Biophysical Society Thematic Meeting - June 28-July 1, 2015

New Biological Frontiers Illuminated by Molecular Sensors and Actuators

Poster Abstracts

17-POS Board 17 Insulin Receptor and IGF1 Receptor Biosensors Employing Between-Domain Fluorescence Tags James D. Johnson , Howard Cen, Søs Skovsø, Tobias Albrecht. University of British Columbia, Vancouver, Canada. The insulin receptor (InsR) is one of the most studied proteins, but fundamental aspects of its biology remain unclear, including the kinetics of insulin receptor activation and internalization from the plasma membrane. To monitor insulin receptor and insulin-like growth factor 1 receptor (IGF1R) dynamics in real time, we employed a novel tagging strategy wherein fluorescent proteins (TagRFP, TagRFP-T, eGFP, eYFP, TagBFP, tagGFP2) were placed at the extracellular region in between two functional domains. Unlike published insulin receptor fusions that have fluorescent proteins attached to their C-termini, our fusion proteins primarily localized to vesicle-like cytoplasmic structures mirroring the pattern we observed for the endogenous insulin receptors in vivo and in vitro. InsRA and InsRB splice variants had overlapping distributions, in contrasting with the conclusions of previous studies with C-terminal tagged receptors. Using insulin secreting pancreatic beta-cells as a model system, we mapped the steady state internalization and trafficking of between-domain-tagged InsRs and endogenous InsRs to organelles labeled with caveolin1 (Cav1), flotillin1 and Lamp1, which bypass compartments positive for clathrin, Rab5a, Rab7, Rab11a, or Rab4a. TIRF imaging of InsRA-eGFP revealed that the phospho-mimetic Cav1-Y14D mutant significantly shortened the lifetime of InsRA domains at the plasma membrane prior internalization. Ratiometric pH sensor fluorescent proteins and Fluorogen Activating Peptides were also developed to track the dynamics of insulin receptor internalization of insulin receptor clusters in living cells in real time using spinning disk confocal microscopy and high-conetnt imaging systems. We expect that these novel approaches to live-cell imaging of tyrosine kinase receptor dynamics will be important for elucidation of insulin and IGF1 signaling and development of high-content screening for molecules that modify insulin receptor trafficking and activity.

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