Tracking Membrane Receptor Dynamics Using Quantum Dot-labeled Ligands and

Quantitative Fluorescence Microscopy

Diane LidkeUK-German Frontiers of Science

The erbB family of Receptor Tyrosine Kinases

Overexpression (and/or mutation) cancer

EGF – erbB1Neuregulin – erbB3/4No ligand for erbB2

erbB1 domainstructure

The erbB signaling network

from Yossi Yarden

EGFR (erbB1/HER1) + EGF

EGF EGF

Domain I

Domain II

Domain III

1ivo H.Ogiso et al., Cell, 110, 775-787 ( 2002)

dimerizationloops

Domain IV

Gur and Yarden Nature Biotechnology 22:169 (2004)

EGF-QD

QDs make it possible to monitor protein dynamics in live cells…

Biotinylated-EGF + Streptavidin QDs = EGF-QD

Quantum Dots

Commercial sources: Quantum Dot Inc., Evident Technologies

Biomolecule (SA)

Polymer Coating

Passivation Shell (ZnS)

SemiconductorNanocrystal (CdSe)

• Broad excitation spectrum • Narrow emission band• Brightness• Photostability• Single molecule sensitivity• Bioconjugates (Streptavidin, Protein A, IgG...)• Non-toxic• Donors for FRET

Monitor EGF binding and internalization in living cells using a combination of Visible Fluorescent fusion proteins and Quantum Dot-labeled ligand.

erbB1

VFP

erbB2

Live cell activation by EFF-QDfunction as “single-molecule” multivalent ligands

Binding leads to uptake

No non-specific binding

Binding leads to activation

Internalization of EGF-QD by erbB1-eGFP CHO cells

• Add EGF-QDs during imaging

• Binding of EGF-QDs induces membrane ruffling and EGF-QD-erbB1 internalization

Kinetics of EGF-QD Binding and Internalization

Binding at plasma membrane reaches a steady-state

Internalization continueslinearly with time

Internalization through clathrin coated pits is rate-limiting step.

erbB3-mCitrine A431 cells

• The EGF-QD binds to the endogenous erbB1 and is internalized

• The erbB1-EGF-QD moves down the filopodia

• The erbB3 remains on the cell surface – it is not internalized with the erbB1

A431-erbB1-eGFP

A431-erbB3-mCitrine

0 50 100 150 200 250Intensity Ch2-T1

0

50

100

150

200

250

Intensity Ch3-T2

Absolute Frequency0 50 100 150 200 250

Scatter R egion123

N um ber P ixels55273928120

Area [ µm x µm ]112.748.00165.63

R elative Area [ % ]8.40.612.4

M ean Intensity C h2-T1452781

M ean Intensity C h3-T2132458

Standard D eviation C h2-T113438

Standard D eviation C h3-T23438

C olocalization C oeffic ient C h2-T1

0.595

C olocalization C oeffic ient C h3-T2

0.954

W eighted C oloc. C oeffic ient C h2-T1

0.865

W eighted Coloc. C oeffic ient C h3-T2

0.984

O verlap C oeffic ient

1.0

C orrelation R

0.85

C orre lation R x R

0.72

0 50 100 150 200 250Intensity Ch2-T1

0

50

100

150

200

250

Intensity Ch3-T2

Absolute Frequency0 50 100 150 200 250

Detection of Hetero-associationDoes erbB2 or erbB3 internalize with EGF activation of

erbB1?

High Colocalization

Low Colocalization

Green

Green

Red

Red

CH

O-e

rbB

1-eG

FPA

431-

erbB

1-eG

FPA

431-

erbB

2-m

YFP

A43

1-er

bB3-

mC

itrin

eA

431-

erbB

2-m

YFP

+ 2C

4

VF

Pn

orm

/QD

no

rm

ErbB2, but not ErbB3, co-internalizes with ErbB1 upon EGF activation

Quantification of Hetero-association

Retrograde Transport

Merge ErbB1-eGFP EGF-QD

What is the transport machinery?

Coupled to retrogradeflow of actin (treadmilling)

Active transport bya motor protein(Myosin VI)

Hasson J. Cell Science 116: 3453-3461 (2003)

Welch et al. Curr. Opin. in Cell Biol. 9: 54 (1997)

Retrograde Transport

A431 cell expressing erbB1-GFP (green) after addition of EGF-QD (red)

Tracking Retrograde Transport

Track loci over time using the “5D Viewer” (Image J plug-in developed by Dr. Rainer Heintzmann) or Matlab/DIPimage routine, which calculates the center of intensity in a region around the maximum in each time step

Typical MSD plots of QD-EGF-ErbB1 retrograde transport on A431 cells under different conditions

These plots can be fit to determine diffusion coefficients and velocities...

time (s)

MS

D (

pixe

ls2

= 0

.01

µm

2 )

Normal

Nocodazole (microtubule disruption)

Cytochalasin D (actin disruption)

PD153035 (erbB1 kinase inhibitor)

MSD = 4D(Δt) + v2(Δt)2

Isolated EGF-QD-erbB1 complexes do not transport

10 nm

A431 cells expressing erbB1-eGFPRoom temperature5 pM EGF-QDExcess unlabeled EGF added after 300 s

activetransport

diffusion

+EGF

Minimum requirement for transport is a liganded dimer

EGF-QD525 (green) and EGF-QD605 (red) are added simultaneously to A431 cells at room temperature.

One green QD and one red QD are seen to merge and then transport together.

Single molecule sensitivityWhen imaged with a CCD camera.

•EGF-QDs bind

•ErbB1 undergoes conformational change

•Stable homodimers form

•Activation (P) leads to binding of adapter protein (blue box)

•Active receptors are transported to the cell body

•Internalization occurs at the base of filopodia

Filopodia serve as sensory organelles for the cell by probing for the presence and concentration of growth factors far from the cell body, coupling remote sensing to cellular response via directed transport of activated receptors.

Lidke et al., JCB 170:619 (2005)

Department of Molecular Biology Max Planck Institute for Biophysical Chemistry

Göttingen, Germany

• Thomas Jovin, Director• Donna Arndt-Jovin, Group Leader• Keith Lidke• Bernd Rieger• Peter Nagy• Janine Post• Rainer Heintzmann