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Multifaceted roles of the transmembrane nuclear envelope protein, Samp1
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0003-1287-0495
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The eukaryotic nuclear envelope (NE), separates the nucleoplasm from cytoplasm and is made up of two concentric lipid membranes, the outer and the inner nuclear membranes (ONM and INM), the nuclear pore complexes (NPCs) and an underlying filamentous nuclear lamina. The INM contains hundreds of unique transmembrane proteins of which only a handful have been characterized. In this thesis, I aimed to understand the functional organization of proteins in the nuclear envelope and I focused on investigating the functions of a recently identified INM transmembrane protein, Samp1. We have developed a novel and robust approach, MCLIP, to identify specific protein-protein interactions taking place in live cells. Using MCLIP, we have shown that Samp1 interacts with proteins of the LINC complex, the nuclear lamina and components of the mitotic spindle. Samp1's specific interactions with a variety of binding partners, suggest that Samp1 plays important roles both in interphase and in mitosis.  We have also shown that Samp1 can provide a binding site at the INM for the GTPase Ran, a master regulator of protein interactions in interphase and in mitosis. Furthermore, we have also investigated the role of Samp1 in cell differentiation using two independent model systems. In human iPSCs, ectopic expression of Samp1 promoted differentiation despite pluripotent culture conditions. In C2C12 myoblast, depletion of Samp1 completely blocked differentiation into myotubes. The two studies complement each other and suggest that Samp1 has a strong differentiation promoting activity. Taken together, the findings in this thesis, give insights on the unexpected and unforeseen roles played by a transmembrane protein in different fundamental cellular process.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2017. , 46 p.
Keyword [en]
Nuclear envelope, transmembrane protein interaction studies, cell differentiation, stem cells, myopathies
National Category
Biochemistry and Molecular Biology Cell Biology Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-141816ISBN: 978-91-7649-577-3 (print)ISBN: 978-91-7649-578-0 (electronic)OAI: oai:DiVA.org:su-141816DiVA: diva2:1089372
Public defence
2017-05-31, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript. Paper 5: Manuscript.

Available from: 2017-05-08 Created: 2017-04-19 Last updated: 2017-06-02Bibliographically 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: 2017-04-27Bibliographically approved
2. Samp1, a RanGTP binding transmembrane protein in the inner nuclear membrane
Open this publication in new window or tab >>Samp1, a RanGTP binding transmembrane protein in the inner nuclear membrane
2016 (English)In: Nucleus, ISSN 1949-1034, E-ISSN 1949-1042, Vol. 7, no 4, 415-423 p.Article in journal (Refereed) Published
Abstract [en]

Samp1 is a transmembrane protein of the inner nuclear membrane (INM), which interacts with the nuclear lamina and the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex in interphase and during mitosis, it localizes to the mitotic spindle. Samp1 was recently found to coprecipitate a protein complex containing Ran, a GTPase with fundamental regulatory functions both in interphase and in mitosis. To investigate the interaction between Samp1 and Ran in further detail, we have designed and expressed recombinant fusion proteins of the Chaetomium thermophilum homolog of Samp1 (Ct. Samp1) and human Ran. Pulldown experiments show that Samp1 binds directly to Ran and that Samp1 binds better to RanGTP compared to RanGDP. Samp1 also preferred RanGTP over RanGDP in living tsBN2 cells. We also show that the Ran binding domain is located between amino acids 75-135 in the nucleoplasmically exposed N-terminal tail of Samp1. This domain is unique for Samp1, without homology in any other proteins in fungi or metazoa. Samp1 is the first known transmembrane protein that binds to Ran and could provide a unique local binding site for RanGTP in the INM. Samp1 overexpression resulted in increased Ran concentrations in the nuclear periphery supporting this idea.

Keyword
EDMD, laminopathies, LINC complex, nucleus, nuclear membrane, Ran
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-135087 (URN)10.1080/19491034.2016.1220465 (DOI)000384442800010 ()27541860 (PubMedID)
Available from: 2016-11-23 Created: 2016-10-31 Last updated: 2017-04-19Bibliographically approved
3. Mitotic spindle stability and correct chromosome segregation is dependent on an integral nuclear membrane protein, Samp1
Open this publication in new window or tab >>Mitotic spindle stability and correct chromosome segregation is dependent on an integral nuclear membrane protein, Samp1
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-141814 (URN)
Available from: 2017-04-18 Created: 2017-04-18 Last updated: 2017-04-27Bibliographically approved
4. An inner nuclear membrane protein induces rapid differentiation of human induced pluripotent stem cells
Open this publication in new window or tab >>An inner nuclear membrane protein induces rapid differentiation of human induced pluripotent stem cells
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(English)Manuscript (preprint) (Other academic)
National Category
Other Chemistry Topics Cell Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-135805 (URN)
Funder
Swedish Research CouncilSwedish Cancer Society
Available from: 2016-11-23 Created: 2016-11-23 Last updated: 2017-04-27Bibliographically approved
5. Spindle associated membrane protein 1 (Samp1) is required for the differentiation of muscle cells
Open this publication in new window or tab >>Spindle associated membrane protein 1 (Samp1) is required for the differentiation of muscle cells
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology Cell Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-141446 (URN)
Available from: 2017-04-04 Created: 2017-04-04 Last updated: 2017-04-27Bibliographically approved

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