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Nuclear envelope protein interaction studies
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0003-1287-0495
2014 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

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. 

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2014.
Keyword [en]
Samp1, Nuclear envelope, protein interactions, chemical crosslinking, proteomics
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-109194ISBN: 978-91-7649-041-9 (print)OAI: oai:DiVA.org:su-109194DiVA: diva2:765086
Presentation
2014-12-16, Heilbronnsalen, Svante Arrhenius väg 16 B, Stockholm, 12:15 (English)
Opponent
Supervisors
Available from: 2014-11-21 Created: 2014-11-14 Last updated: 2015-03-17Bibliographically approved
List of papers
1. MCLIP, an effective method to detect interactions of transmembrane proteins of the nuclear envelope in live cells
Open this publication in new window or tab >>MCLIP, an effective method to detect interactions of transmembrane proteins of the nuclear envelope in live cells
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2014 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, no 10, 2399-2403 p.Article in journal (Refereed) Published
Abstract [en]

Investigating interactions of proteins in the nuclear envelope (NE) using co-immunoprecipitation (Co-IP) has previously been difficult or even impossible due to their inherent resistance to extraction. We have developed a novel method, MCLIP (Membrane protein Cross-Link ImmunoPrecipitation), which takes advantage of a cell permeable crosslinker to enable effective detection and analysis of specific interactions of NE proteins in live cells using Western blot. Using MCLIP we show that, in U2OS cells, the integral inner nuclear membrane protein Samp1 interacts with Lamin B1, the LINC (Linker of nucleoskeleton and cytoskeleton) complex protein, Sun1 and the soluble small GTPase Ran. The results show that the previously detected in vitro interaction between Samp1 and Emerin also takes place in live cells. In vitro pull down experiments show, that the nucleoplasmic domains of Samp1 and Emerin can bind directly to each other. We also, show that MCLIP is suitable to coprecipitate protein interactions in different stages of the cell cycle.

Keyword
Samp1, Nuclear envelope, Nuclear membrane, Crosslinking, CoIP, Protein–protein interaction
National Category
Chemical Sciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-109181 (URN)10.1016/j.bbamem.2014.06.008 (DOI)000340975600005 ()
Funder
Swedish Research Council, 621-2010-448Swedish Cancer Society, 110590
Available from: 2014-11-14 Created: 2014-11-14 Last updated: 2017-10-24Bibliographically approved
2. The integral nuclear membrane protein, Samp1 modulates importin-β and NuMA in the mitotic spindle
Open this publication in new window or tab >>The integral nuclear membrane protein, Samp1 modulates importin-β and NuMA in the mitotic spindle
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(English)Manuscript (preprint) (Other academic)
Keyword
Samp1, mitotic spindle, centrosome, LINC complex, Lamina, Sun1, mitosis
National Category
Biological Sciences Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-109192 (URN)
Funder
Swedish Research Council, 621-2010-448Swedish Cancer Society, 110590
Available from: 2014-11-14 Created: 2014-11-14 Last updated: 2016-01-29Bibliographically approved

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