Differentiating Membrane-Associated And Soluble Protein Populations Within The Nuclear Envelope Via Fluorescence Fluctuation Spectroscopy

The nuclear envelope (NE) consists of two membranes separated by the ∼40 nm wide lumen. This thin structure, being well below the resolution limit, presents a challenge to differentiating membrane-associated and luminal protein populations within the NE of living cells. Distinguishing these two populations is further complicated by the observation that diffusion time, a commonly used marker for membrane association in other cellular compartments, shows a strong dependence on molecular size within the lumen. Thus, a decrease in proteins mobility within the NE may be caused by the formation of large soluble complexes or association with the membranes. We recently observed a striking divergence in the temperature dependence of mobility for luminal and membrane-associated proteins within the NE, which provides a reliable marker for differentiating these two populations. Furthermore, previously reported nuclear membrane undulations introduce an additional fluctuation signal to luminal proteins while not affecting membrane-associated proteins. We demonstrate that this effect providing a second, independent marker distinguishing luminal and membrane-associated protein. These two independent assays for membrane-association are applied to proteins of biological interest. The luminal domain of SUN2 was previously shown to exist as a mixture of fast diffusing monomers and slowly diffusing trimers. We apply our tools to determine whether this decrease in mobility is caused solely by the three-fold increase in molecular weight or by membrane association. Finally, we use these techniques to identify the extent of membrane association for the AAA+ ATPase torsinA and explore the effect of tagging the fluorescent label to its N- or C-terminus. This work has been supported by a grant from the National Institutes of Health (R01 GM64589).