Opponent: Professor Katharine S. Ullman, Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah.

Abstract

The nucleus, a hallmark in eukaryotic cells, contains the genome separating it from molecules in the cytoplasm. The nucleus is surrounded by a nuclear envelope consisting of two concentric membranes, the outer nuclear membrane and the inner nuclear membrane, the nuclear lamina and nuclear pore complexes. The cytoskeleton is physically connected with the nucleoskeleton by the LINC complexes, spanning the nuclear envelope. In this way the cell surface is linked directly to chromatin. There are hundreds of unique inner nuclear membrane proteins, but today we only know the functions of a handful. The best characterized inner nuclear membrane proteins are involved in chromatin organisation and gene regulation.

This thesis focuses on Samp1, an integral membrane protein that localizes to the inner nuclear membrane during interphase. During mitosis, a fraction localizes to the mitotic spindle, which is responsible for accurate segregation of chromosomes.

It is difficult to investigate inner nuclear membrane protein-protein interactions, because transmembrane proteins are often associated with the “hard-to-solubulize” nuclear lamina. MCLIP was developed as a method to detect interactions between proteins of the nuclear envelope in live cells. MCLIP has been valuable in identifying interaction partners of Samp1. In interphase, Samp1 distributes in distinct micro domains of the inner nuclear membrane and interacts with the nuclear lamina, emerin and the LINC complex protein SUN1, suggesting that Samp1 might have a functional role associated with both the nucleoskeleton and cytoskeleton.

In mitosis Samp1 distributes in filamentous membrane structures partially overlapping with kinetochore microtubules of the mitotic spindle. Samp1 binds directly to γ-tubulin and recruits γ-tubulin and Haus6 to the mitotic spindle and thus contributes to spindle assembly. Samp1 also interacts with Aurora B, a kinase important for k-fiber error correction at the kinetochores. Depletion of Samp1 caused an increased activation and distribution of Aurora B at the metaphase plate, decreased formation of stable k-fibers, metaphase prolongation and increased chromosome mis-segregation. Samp1 is the first transmembrane protein found to be involved in mitotic spindle assembly and stability, important for correct segregation of chromosomes.