Identifying the Axial Location of Proteins at the Nuclear Envelope with Nanometer Resolution

Abstract

The nuclear envelope (NE) separates the cytoplasm and nucleus by a double-layered membrane consisting of the inner nuclear membrane (INM) and outer nuclear membrane (ONM). The INM and ONM are separated by tens of nanometers. Specific INM and ONM proteins form LINC (linker of nucleoskeleton and cytoskeleton) complexes, which span the nuclear envelope and mechanically couple the nucleoskeleton and cytoskeleton. It has been suggested that LINC complexes define the spacing of the INM and ONM, which might be modulated by a wide range of diseases associated with LINC complexes. To test this hypothesis in future work we are seeking to directly measure the distance between the INM and ONM in live cells. We axially scan the two-photon point spread function through a cell with a fluorescently-labeled protein residing on a nuclear membrane. The scan generates an intensity profile that reveals the spatial location of the proteins along the scan path. To measure the axial distance separating two protein systems we label each with a distinct color and perform a dual color z-scan, which simultaneously determines the location of each protein species. We experimentally verify that dual-color z-scan is capable of identifying the axial separation of membrane proteins with a few nanometer uncertainty using model systems. We also perform direct measurements of the distance between the INM and ONM on individual cells. In addition, we extend our studies to the translocation of transmembrane proteins from the ONM to the INM. This work has been supported by a grant from the National Institutes of Health (R01 GM064589).