Fig 2: Disruption of actin filaments abolishes the difference between the diffusion coefficients of ErbB2 and ErbB3. CHO-ErbB2-ErbB3 cells were treated with 1 µM latrunculin B for 10 min followed by labeling these treated and control cells with fluorescent anti-ErbB2 and anti-ErbB3 Fab at an equimolar ratio (10–10 µg/mL). The ratio of the diffusion coefficients of ErbB2 and ErbB3 are plotted along with their standard error of the means.

Fig 3: Model for the homo- and heteroclustering of ErbB3. The model encompasses all the evidence from tracking and co-localization measurements. ErbB3 is assumed to have a closed conformation in which the ligand binding site is blocked, and the dimerization arm is not exposed. The closed conformation is in equilibrium with the extended conformation, in which the ligand binding site is open, and the dimerization arm is exposed. Both liganded and unliganded ErbB3 in the extended conformation are assumed to undergo homodimerization, leading to the formation of bone fide homodimers. Besides these structures, looser ErbB3 homodimers may also form. ErbB2 competes with the formation of these bona fide ErbB3 homodimers, but it also facilitates the generation of ErbB3 homodimers (protein symbols surrounded by a dashed ellipse). The structure of ErbB2-induced ErbB3 homodimers is unclear. They can be bona fide ErbB3 homodimers or looser molecular associations. Formation of ErbB3/ErbB2 heterodimers is mediated by the dimerization arm of ErbB2 both in quiescent and HRG-stimulated cells because they are completely absent from PRT-pretreated samples. To see this figure in color, go online.

Fig 4: Experimental strategy for detection of ErbB3 dimerization. (A) The laser power/pulse duration and image acquisition protocol applied in two-color TOCCSL experiments is indicated. The inset shows a zoom into the timing protocol with a delay time of 20 ms between recordings of the green and the red color channel. (B) ErbB3-positive CHO cells growing on fibronectin coated coverslips were quantitatively decorated with equimolar amounts of AF488- and AF647-labeled monovalent antibody fragments (AF488-ErbB3 Fab and AF647-ErbB3 Fab). A defined CHO cell region was illuminated in TIR mode by using an aperture (dashed area) in the excitation pathway. Emission was split into two-color channels and imaged on the same electron-multiplying charge-coupled device camera. The average ErbB3 surface density was too high to allow for the resolution of single entities (i), yielding many ErbB3 molecules per camera pixel (see sketch below). After applying a 0.7-s long photobleaching pulse, all fluorophores were ablated as can be seen in the image recorded 10 ms after bleaching (ii). At the onset of the recovery process after 5–10 s, single probes that have diffused from the masked region into the central field of view can be seen as diffraction limited signals (iii). Single diffraction limited signals were detected in both color channels (green and red circles), and homoassociation was determined by the co-localization of signals within a radius of 160 nm (yellow circles). After the TOCCSL image, an additional image sequence was recorded and allowed for tracking of co-localized and individual ErbB3 signals for the subsequent determination of diffusion constants (laser pulses iv in the upper panel). (C) A sketch showing the effect of the photobleaching protocol on putative ErbB3 homodimers is shown. To see this figure in color, go online.

Fig 5: The fraction of ErbB3 associated to homo- and heterodimers in CHO-ErbB3 and CHO-ErbB2-ErbB3 cells determined by two-color TOCCSL. The mean (± standard error of the mean) of the homo- (black bars) (A and B) and heterodimeric fractions (white bars) (C) of ErbB3 are shown under different treatment conditions (HRG, heregulin; PRT, pertuzumab). In TOCCSL recordings, co-localized signals were counted, the respective numbers were corrected for labeling and for the different diffusion constants of monomers and dimers, and finally, the fraction of ErbB3 in dimers was calculated (see Supporting Materials and Methods for details of the correction). The corrected values, shown in the figure, and the raw data can be found in Table S2.