Transcript Tracking Membrane Receptor Dynamics Using Quantum Dot

Tracking Membrane Receptor Dynamics Using
Quantum Dot-labeled Ligands and Quantitative
Fluorescence Microscopy
Diane Lidke
UK-German Frontiers of Science
The erbB family of Receptor Tyrosine Kinases
EGF – erbB1
Neuregulin – erbB3/4
No ligand for erbB2
Overexpression (and/or mutation)  cancer
erbB1 domain
structure
The erbB signaling network
from Yossi Yarden
EGFR (erbB1/HER1) + EGF
Domain II
Domain I
EGF
EGF
Domain III
Domain IV
dimerization
loops
1ivo H.Ogiso et al., Cell, 110, 775-787 ( 2002)
QDs make it possible to monitor protein dynamics in live cells…
EGF-QD
Gur and Yarden
Nature Biotechnology
22:169 (2004)
Biotinylated-EGF + Streptavidin QDs = EGF-QD
Quantum Dots
Biomolecule (SA)
Polymer Coating
Passivation Shell (ZnS)
Semiconductor
Nanocrystal (CdSe)
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Broad excitation spectrum
Narrow emission band
Brightness
Photostability
Single molecule sensitivity
Bioconjugates (Streptavidin,
Protein A, IgG...)
• Non-toxic
• Donors for FRET
Commercial sources: Quantum Dot Inc., Evident Technologies
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-QD
function as “single-molecule”  multivalent ligands
No non-specific
binding
Binding leads to uptake
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-QDerbB1 internalization
Kinetics of EGF-QD Binding and Internalization
Binding at plasma membrane
reaches a steady-state
Internalization continues
linearly 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
Detection of Hetero-association
Does erbB2 or erbB3 internalize with EGF activation of erbB1?
A431-erbB1-eGFP
I nt ensit y Ch3- T2
High Colocalization
250
Red
200
150
100
50
0
0
A431-erbB3-mCitrine
50
100
50
Scatter Region
1
2
3
Number Pixels
5527
392
8120
I nt ens it y
150
I nt ensit y Ch2- T1
Green
0
100
150
Absolute Frequency
Area [ µm x µm ]
112.74
8.00
165.63
200
250
200
Relative Area [ %]
8.4
0.6
12.4
250
Mean Intensity Ch2-T1
45
27
81
Ch3- T2
Low Colocalization
250
Red
200
150
100
50
0
0
0
50
50
100
I nt ens it y
150
Ch2- T1
Green
100
150
Absolute Frequency
200
200
250
250
Mean Intens
13
24
58
Quantification of Hetero-association
0.4
VFPnorm/QDnorm
0.3
0.2
0.1
0.0
ErbB2, but not ErbB3, co-internalizes with ErbB1 upon EGF activation
Retrograde Transport
Merge
ErbB1-eGFP
EGF-QD
What is the transport machinery?
Coupled to retrograde
flow of actin (treadmilling)
Welch et al. Curr. Opin. in
Cell Biol. 9: 54 (1997)
Active transport by
a motor protein
(Myosin VI)
Hasson J. Cell Science
116: 3453-3461 (2003)
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
MSD (pixels2 = 0.01 µm2)
i
250
Normal
Nocodazole (microtubule disruption)
Cytochalasin D (actin disruption)
PD153035 (erbB1 kinase inhibitor)
200
MSD = 4D(Δt) + v2(Δt)2
150
100
50
0
0
D time (s)
50
These plots can be fit to determine diffusion coefficients and velocities...
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Isolated EGF-QD-erbB1 complexes do not
transport
10 nm
active
transport
A431 cells expressing erbB1-eGFP
Room temperature
5 pM EGF-QD
Excess unlabeled EGF added after 300 s
+EGF
diffusion
Minimum requirement for transport is a liganded dimer
EGF-QD525 (green) and EGF-QD605
(red) are added simultaneously to
A431 cells at room temperature.
Single molecule sensitivity
When imaged with a CCD camera.
One green QD and one red
QD are seen to merge and
then transport together.
•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
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Thomas Jovin, Director
Donna Arndt-Jovin, Group Leader
Keith Lidke
Bernd Rieger
Peter Nagy
Janine Post
Rainer Heintzmann