The Functional Organization of Nuclear Envelope Proteins

Abstract: In eukaryotic cells, the nucleus is enclosed by a double lipid membrane, termed the nuclear envelope (NE). The NE consists of the outer nuclear membrane (ONM), the inner nuclear membrane (INM), the nuclear pore complexes (NPCs) and the nuclear lamina. Recently it has been realized that the NE proteins not only serve structural functions but are also involved in a diverse group of genetic diseases collectively termed laminopathies or envelopathies. So far, only a few NE proteins have been characterized in detail. Here, we have identified and investigated a novel transmembrane protein from the NE, which is highly conserved in evolution. We termed the protein, Spindle associated membrane protein 1 (Samp1). During mitosis, a subpopulation of Samp1 is concentrated in the mitotic spindle. Samp1 has four transmembrane domains and is specifically localized to the INM. The N-terminal half of Samp1 contains a Zinc finger domain and is exposed in the nucleoplasm. Over expression of Zinc finger mutants of Samp1 gave an abnormal phenotype characterized by disruption of the localization of endogenous Samp1 and a specific set of NE proteins, suggesting that Samp1 is functionally associated with LINC complex and A-type lamina network proteins. After posttranscriptional silencing of Samp1 expression we showed that Samp1 is required for correct localization of Emerin to the NE. We also showed that Samp1 interacts with Emerin in live cells and that this interaction can occur by direct binding. The fact that the interaction between Emerin and Samp1 depended on Zinc, supports the idea that Samp1 has functional Zinc finger(s). Posttranscriptional silencing of Samp1 gave rise to an increase in the distance between the centrosome and the NE, suggesting that Samp1 is functionally associated with the microtubule cytoskeleton, most likely mediated via the LINC complexes. Using high-resolution fluorescence microscopy we showed that Samp1 is distributed in a distinct pattern in the NE and partially colocalized with the LINC complex protein, Sun1. We also showed that the Samp1 can interact with Sun1 in live cells. We developed a novel method, Membrane protein Cross-Link ImmunoPrecipitation (MCLIP) that enables detection of specific interactions of NE proteins in live cells. Using MCLIP we identified specific interaction partners of Samp1 in U2OS cells. Human induced pluripotent stem cells (hiPSCs) displayed increased expression of Samp1 during differentiation. Over expression of YFP-Samp1 induced a rapid differentiation of hiPSCs into neurons. The medium from