The eukaryotic cell is defined by the nucleus, which is delimited by a double membrane structure termed the nuclear envelope (NE). The NE is implicated in a multitude of different processes, for example chromatin organization. During mitosis in higher eukaryotes the nucleus is disassembled to allow the formation of the mitotic spindle, which segregates the duplicated chromosomes between daughter cells. We have characterized a novel transmembrane protein of the inner nuclear membrane. Because of its distribution along spindle microtubule during mitosis, we termed the protein Samp1 (Spindle associated membrane protein 1). Samp1 is the founding member of transmembrane proteins that define a novel membrane domain that we have termed the SE (spindle endomembrane). Furthermore, we have shown that in interphase Samp1 specifically interacts with the centrosome and A-type lamina network proteins. Moreover, Samp1 contains an evolutionary highly conserved N-terminal tail containing two putative zinc fingers.

Recent studies indicate local caspase activity in dendrites or axons during development and in neurodegenerative disorders. Here I present the development of a novel and unique system to monitor protease activity at sub-cellular resolution in live cells. This system relies on a cleavable FRET sensor that is anchored to the cytoskeleton. Using this system we demonstrate local caspase activation of the soma in neuronaly differentiated cells. We also used the anchored FRET sensors to monitor caspase activation after treatment with the Alzheimer’s decease related amyloid-β peptide.

Moreover we have improved a NF-ĸB decoy delivery system. The system consists of a cell penetrating peptide, transportan-10, covalently linked to a peptide nucleic acid sequence that hybridizes with a nonanucleotide sequence in the decoy. We show that this system effectively delivered the decoy and inhibited an inflammatory response in primary rat glial cells.

The nuclear envelope (NE) consists of two concentric membranes, the outer nuclear membrane (ONM) and the inner nuclear membrane (INM). The LINC (linker of nucleoskeleton and cytoskeleton) complex spans both the ONM and the INM connecting the cytoskeleton to the nucleoskeleton and chromatin. Only a few of the known INM proteins have been functionally characterized and shown to have important roles in chromatin organisation. Defects in the genes coding for proteins in the INM and the nuclear lamina give rise to serious human diseases, called envelopathies.

In 2009 (Buch et al. 2009) our group made two major discoveries. We showed for the first time, that an integral INM protein distributed along the microtubules of the mitotic spindle. This protein was therefore named Samp1, Spindle Associated Membrane Protein 1. The second discovery was that depletion of Samp1 caused detachment of the centrosome from the NE, suggesting that Samp1 is associated with the microtubule cytoskeleton both in interphase and mitosis.

In this thesis we continued to investigate Samp1´s role during interphase. We also wanted to investigate the localisation of Samp1 in the mitotic spindle and possible function during mitosis. We show that the expression of Samp1 mutants and depletion of Samp1 affects the distribution and organisation of A-type lamins, the LINC complex protein Sun1 and the LINC complex associated protein emerin. Thus, in interphase Samp1 is functionally connected to the LINC complex and the A-type lamina network. The LINC complex can help explain how the centrosomes detach from the NE in Samp1 depleted cells. In mitotic cells, we found that depletion of Samp1 caused prolonged metaphase and aberrant mitotic phenotypes such as bi-nucleation, enlarged nuclei and micronuclei. We also showed that Samp1 interacts with RanGTPase and importin-β, which are key players in assembling the mitotic spindle. Samp1 also modulates the levels of importin-β and NuMA in the mitotic spindle, which could explain the mitotic phenotypes that we se in Samp1 depleted cells. Here we present evidence showing, for the first time, that an INM protein is present on kinetochore microtubules and have an essential role for correct chromosome segregation and spindle assembly.

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 organization 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-solubilize” 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.