Nuclear envelope protein interaction studies

Abstract: The nuclear envelope (NE) separating the nucleoplasm from cytoplasm consists of two concentric lipid membranes, the outer (ONM) and inner (INM) nuclear membranes, the nuclear pore complexes (NPCs) and an underlying nuclear lamina network. The INM contains more than 100 unique transmembrane proteins of which only a few have been characterized. This thesis is focused on one of these INM proteins, Samp1 (Spindle associated membrane protein 1)Protein-protein interactions in the NE have been difficult to study due to the resistance of NE proteins to extraction. We have established a reversible in vivo crosslinking immunoprecipitation method called, MCLIP (Membrane protein Cross-Link ImmunoPrecipitation) to overcome this problem. Using MCLIP we were able to show that, Samp1 specifically interacts with Emerin, Lamin B1, Sun1 and the small GTPase Ran. We also showed that, the nucleoplasmic domain of Samp1 and Emerin can interact with each other directly.Furthermore, we investigated the functional role of Samp1 in mitosis. Samp1 depletion gave rise to aneuploid phenotypes and signs of destabilization of the mitotic spindle. Using MCLIP, in mitotic cells, we showed that, Samp1 interacts with Ran and Importin-β, two key players of mitotic spindle assembly. We observed that, Samp1 modulates the level of Importin-β and NuMA in the mitotic spindle, which may explain the mitotic defects and aberrant phenotypes observed in Samp1 depleted cells. These findings show that Samp1 plays an important role in spindle stabilization and chromosome segregation.