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

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)

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

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

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

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.

Di-4-ANEPPDHQ Reveals Lipid Polarity in Polarized Migrating Neutrophils

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

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

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

Fertilization of Di-8-ANEPPS Stained Sea Urchin Egg Andrew Millard and Mark Terasaki

SHG TPEF

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

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)

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)