SummaryFluorescence imaging of two independently labelled proteins is commonly used to determine their co-localization in cells. Antibody-mediated crosslinking can mediate the patching of such proteins at the cell surface, and their co-localization can serve to determine complex formation among them. However, manual analysis of such studies is both tedious and subjective. Here we present a digital co-localization analysis that is independent of the fluorescence intensity, is highly consistent and reproducible between observers, and dramatically reduces the analysis time. The approach presented is based on a segmentation procedure that creates binary objects, and then determines whether objects belonging to two different groups (e.g. greenand red-labelled) are co-localized. Two methods are used to determine co-localization. The 'overlap' analysis defines two objects as co-localized if the centre of mass of one falls within the area of the other. The 'nearest-neighbour distance' analysis considers two objects as co-localized if their centres are within a threshold distance determined by the imaging modality. To test the significance of the results, the analysis of the actual images is tested against randomized images generated by a method that creates images with uncorrelated distributions of objects from the two groups. The applicability of the algorithms presented to study protein interactions in live cells is demonstrated by co-patching studies on influenza haemagglutinin mutants that do or do not associate into mutual oligomers at the cell surface via binding to AP-2 adaptor complexes. The approach presented is potentially applicable to studies of co-localization by other methods (e.g. electron microscopy), and the nearest-neighbour distance method can also be adapted to study phenomena of correlated placement.

Erythropoietin receptor (EpoR) activation is crucial for mature red blood cell production. The murine EpoR can also be activated by the envelope protein of the polycythemic (P) spleen focus forming virus (SFFV), gp55-P. Due to differences in the TM sequence, gp55 of the anemic (A) strain SFFV, gp55-A, cannot efficiently activate the EpoR. Using antibody-mediated immunofluorescence co-patching, we show that the majority of EpoR forms hetero-oligomers at the cell surface with gp55-P and, surprisingly, with gp55-A. The EpoR TM domain is targeted by gp55-P and -A, as only chimeric receptors containing EpoR TM sequences oligomerized with gp55 proteins. Both gp55-P and gp55-A are homodimers on the cell surface, as shown by co-patching. However, when the homomeric interactions of the isolated TM domains were assayed by TOXCAT bacterial reporter system, only the TM sequence of gp55-P was dimerized. Thus, homo-oligomerization of gp55 proteins is insufficient for full EpoR activation, and a correct conformation of the dimer in the TM region is required. This is supported by the failure of gp55-A3 P, a mutant protein whose TM domain can homo-oligomerize, to fully activate EpoR. As unliganded EpoR forms TM-dependent but inactive homodimers, we propose that the EpoR can be activated to different extents by homodimeric gp55 proteins, depending on the conformation of the gp55 protein dimer in the TM region.

Signaling via interleukin-2 (IL-2) and interleukin-9 receptors (IL-2R and IL-9R) involves heteromeric interactions between