Monte Carlo simulations for the evaluation of oligomerization data in TOCCSL experiments.

The interplay and communication between cells build the foundation of life. Many signaling processes at the cell surface and inside the cell, as well as the cellular function itself, depend on protein-protein interactions and the oligomerization of proteins. In the past, we presented an approach to single out interactions of fluorescently labeled membrane proteins by combining photobleaching and single-molecule microscopy. With this approach, termed "thinning out clusters while conserving the stoichiometry of labeling" (TOCCSL), oligomerization can be detected even at physiologically high surface densities of fluorescently labeled proteins. In TOCCSL, an aperture-restricted region of the plasma membrane is irreversibly photobleached by applying a high-intensity laser pulse. During a recovery time, in which illumination is turned off, nonphotobleached molecules from the nonilluminated area of the plasma membrane re-populate the aperture-restricted region. At the onset of this recovery process, these molecules can be detected as well-separated, diffraction-limited signals and their oligomerization state can be quantified. Here, we used extensive Monte Carlo simulations to provide a theoretical framework for quantitative interpretation of TOCCSL measurements. We determined the influence of experimental parameters and intrinsic characteristics of the investigated system on the outcome of a TOCCSL experiment. We identified the diffraction-affected laser intensity profile and the diffusion of molecules at the aperture edges during photobleaching as major sources of generating partially photobleached oligomers. They are falsely detected as lower-order oligomers and, hence, higher-order oligomers might be prevented from detection. The amount of partially photobleached oligomers that are analyzed depends on the photobleaching and the recovery time, on the mobility of molecules and-for mixed populations of oligomers-on mobility differences between different kinds of oligomers. Moreover, we quantified random colocalizations of molecules after recovery, which are falsely detected as higher-order oligomers.

Homo- and Heteroassociations Drive Activation of ErbB3.

Dimerization or the formation of higher-order oligomers is required for the activation of ErbB receptor tyrosine kinases. The heregulin (HRG) receptor, ErbB3, must heterodimerize with other members of the family, preferentially ErbB2, to form a functional signal transducing complex. Here, we applied single molecule imaging capable of detecting long-lived and mobile associations to measure their stoichiometry and mobility and analyzed data from experiments globally, taking the different lateral mobility of monomeric and dimeric molecular species into account. Although ErbB3 was largely monomeric in the absence of stimulation and ErbB2 co-expression, a small fraction was present as constitutive homodimers exhibiting a ∼40% lower mobility than monomers. HRG stimulation increased the homodimeric fraction of ErbB3 significantly and reduced the mobility of homodimers fourfold compared to constitutive homodimers. Expression of ErbB2 elevated the homodimeric fraction of ErbB3 even in unstimulated cells and induced a ∼2-fold reduction in the lateral mobility of ErbB3 homodimers. The mobility of ErbB2 was significantly lower than that of ErbB3, and HRG induced a less pronounced decrease in the diffusion coefficient of all ErbB2 molecules and ErbB3/ErbB2 heterodimers than in the mobility of ErbB3. The slower diffusion of ErbB2 compared to ErbB3 was abolished by depolymerizing actin filaments, whereas ErbB2 expression induced a substantial rearrangement of microfilaments, implying a bidirectional interaction between ErbB2 and actin. HRG stimulation of cells co-expressing ErbB3 and ErbB2 led to the formation of ErbB3 homodimers and ErbB3/ErbB2 heterodimers in a competitive fashion. Although pertuzumab, an antibody binding to the dimerization arm of ErbB2, completely abolished the formation of constitutive and HRG-induced ErbB3/ErbB2 heterodimers, it only slightly blocked ErbB3 homodimerization. The results imply that a dynamic equilibrium exists between constitutive and ligand-induced homo- and heterodimers capable of shaping transmembrane signaling.