The fluid mosaic model of Singer and Nicolson correctly predicted that the plasma membrane (PM) forms a lipid bi-layer containing many integral trans-membrane proteins. This model also suggested that most of these proteins were randomly dispersed and freely diffusing moieties. Initially, this view of a dynamic and rather unorganized membrane was supported by early observations of the cell surfaces using the light microscope. However, recent studies on the PM below the diffraction limit of visible light (~ 250 nm) revealed that, at nanoscale dimensions, membranes are highly organized and compartmentalized structures. Lymphocytes are particularly useful to study this nanoscale membrane organization because they grow as single cells and are not permanently engaged in cell:cell contacts within a tissue that can influence membrane organization. In this review, we describe the methods that can be used to better study the protein:protein interaction and nanoscale organization of lymphocyte membrane proteins, with a focus on the B cell antigen receptor (BCR). Furthermore, we discuss the factors that may generate and maintain these membrane structures.

During the preparation of figures for the above article, we inadvertently rotated the astrin blot in Figure 3F by 180 , and we inserted incorrect images in Figures 3H and 4F. The correct and incorrect images were derived from experiments conducted in parallel and are quite similar in appearance. We mislabeled them when we retrieved them for figure preparation. In Figure 3H, we inserted an incorrect image for astrin. In Figure 4F, we inserted an incorrect image for raptor-astrin PLA under arsenite treatment in the right panel. Furthermore, we used DAPI for nuclear staining in PLA but instead indicated Hoechst in the figure legends. These errors do not affect the results or interpretation of any of these experiments. The corrected images have been inserted into the figures below, and the figures and figure legends of PLA panels have been corrected online. We regret these errors and apologize for the inconvenience that they may have caused.

Introduction Activating FLT3 mutations are found in 30% of AML patients. Internal tandem duplication (ITD) mutations are most common, and are associated with poor prognosis. FLT3 tyrosine kinase inhibitors (TKI) were shown to be effective in clinical trials. However, complete remissions are rare, responses are short-lived, and the majority of patients display primary or secondary resistance to FLT3 inhibition. In FLT3 kinase inhibitor resistant, FLT3-ITD positive cell lines, we identified a direct interaction between FLT3-ITD and IL-3Rβc. We therefore sought to characterize this interaction and determine its' role in FLT3-ITD oncogenic signaling. Methods Drug sensitive BA/F3 FLT3-ITD cells and sublines resistant to FLT3 TKIs were subjected to analysis of activated IL-3Rβc - JAK1/2 - STAT signaling by PCR, Western-Blot (WB) and Immunoprecipitation (IP). Interaction studies were performed in vitro using IVT FLT3-ITD and GST IL-3Rβc, and in cell lines using γ2A cells transfected with human FLT3-ITD and human IL-3Rβc, human MOLM13 and MV4-11 AML cell lines, and primary AML patient samples by WB, IP and proximity ligation assay (PLA). Mapping experiments were performed in mouse embryonic fibroblasts (MEF) transduced with FLT3-ITD and flag-IL-3Rβc full-length or cytoplasmic Y to F mutant constructs. Knockdown-experiments were performed in BA/F3 cells with stably transfected FLT3-ITD and inducable IL-3Rβc shRNA. Results In TKI resistant FLT3-ITD positive cell-lines that did not harbor secondary FLT3 mutations inducing TKI resistance, we observed phosphorylation of IL-3Rβc in 5/16 resistant lines (30%) in the presence of FLT3 TKI. In these cells, IL-3Rβc phosphorylation was mediated by an activating JAK1 V658F mutation that bypasses FLT3-ITD dependent IL-3Rβc phosphorylation. Thus, IL-3Rβc in FLT3-ITD expressing cells mediates JAK1/2-dependent TKI resistance. Of note in inhibitor sensitive cells, IL-3Rβc interacted with and was phosphorylated by FLT3-ITD in a JAK1/2 independent manner, suggesting that IL-3Rβc participates in FLT3-ITD dependent oncogeneic signaling. Indeed, in IL-3Rβc and JAK2 deficient γ2A cells, expression of human FLT3-ITD was sufficient to induce interaction with and phosphorylation of human IL-3Rβc. In human FLT3-ITD-positive AML cell lines MOLM13 and MV4-11, IL-3Rβc phosphorylation occurred in a FLT3-ITD dependent fashion and IL-3Rβc interacted with FLT3-ITD. Proximity ligation assay (PLA) experiments detected FTL3-ITD and IL-3Rβc in close proximity suggesting a direct interaction of both proteins in MOLM13 and MV4-11 cells, as well as in primary cells from FLT3-ITD positive AML patients. Interaction studies revealed that binding to FLT3-ITD occurred independent of IL-3Rβc cytoplasmic tyrosines, and phosphorylation of IL-3Rβc by FLT3-ITD did not require the presence of IL-3Rαc. Preliminary results from knock down experiments in FLT3-ITD expressing BA/F3 cells indicated that knock-down of IL-3Rβc renders cells more susceptible to FLT3 TKI inhibition. Conclusion These data suggest an entirely novel model of FLT3-ITD "physiologically" employing IL-3Rβc as signaling intermediate, and IL-3Rβc serving as a signaling module mediating JAK1/2-dependent TKI resistance. These findings point toward the significance of IL-3Rβc for FLT3-ITD dependent transformation and treatment resistance, supporting the relevance of IL-3Rβc as a possible treatment target in FLT3-ITD positive AML. Disclosures von Bubnoff: Novartis: Research Funding; BMS: Speakers Bureau.