FIG 3.

Characterization of erbB3 diffusion by single particle tracking (SPT). (A) Configuration of QD probes bound to hypothetical dimer structures: an erbB3 homodimer tracked with two QD-HRG probes (top) and an erbB3/erbB2 heterodimer tracked with a QD655-HRG probe and a QD585 anti-HA Fab probe (bottom). Structural information from erbB1 dimers, erbB2, Fab fragments, and reported QD diameters (55) was used to estimate the distance between QDs in a dimer pair. Homology modeling indicates that erbB3 homodimer distances would be comparable to those of erbB1 (43). (B) For the top panel, SKBR3 cells were stimulated for 2 min with 12 nM unlabeled HRG, biotinylated HRG (bHRG), or biotinylated HRG conjugated to QDs (QD-HRG). erbB3 phosphorylation was assessed by immunoblotting with the phospho-specific PY1289 erbB3 antibody and normalizing to the total erbB3. The lower panel shows that binding of the QD-conjugated anti-HA Fab to HA-erbB3 does not interfere with erbB3 phosphorylation. To ensure that steric hindrance of the QDs do not disrupt erbB3 activation, serum-starved CHO-HA-erbB3 cells were incubated for 30 min with or without QD anti-HA Fab and then stimulated with 12 nM HRG for 2 min. (C) Diffusion coefficients of ligand-bound erbB3 tracked on the surface of SKBR3 cells with QD-HRG with or without 30 min pretreatment with 500 nM lapatinib. In SKBR3 cells transiently transfected with HA-erbB3, unliganded erbB3 was tracked with QD anti-HA and liganded erbB3 was tracked with QD anti-HA plus 100 nM unlabeled HRG or QD-HRG. (D) Diffusion coefficients of unliganded or liganded HA-erbB3 (wild type or kinase dead [K723M]) tracked on the surface of CHO cells. The diffusion coefficients shown in panels C and D were calculated by fitting the ensemble mean square displacement (MSD) of each experiment, and the average of at least three experiments was plotted (±the standard error of the mean). P values are based on statistical analysis using the Student t test.