Super-resolved and dynamic imaging of membrane proteins in plant cells reveal contrasting kinetic profiles and multiple confinement mechanisms.

Abstract

Dear Editor, Microscopic techniques allow either a global mobility analysis of proteins with fluorescence recovery after photobleaching (FRAP) or single-protein mobility characterization with single-particle or quantum dot tracking.For instance,total internal reflection fluorescence microscopy allowed single-particle tracking (SPT) of Arabidopsis plasma membrane (PM) proteins,revealing their heterogeneous distribution,low lateral diffusion,and dynamic properties in response to salt stress (Li et al.,2011).Studies of SPT based on green fluorescent protein are unfortunately restricted by the density of proteins at the surface,since diffracted emission fluorescence prevents tracking of individual proteins separated by less than 1 μm.The recent emergence of high-density SPT techniques based on temporal emission decorrelation,such as single-particle tracking with photoactivated localization microscopy (sptPALM),allowed the diffraction limit of classic light microscopy to be broken and reach nanometerlevel spatial resolutions (Manley et al.,2008).Application of these techniques has rendered possible the characterization of the structural and dynamic heterogeneity of PM proteins with an accuracy of ～20-80 nm (Rossier et al.,2012).Such super-resolved dynamic imaging of membrane proteins has not yet been applied to any plant system.Here,we report the first use in plants of the live-cell sptPALM technique,providing a high-density super-resolved nanoscale map of individual membrane-protein motions.