Mapping dynamic protein interactions to insulin secretory granule behavior with TIRF-FRET.
Biological processes are governed by extensive networks of dynamic molecular interactions. Yet, establishing a spatial and temporal map of these interactions and their direct relationship to specific cell functions has remained a challenge. Here, we implement sensitized emission Förster resonance energy transfer (FRET) stoichiometry under total internal reflection fluorescence (TIRF) microscopy. We demonstrate through quantitative analysis and modeling that evanescent fields must be precisely matched between FRET excitation wavelengths to isolate dynamic interactions between bimolecular FRET pairs that are not entirely membrane-delimited. We then use TIRF-FRET to monitor the behavior of individual insulin-containing secretory granules at the plasma membrane of living cells, while simultaneously tracking the dynamic interaction between the GTPase Rab27A and its effector Slp4A, on those same granules. Notably, insulin granules that underwent exocytosis demonstrated a specific increase in Rab27A-GTP/Slp4A FRET in the 5 s before membrane fusion, which coincided temporally with an increase in granule displacement and mobility. These results demonstrate an initial spatiotemporal mapping of a dynamic protein-protein interaction on individual secretory granules that is linked to a specific granule behavior in living cells.
Lam
Alice D
AD
Department of Molecular and Integrative Physiology and Neuroscience Program, University of Michigan, Ann Arbor, Michigan, USA.
Ismail
Sahar
S
Wu
Ray
R
Yizhar
Ofer
O
Passmore
Daniel R
DR
Ernst
Stephen A
SA
Stuenkel
Edward L
EL
D000818:Animals; D002138:Calibration; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D022142:Secretory Vesicles / Q000378:metabolism*; D012995:Solubility; D014162:Transfection; D033921:Vesicular Transport Proteins / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000075745:rab27 GTP-Binding Proteins
D005456:Fluorescent Dyes; D007328:Insulin; D033921:Vesicular Transport Proteins; D000075745:rab27 GTP-Binding Proteins; C512962:Rab27a protein, mouse; D020691:rab GTP-Binding Proteins