Fluorescence imaging of pancreatic beta cells reveals receptor localization and informs mathematical model of cell signaling

Introduction:: Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling through the prolactin receptor (PRLR) has been shown to drive pancreatic beta cell proliferation. While advances have been made in understanding control of cell-cycle progression in mouse and human beta cells, the network of intracellular signal transduction linking extracellular nutrient and growth factor actions at the beta cell surface to cell-cycle machinery is poorly understood. Because a deficit of functional insulin-secreting beta cells underlies both Type 1 and Type 2 diabetes, elucidating the molecular mechanisms of PRLR signaling in beta cell survival is critical to harnessing the proliferative potential of the pathway for therapeutic development. Here, we perform live, quantitative imaging to build a mathematical model of PRLR and STAT5 dynamics.

Materials and Methods:: This work builds upon a published computational model of prolactin-mediated JAK-STAT signaling in pancreatic beta cells, which uses ordinary differential equations to simulate biochemical reactions of ligand-receptor binding, phosphorylation, dimerization, and trafficking of PRLR and STAT5 between spatial compartments. In parallel to our work advancing the model development, we engineered expression vectors that fuse fluorescent proteins to PRLR and STAT5 for use in fluorescence fluctuation spectroscopy (FFS) experiments in cultured cells. FFS measurements quantitatively capture the dynamics of fluorescently tagged proteins of interest, yielding data that can be integrated into the computational model as parameter values. Live and fixed cell imaging, as well as immunofluorescence in mouse pancreatic islets, was used to determine the localization patterns of PRLR.

Results, Conclusions, and Discussions:: Our imaging experiments in both live cells and fixed pancreatic islets reveal large intracellular stores of PRLR relative to their expression at the cell membrane. In live cells, transiently transfected with our expression vectors, this result was observed for all vector designs (Figure 1A). Immunofluorescence in mouse islets confirmed the high levels of intracellular PRLR relative to the membrane (Figure 1B). These results suggest that PRLR is internalized and perhaps capable of signaling from cell surface and intracellular populations. Using these results, we expanded the base model to include internalization and recycling reactions that allow PRLR to be compartmentalized not only at the cell surface but also in endosomes. This has implications for both sensing extracellular signals and the composition of receptor complexes within the separate compartments, further impacting STAT5 compartmentalization.

We examined PRLR and STAT5 dynamics, including both PRLR localization within endosomes and its expression at the cell surface. We demonstrated the ability of FFS to quantitatively capture dynamics of fluorescently tagged PRLR and STAT5 under ligand stimulation, which is relevant to understanding the role of JAK-STAT signaling in beta cell proliferation. Our predictive model can integrate the multi-dimensional and multi-scale data intrinsic to cell signaling pathways into frameworks that can be used to expand our molecular understanding of biological signaling networks. This provides a powerful approach to quantitatively study pancreatic beta cell signaling, iterating between computational and experimental techniques, to improve our quantitative understanding of pancreatic beta cells, and to provide the foundational knowledge needed for developing novel therapeutic approaches.


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