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MCLIP, an effective method to detect interactions of transmembrane proteins of the nuclear envelope in live cells
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.ORCID-id: 0000-0003-1287-0495
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.ORCID-id: 0000-0002-3481-1106
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.ORCID-id: 0000-0003-1476-6675
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
Vise andre og tillknytning
2014 (engelsk)Inngår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, nr 10, s. 2399-2403Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
2014. Vol. 1838, nr 10, s. 2399-2403
Emneord [en]
Samp1, Nuclear envelope, Nuclear membrane, Crosslinking, CoIP, Protein–protein interaction
HSV kategori
Forskningsprogram
neurokemi med molekylär neurobiologi
Identifikatorer
URN: urn:nbn:se:su:diva-109181DOI: 10.1016/j.bbamem.2014.06.008ISI: 000340975600005OAI: oai:DiVA.org:su-109181DiVA, id: diva2:763439
Forskningsfinansiär
Swedish Research Council, 621-2010-448Swedish Cancer Society, 110590Tilgjengelig fra: 2014-11-14 Laget: 2014-11-14 Sist oppdatert: 2018-03-20bibliografisk kontrollert
Inngår i avhandling
1. Nuclear envelope protein interaction studies
Åpne denne publikasjonen i ny fane eller vindu >>Nuclear envelope protein interaction studies
2014 (engelsk)Licentiatavhandling, med artikler (Annet vitenskapelig)
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. 

sted, utgiver, år, opplag, sider
Stockholm: Department of Neurochemistry, Stockholm University, 2014
Emneord
Samp1, Nuclear envelope, protein interactions, chemical crosslinking, proteomics
HSV kategori
Forskningsprogram
neurokemi med molekylär neurobiologi
Identifikatorer
urn:nbn:se:su:diva-109194 (URN)978-91-7649-041-9 (ISBN)
Presentation
2014-12-16, Heilbronnsalen, Svante Arrhenius väg 16 B, Stockholm, 12:15 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2014-11-21 Laget: 2014-11-14 Sist oppdatert: 2015-03-17bibliografisk kontrollert
2. Identification and characterization of nuclear envelope protein interactions
Åpne denne publikasjonen i ny fane eller vindu >>Identification and characterization of nuclear envelope protein interactions
2015 (engelsk)Licentiatavhandling, med artikler (Annet vitenskapelig)
Abstract [en]

The Nuclear envelope which surrounds the chromatin of eukaryotic cells contains more than a hundred transmembrane proteins. Mutations in some genes encoding nuclear envelope proteins give rise to human diseases including neurological disorders. The function of many nuclear envelope proteins is not well established. This is partly because nuclear envelope proteins and their interactions are difficult to study due to the inherent resistance to extraction of nuclear envelope proteins. We have developed a novel method called MCLIP, to identify interacting partners of nuclear envelope proteins in live cells. Using MCLIP, we found three new binding partners of the inner nuclear membrane protein Samp1: the intermediate filament protein Lamin B1, the LINC complex protein Sun1 and the G-protein Ran. Furthermore, using in vitro studies, we show that Samp1 binds both Emerin and Ran directly. We have also studied the interaction between Samp1 and Ran in detail.

sted, utgiver, år, opplag, sider
Stockholm: Department of Neurochemistry, Stockholm University, 2015. s. 48
Emneord
Samp1, MCLIP, Nuclear envelope, Ran, Emerin
HSV kategori
Forskningsprogram
neurokemi med molekylär neurobiologi
Identifikatorer
urn:nbn:se:su:diva-122052 (URN)978-91-7649-289-5 (ISBN)
Presentation
2015-11-04, Heilbronnsalen, C458, Svante Arrhenius väg 16 B, Stockholm, 13:00 (engelsk)
Opponent
Veileder
Forskningsfinansiär
Swedish Research Council, 621-2010-448Swedish Cancer Society, 110590Stiftelsen Olle Engkvist Byggmästare
Tilgjengelig fra: 2015-10-23 Laget: 2015-10-21 Sist oppdatert: 2018-01-11bibliografisk kontrollert
3. Multifaceted roles of the transmembrane nuclear envelope protein, Samp1
Åpne denne publikasjonen i ny fane eller vindu >>Multifaceted roles of the transmembrane nuclear envelope protein, Samp1
2017 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: Department of Neurochemistry, Stockholm University, 2017. s. 46
Emneord
Nuclear envelope, transmembrane protein interaction studies, cell differentiation, stem cells, myopathies
HSV kategori
Forskningsprogram
neurokemi med molekylär neurobiologi
Identifikatorer
urn:nbn:se:su:diva-141816 (URN)978-91-7649-577-3 (ISBN)978-91-7649-578-0 (ISBN)
Disputas
2017-05-31, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad

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.

Tilgjengelig fra: 2017-05-08 Laget: 2017-04-19 Sist oppdatert: 2018-06-19bibliografisk kontrollert
4. Identification and characterization of protein-protein interactions in the nuclear envelope
Åpne denne publikasjonen i ny fane eller vindu >>Identification and characterization of protein-protein interactions in the nuclear envelope
2017 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

The nuclear envelope forms the interface between the nucleus and the cytoplasm. The nuclear envelope consists of the two concentric lipid membranes, the nuclear pores and the nuclear lamina. The inner nuclear membrane contains hundreds of unique transmembrane proteins showing high tissue diversity. Mutations of some proteins in the nuclear envelope give rise to a broad spectrum of diseases called envelopathies or laminopathies. In this thesis, I aimed to study the functional organization of the nuclear envelope by identifying and characterizing interactions between the nuclear envelope proteins. For this, we developed a novel method called the Membrane Protein Crosslink Immuno-Precipitation, which enable identification of protein-protein interactions in the nuclear envelope in live cells. We identified several novel interactions of the inner nuclear membrane protein, Samp1, and studied the interaction between the Samp1 and the nuclear GTPase, Ran in detail. Samp1 can bind to Ran and is thus the first known transmembrane Ran binding protein and Samp1 might provide a local binding site for Ran in the inner nuclear membrane. We found that Samp1 also binds to the inner nuclear membrane protein, Emerin and Ran can regulate the Samp1-Emerin interaction in the nuclear envelope. During mitosis, Samp1 distributes in the mitotic spindle. Therefore, we investigated a possible functional role of Samp1 in the mitotic machinery. Samp1 depletion resulted in aneuploid phenotypes, metaphase prolongation and decreased distribution of γ-tubulin and β-tubulin in the mitotic spindle. We found that Samp1 can bind to γ-tubulin, which is essential for the microtubule nucleation and hence for the spindle stability. The new interesting features of Samp1 provide insights on the unforeseen functions of the nuclear envelope proteins.

sted, utgiver, år, opplag, sider
Stockholm: Department of Neurochemistry, Stockholm University, 2017
Emneord
Nuclear envelope, Samp1, Emerin, Ran and transmembrane proteins
HSV kategori
Forskningsprogram
neurokemi med molekylär neurobiologi
Identifikatorer
urn:nbn:se:su:diva-148432 (URN)978-91-7797-029-3 (ISBN)978-91-7797-030-9 (ISBN)
Disputas
2017-12-08, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad

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

Tilgjengelig fra: 2017-11-15 Laget: 2017-10-24 Sist oppdatert: 2019-12-09bibliografisk kontrollert
5. Characterization of the inner nuclear membrane protein Samp1, during interphase and mitosis
Åpne denne publikasjonen i ny fane eller vindu >>Characterization of the inner nuclear membrane protein Samp1, during interphase and mitosis
2018 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

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.

 

 

sted, utgiver, år, opplag, sider
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2018. s. 64
Emneord
Samp1, LINC complex, nuclear lamina, MCLIP, nuclear envelope, spindle assembly, spindle stability, k-fiber, gamma tubulin, Aurora B, Haus6, Augmin
HSV kategori
Forskningsprogram
neurokemi med molekylär neurobiologi
Identifikatorer
urn:nbn:se:su:diva-154093 (URN)978-91-7797-199-3 (ISBN)978-91-7797-200-6 (ISBN)
Disputas
2018-05-03, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Tilgjengelig fra: 2018-04-10 Laget: 2018-03-14 Sist oppdatert: 2018-04-04bibliografisk kontrollert

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