Actin Corrals G-Protein Coupled Receptors in Ciliary Membrane

Abstract

Current understanding of the mechanisms underlying G-Protein Coupled Receptor (GPCR) transport within primary cilium is limited and several competing models exist, including motor-driven transport, local binding and free diffusion. Our analysis of the mean square displacement (MSD) of single GPCRs within ciliary membranes as a function of time step magnitude showed predominant mode of transport is diffusion with transient confinement to membrane sub-regions. The mechanism of this confinement is not well understood, and we hypothesize that actin filaments delimit the membrane sub-regions. We performed super-resolution high speed imaging of single GPCRs transfected into murine inner-medullary collecting duct (IMCD3) cells using quantum-dots. Latrunculin A, Cytochalaisin D, and Jasplakinolide was used to alter actin dynamics in primary cilium. The MSD(tau) analysis revealed free and fast local diffusion, followed by confinement to membrane sub-regions, slowing diffusion ∼15 fold. The average diffusion coefficient of the free local diffusion was [2.53 ± 0.42 micro m2 s−1], which is an order of magnitude higher than previous measurements of GPCR diffusion coefficient inside cilium. The average diffusion coefficient of the slow confined diffusion was [0.18 ± 0.03 mm2 s−1]. The size of the corral is calculated to be approximately 260nm. Disruption of actin by Latrunculin-A and Cytochalasin D resulted in increased diffusion coefficient and MSD. Latrunculin A treatment resulted in a 1.53±0.19 fold increase in the diffusion coefficient above vehicle control. Enhancing actin polymerization with Jasplakinolide significantly decreased the diffusion coefficient and MSD(tau). Fast imaging and the MSD analysis following altered actin dynamics demonstrates that the predominant modes of GPCR movement within the ciliary compartment is free local diffusion in highly fluid membrane, followed by transient confinement to membrane sub-regions delimited by actin. Ongoing studies aim to understand the physiological role of the ciliary membrane compartmentalization.