600 650 700
0
2x106
4x106
>550 nm Emission
Q = 0.04
Q = 0.08
4039
= 1.47ns (2500 M) = 1.60ns (7.7 m) = 1.60ns (1.5 M) = 1.65ns (0.25 m) = 1.75ns (0 M)
0
5x106
1x107
>590 nm Emission
Q = 0.03
4042
= 1.83ns (2500 M) = 1.80ns (7.7 M)
nsns = 2.09ns (0 M)
Flu
orescence
0
2x106
4x106
>590 nm Emission
Q = 0.12
Q = 0.03
Q = 0.15
= 1.96ns (24.0 M) = 1.93ns (2500 M)
= 2.05ns (7.6 M) = 2.13ns (1.6 M)
ns = 2.23ns (0 M) 4045
N+
N
NH
O N
O
O
Br
JPW-4045
Flu
ores
cenc
e
Environmentally-sensitive fluorescent labels
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Di-4-ANEPPDHQ Emission Spectrum in LUVs
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
500 540 580 620 660 700 740
norm
aliz
ed in
tens
ity
DOPC, peak 630 DPPC7:chol3, peak 582
N+
N
OHN+
OH
Br
Br
Characterization and Application of a New Optical Probe for Membrane Lipid Domains
Lei Jin et al., Biophys. J., 2006
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mergeSHG TPF
540/50
675/50
SHG and 2PF images of GUVs stained by di-4-ANEPPDHQ.The dye shows stronger SHG in the liquid disordered phase,
corresponding to the red-shifted 2PF.
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Di-4-ANEPPDHQ Reveals Lipid Polarity in Polarized Migrating Neutrophils
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Table 1. Dye emission maxima in DDPC/Cholesterol vs. DOPC vesicles
maxEmλ (nm)
Name Structure Cholesterol/DPPC
(30:70) DOPC
JPW-6008 N
N
2BrN
629 668
JPW-4090 N
N
2Br
N
667 708
JPW-6003 N
N
2Br
N
665 712
JPW-6023 N
N
2Br
N
630 676
PY-2045 N
N
2BrN
596 639
PY-1261 S
N
N
2Br S N
654 690
PY-1266 S
N
N
2Br S N
662 696
PY-1237 S N
N
N
2Br
HO
OH
606 594
PY-1284 S N
N
N
2Br
614 596
PY-2030 O
N
N
2Br O N
NA 698
Newly synthesized dyes andtheir emission maxima in liquid ordered and liquid disordered lipid membranes
Ping Yan and Joe Wuskell
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py-1266 (Emission)
0.E+00
1.E+06
2.E+06
3.E+06
4.E+06
550 600 650 700 750 800
wavelength (nm)
EM
(c
ps) DPPC:Cholesterol
DOPC
Br
Br
SN
+
N+
SN
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PY-1237 (Emission)
0.E+00
5.E+05
1.E+06
2.E+06
2.E+06
520 560 600 640 680 720
wavelength (nm)
EM
(c
ps)
DPPC:Cholesterol
DOPC
Br
Br
SN
+
N+
OHOH
N
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Fertilization of Di-8-ANEPPS Stained Sea Urchin Egg Andrew Millard and Mark Terasaki
SHG TPEF
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Unique Contrast Patterns from Resonance Enhanced Chiral SHG of Cell Membranes
Ping Yan et al., J. Am. Chem. Soc., 2006
Monomer Racemic Chiral Monomer Racemic Chiral
SHG 2PF 2PF2PFSHGSHG
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O
OHO
OHOH
O
OH O
OOH
O
O
OO2N
O
OHO
OHOH
O
OHO
OHOH
O
Dextran, MW 8,500-11,500
O O
I
NaO
I I
I
Cl
Cl
Cl
ClO
O
NH3
+
Br
O O
I
NaO
I I
I
Cl
Cl
Cl
Cl COONa
Rose Bengal, disodium salt
ClO
OO2N
Br NH3
+ Br
DMSO/Pyridine,DMAP, 0 oC, 9 h
H2O, Acetone, 100 oC, 2 h
O
OHO
OHOH
O
OH O
OOH
O
NH
O
O
O
I
NaO
I I
I
Cl
Cl
Cl
ClO
O
TEA, DMSO
RT, 17 h
Functionalization of 10KD Dextran forCovalent Linkage to Rose Bengal
(Ping Yan)
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Short Term Goals
• Develop covalent labeling reagents from the most environmentally sensitive of our new chromophores (Ping Yan, Joe Wuskell)
• Support Nanofabrication with improved intracellular photosensitizers (Ping Yan)
• Determine the mechanism of 2PF increase and SHG decrease upon fusion of exocytic vesicles (Aifang Xie)