1. Tracking Membrane Receptor Dynamics Using Quantum Dot-labeled Ligands and Quantitative Fluorescence Microscopy Diane Lidke UK-German Frontiers of Science

2. The erbB family of Receptor Tyrosine Kinases Overexpression (and/or mutation)  cancer EGF – erbB1 Neuregulin – erbB3/4 No ligand for erbB2 erbB1 domain structure

3. The erbB signaling network from Yossi Yarden

4. EGFR (erbB1/HER1) + EGF EGF Domain I Domain II Domain III 1ivo H.Ogiso et al., Cell, 110, 775-787 ( 2002) dimerization loops Domain IV

5. 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

6. Quantum Dots Commercial sources: Quantum Dot Inc., Evident Technologies Biomolecule (SA) Polymer Coating Passivation Shell (ZnS) Semiconductor Nanocrystal (CdSe) Broad excitation spectrum Narrow emission band Brightness Photostability Single molecule sensitivity Bioconjugates (Streptavidin, Protein A, IgG...) Non-toxic Donors for FRET

7. Monitor EGF binding and internalization in living cells using a combination of Visible Fluorescent fusion proteins and Quantum Dot-labeled ligand. erbB1 VFP erbB2

8. Live cell activation by EFF-QD function as “single-molecule”  multivalent ligands Binding leads to uptake No non-specific binding Binding leads to activation

9. 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

10. 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.

11. 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

12. A431-erbB1-eGFP A431-erbB3-mCitrine Detection of Hetero-association Does erbB2 or erbB3 internalize with EGF activation of erbB1? High Colocalization Low Colocalization Green Red

13. CHO-erbB1-eGFP A431-erbB1-eGFP A431-erbB2-mYFP A431-erbB3-mCitrine A431-erbB2-mYFP + 2C4 VFP norm /QD norm ErbB2, but not ErbB3, co-internalizes with ErbB1 upon EGF activation Quantification of Hetero-association

14. Retrograde Transport Merge ErbB1-eGFP EGF-QD

15. What is the transport machinery? Coupled to retrograde flow of actin (treadmilling) Active transport by a motor protein (Myosin VI) Hasson J. Cell Science 116: 3453-3461 (2003) Welch et al. Curr. Opin. in Cell Biol. 9: 54 (1997)

16. Retrograde Transport A431 cell expressing erbB1-GFP (green) after addition of EGF-QD (red)

17. 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

18. 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) MSD (pixels 2 = 0.01 µm 2 ) Normal Nocodazole (microtubule disruption) Cytochalasin D (actin disruption) PD153035 (erbB1 kinase inhibitor) MSD = 4D( Δ t) + v 2 ( Δ t) 2

19. Isolated EGF-QD-erbB1 complexes do not transport 10 nm A431 cells expressing erbB1-eGFP Room temperature 5 pM EGF-QD Excess unlabeled EGF added after 300 s active transport diffusion +EGF

20. 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 sensitivity When imaged with a CCD camera.

21. 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)

22. 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