Organization and dynamics of the serotonin1A receptor in live cells using fluorescence microscopy

摘要

It is important to understand the dynamic organization of membrane-bound molecules in order to arrive at a comprehensive view of cellular signaling mediated by membrane-bound receptors.¹ We addressed the organization and dynamics of the human serotonin1A receptor fused to enhanced yellow fluorescent protein expressed in CHO cells. Serotonin1A receptors are prototypical members of the G-protein coupled receptor superfamily and represent a prime target for therapeutic actions of several anxiolytic and antidepressant drugs.² Interestingly, we observed significant retention in fluorescence of serotonin1A receptors upon Triton X-100 treatment of intact cells at low temperature demonstrating their detergent insolubility.³ We analyzed the role of cholesterol in the plasma membrane organization of the serotonin1A receptor by fluorescence recovery after photobleaching (FRAP) measurements with varying bleach spot sizes. Our results show that lateral diffusion parameters of serotonin1A receptors are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin1A receptors in the plasma membrane.⁴ Our recent work using z-scan fluorescence correlation spectroscopy (zFCS) provides novel insight on the effects of cholesterol depletion and actin cytoskeleton destabilization on receptor confinement.⁵ Interestingly, results from FRAP measurements performed under conditions of mild cytoskeletal destabilization suggest that receptor signaling is correlated with receptor mobility, in agreement with the ‘mobile receptor hypothesis’.⁶ In addition, we developed a novel microscopy-based image reconstruction approach to quantitatively monitor dynamic changes in actin cytoskeletal network upon signaling.⁷ We recently proposed utilizing Homo-FRET in live cells, that the serotonin1A-nreceptor is present as constitutive oligomers and implicated the presence of higher-order oligomers.⁸ Taken together, these results on the cellular organization and dynamics of the serotonin1A receptor would be valuable in understanding the function of the receptor in healthy and diseased states.