From Mellman's group in Genentech. Nicely done, TRIF and quantum dot conjugated antibody!
Preparation of Fab-QDs and tagging single EGFRs with the conjugate
AntiEGFR–Fab-biotin was produced by pepsin digestion of an anti-EGFR IgG (AB-11; clone 199.12, LabVision) followed by reduction of the resulting F(ab′)2 to yield a Fab′ fragment. The Fab′ was then conjugated with biotin through the unique -SH at its carboxy-terminal end. To prepare antiEGFR–Fab-QD conjugates, a solution of 0.7 nM antiEGFR–Fab-biotin in 1 ml of DMEM/F12 was added dropwise to 1 ml of 2 nM QD605-streptavidin conjugate (Invitrogen). Cell monolayers (50% confluent) previously serum starved for >1 h were incubated with 500 μl of the final antiEGFR–Fab-QD solution at 25 °C for ~2 min. The cells were then washed twice with PBS at 25 °C, placed in serum-free medium, and loaded onto the microscope stage at 37 °C in 5% CO2 for imaging.
Imaging system and analysis
Total internal reflection fluorescence microscope (TIRFM) imaging was performed using a Nikon Eclipse TE2000 inverted microscope with 100×/1.49NA Plan Apo objectives (Nikon). The sample was illuminated by the 488 nm line of a solid-state laser (Andor Technology) and images were acquired by the iXon back-illuminated EMCCD camera (Andor Technology). Individual trajectories of antiEGFR–Fab-QD were constructed by manual tracking plug-in in Image-J in parallel with the tracking module in Imaris (Bitplane). 10Hz, 100 ms per frame, not that fast.
Couple interesting points in the paper:
- "We tagged a small fraction of individual EGFR molecules with a conjugate of one CdSe quantum dot linked to one anti-EGFR antibody Fab fragment (antiEGFR–Fab) (Fig. 1a)."
- "Single antiEGFR–Fab-quantum-dot (QD)-bound EGFRs (EGFR–Fab-QD) were visualized by total internal reflection fluorescence microscopy illuminated for <10 min to minimize the appearance of prolonged quantum dot dark states"
- "Assuming monomer–dimer equilibrium, the Kd values for DelC and DelD were 2.9- and 14.2-fold greater than wild type (,10% errors; Supplementary Information 3). Thus, the structural stability of spontaneously formed dimers was also derived largely from the dimerization arms."
- "Thus, although ligand was necessary for receptor activation 5,6, it did not stabilize the dimer state27, contrary to the conventional view."
- "The frequency of spontaneous dimerization increased with receptor expression and was also location-dependent in the membrane of EGFR-overexpressing cells (A431 and BT20 cells; Fig. 3a)."
- "The peripheral enrichment of both dimerization and ligand binding suggested that EGF bound preferentially to dimers 12,14."
- "For more than two decades, ligand binding and receptor dimerization were known to be critical steps in the EGFR activation process, but the exact relationship between these events was uncertain30. By analysing single receptors on living cells, we demonstrated that un-liganded EGFR fluctuates continuously between monomer and dimer states. Pre-formed dimers are primed for ligand binding and signaling, and in certain cell lines the distribution of dimers, and thus receptor responsiveness, are asymmetric. This asymmetry is related to both receptor expression level and the organization of the actin cytoskeleton 28,29."
Nature. 2010 Mar 7. [Epub ahead of print]
Spatial control of EGF receptor activation by reversible dimerization on living cells.
Chung I, Akita R, Vandlen R, Toomre D, Schlessinger J, Mellman I.
Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.
Epidermal growth factor receptor (EGFR) is a type I receptor tyrosine kinase, the deregulation of which has been implicated in a variety of human carcinomas. EGFR signalling is preceded by receptor dimerization, typically thought to result from a ligand-induced conformational change in the ectodomain that exposes a loop (dimerization arm) required for receptor association. Ligand binding may also trigger allosteric changes in the cytoplasmic domain of the receptor that is crucial for signalling. Despite these insights, ensemble-averaging approaches have not determined the precise mechanism of receptor activation in situ. Using quantum-dot-based optical tracking of single molecules combined with a novel time-dependent diffusivity analysis, here we present the dimerization dynamics of individual EGFRs on living cells. Before ligand addition, EGFRs spontaneously formed finite-lifetime dimers kinetically stabilized by their dimerization arms. The dimers were primed both for ligand binding and for signalling, such that after EGF addition they rapidly showed a very slow diffusivity state that correlated with activation. Although the kinetic stability of unliganded dimers was in principle sufficient for EGF-independent activation, ligand binding was still required for signalling. Interestingly, dimers were enriched in the cell periphery in an actin- and receptor-expression-dependent fashion, resulting in a peripheral enhancement of EGF-induced signalling that may enable polarized responses to growth factors.
F1000 review http://f1000biology.com/article/id/2502956/evaluation
Kermit Carraway
University of Miami School of Medicine, United States of America
Cell Biology
The authors report the interesting finding that epidermal growth factor receptors (EGFRs) are spatially regulated in tumor cells with or without binding and activation by ligand.
Using quantum dot tracking of single EGFRs on living tumor cells, the authors have validated the emerging finding that the ErbB receptors undergo reversible dimerization in the absence of ligand. These dimers are stabilized via their dimerization arms and are primed for ligand binding, which is required for kinase activation and downstream signaling. Importantly, the dimers are localized asymmetrically at the periphery of these tumor cells, which may be important to their contributions to cell motility and tumor metastasis.