Super-Resolution Imaging and Single Molecule Tracking of the Nuclear Protein Emerin

Abstract

Emerin is an integral protein of the nuclear membrane expressed in most somatic cells. When under-expressed or mutated, it leads to Emery-Dreifuss Muscular Dystrophy. This disease is characterized by cardiac abnormalities and progressive muscle wasting. The molecular bases of these phenotypes are not well defined. Previous attempts at characterizing emerin localization and diffusional mobility have relied on ensemble confocal fluorescence microscopy. This approach did not reveal differences between normal and mutated versions of emerin, perhaps due to the limited optical resolution of confocal and FRAP techniques. Here we employ single molecule tracking and super-resolution optical imaging of emerin to further characterize emerin diffusion behaviors and localization at the nanometer scale.We performed single protein tracking of photoactivable-TagRFP-emerin fusions in live cells and study their dynamics in the nuclear membrane. Diffusion analyses indicate the co-existence of four diffusing sub-populations. One emerin sub-population is associated with the endoplasmic reticulum diffusion, two are located exclusively to the nuclear membrane, and a nearly immobile sub-population also locates to the nuclear membrane. Single molecule pulse-chase experiments reveal that the two nuclear-exclusive sub-populations accumulate at different rates, providing further evidence of their mutual uniqueness and their differential distribution between the inner and outer nuclear membrane. Further studies with disease-associated emerin mutants revealed that a single point emerin mutant has a 250% increase in diffusion coefficient for one of the nuclear membrane sub-populations when compared to the wild type.We also map the 3D nanoscale organization of emerin at the nuclear membrane using super-resolution imaging of SNAP-tag-emerin fusions. Analysis of emerin clusters was performed with 3D Ripley's K-function to compare the distribution of wild-type and mutated versions of emerin.