ARTICLES 198 VOLUME 22 NUMBER 2 FEBRUARY 2004 NATURE BIOTECHNOLOGY The erbB family of RTKs includes erbB1 (the classical EGF receptor, EGFR), erbB2, erbB3 and erbB4 1 . Activation of these transmembrane proteins initiates signaling cascades controlling numerous cellular processes such as DNA replication and division 1,2 . Ligand binding leads to auto- and transactivation of cytoplasmic protein kinase domains in response to the formation 3 or reorientation 4–6 of homo- and heteroassociated RTK monomers. The fate of the activated recep- tors is complex: endocytosis via coated pits, covalent modification (deactivation by enzymatic dephosphorylation and ubiquitination), and endosomal trafficking leading to proteosomal and/or lysosomal degradation or recycling to the plasma membrane. The overexpression and/or unrestrained activation of the erbB family, particularly erbB2, are implicated in many types of cancer 1,7 . We show here that QDs bearing natural ligands as effector molecules provide the means for prolonged real-time visualizations of the multiple steps in signaling mechanisms in living cells, resulting in detailed movies of the processes involved. This capability has important implications for biotechnological screening and assay strategies. For the studies reported here, biotinylated EGF was bound to com- mercial QDs with bioconjugated streptavidin 8 . QDs provide unique advantages for cellular imaging: (i) a very high and uniform brilliance, permitting detection down to the single nanoparticle level and reliable quantification of binding and transport phenomena, (ii) photostabil- ity, allowing imaging over prolonged periods, (iii) a broad excitation spectrum, allowing the simultaneous excitation of enhanced green flu- orescent protein (eGFP) and QDs, (iv) a very narrow emission band conveniently red-shifted to the fluorescence of the VFPs and (v) the high biochemical stability and specificity, low background and adjustable stoichiometry (ligands/QD) afforded by the biotin-strepta- vidin system. This study adds to the list: (vi) ligand compliance, the noninterference of the QD with the biochemical specificity of an attached ligand and (vi) steric compatibility, for example, the ability of two QD-ligands to bind to the two monomers of a dimeric RTK. The formation of homo- and heterodimers and higher order oligomers of the erbB proteins is regarded as an essential feature of the signaling mechanism 3,9,10 , although characterization of these protein- protein interactions on and in living cells has proven difficult. The cen- tral dogma featuring a ligand-induced dimerization as an obligatory intervening step 3 has been reinforced by recent crystallographic struc- tures of isolated 11–14 and growth factor-liganded 15 erbB ectodomains, revealing homodimers stabilized by interdomain and multiple ligand- ligand contacts (erbB1). Crystallographic evidence for heterodimers is as yet unavailable. However, numerous studies of intact RTKs in their cellular environment 4,5,16–18 provide evidence for the existence of pre- formed, that is, ligand-free, homo- and heterodimers. Thus, in addi- tion to the dramatic ligand-induced conformational transitions, it is possible that the activation mechanism may also involve monomer- monomer rearrangements—for example, via transverse rotational motions 4,5 —in preexisting dimeric or heterotetrameric 6,19 complexes. The latter could be stabilized via interactions between the transmem- brane receptor domains 20 . These considerations and reports of the preferential formation of heterodimers 21 with a reduced internaliza- 1 Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany. 2 Laboratorio de Electrónica Cuántica, Departamento de Física, Facultad de Ciencias Exactas y Naturales, Intendente Güiraldes 2160, Pabellón I, Ciudad Universitaria, Universidad de Buenos Aires, C1428EHA Buenos Aires, Argentina. 3 Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón II 3er piso, Ciudad Universitaria, C1428EHA Buenos Aires, Argentina. Correspondence should be addressed to D.S.L. (dlidke@gwdg.de) or T.M.J. (tjovin@gwdg.de). Published online 4 January 2004; doi:10.1038/nbt929 Quantum dot ligands provide new insights into erbB/HER receptor–mediated signal transduction Diane S Lidke 1 , Peter Nagy 1 , Rainer Heintzmann 1 , Donna J Arndt-Jovin 1 , Janine N Post 1 , Hernan E Grecco 2 , Elizabeth A Jares-Erijman 3 & Thomas M Jovin 1 The erbB/HER family of transmembrane receptor tyrosine kinases (RTKs) mediate cellular responses to epidermal growth factor (EGF) and related ligands. We have imaged the early stages of RTK-dependent signaling in living cells using: (i) stable expression of erbB1/2/3 fused with visible fluorescent proteins (VFPs), (ii) fluorescent quantum dots (QDs) bearing epidermal growth factor (EGF-QD) and (iii) continuous confocal laser scanning microscopy and flow cytometry. Here we demonstrate that EGF-QDs are highly specific and potent in the binding and activation of the EGF receptor (erbB1), being rapidly internalized into endosomes that exhibit active trafficking and extensive fusion. EGF-QDs bound to erbB1 expressed on filopodia revealed a previously unreported mechanism of retrograde transport to the cell body. When erbB2-monomeric yellow fluorescent protein (mYFP) or erbB3-monomeric Citrine (mCitrine) were coexpressed with erbB1, the rates and extent of endocytosis of EGF-QD and the RTK-VFP demonstrated that erbB2 but not erbB3 heterodimerizes with erbB1 after EGF stimulation, thereby modulating EGF-induced signaling. QD-ligands will find widespread use in basic research and biotechnological developments. © 2004 Nature Publishing Group http://www.nature.com/naturebiotechnology