10-16 June 2018
Dalhousie University
America/Halifax timezone
Welcome to the 2018 CAP Congress Program website! / Bienvenue au siteweb du programme du Congrès de l'ACP 2018!

Single-Particle Tracking Reveals Diverse Diffusion Regimes of Individual M2 Receptors and Gi Proteins in Live Cells (G)

12 Jun 2018, 16:30
15m
SUB 224 (cap.50) (Dalhousie University)

SUB 224 (cap.50)

Dalhousie University

Oral (Graduate Student) / Orale (Étudiant(e) du 2e ou 3e cycle) Physics in Medicine and Biology / Physique en médecine et en biologie (DPMB-DPMB) T4-7 Physics of Biosensing (DPMB) | Physique de la biodétection (DPMB)

Speaker

Yuchong Li (University of Toronto Mississauga)

Description

G protein coupled receptors (GPCRs) are a superfamily of membrane receptors known for high signal transduction efficiencies. One of the key aspects of the GPCR signaling mechanism is the coupling interaction between the receptor and the G protein in response to external stimuli. We examined the pre-stimulus receptor-G protein coupling state by single-particle tracking (SPT) of M$_2$ muscarinic receptors and G$_i$ proteins in live cells.
M$_2$ receptors and G$_i$ proteins were genetically fused with fluorescent proteins (GFP and/or mCherry), expressed in CHO cells, and imaged on a Total Internal Reflection Fluorescence (TIRF) microscope. Single particles were identified in each frame of the TIRF movies and tracked using the TrackMate software. Mean-squared displacement (MSD) functions were computed for each single-particle trajectory. The diffusion parameters for receptors and G proteins were obtained by fitting their MSD functions to appropriate diffusion models.
Both the M$_2$ receptors and the G$_i$ proteins exhibited significant fractions of confined diffusion (compatible with the membrane compartment formed by actin microfilament-based meshwork) and active transportation (compatible with the rate of myosin trafficking along actin microfilaments). The motions of the M$_2$ receptors and of the G$_i$ proteins were distinctive from each other in the basal state of receptors, but they became similar when the receptors were activated by the agonist. Corroborated with dual-color fluorescence correlation spectroscopy measurements performed on the same samples, the SPT results supported a transient recruitment model without a stable pre-stimulus coupled complex.

Primary author

Yuchong Li (University of Toronto Mississauga)

Co-authors

Dr James Wells (Dept. of Pharmacology and Pharmaceutical Sciences, University of Toronto) Claudiu Gradinaru (University of Toronto)

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