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Dynamic properties of nuclear pore complex proteins in gp210 deficient cells
Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institute.
Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Stockholm University.
Södertörn University, School of Chemistry, Biology, Geography and Environmental Science.
2004 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 572, no 1-3, 261-265 p.Article in journal (Refereed) Published
Abstract [en]

Gp210, an integral membrane protein of the nuclear pore complex (NPC), is believed to be involved in NPC biogenesis. To test this hypothesis, we have investigated dynamic properties of the NPC and distribution of NPC proteins in NIH/ 3T3 cells lacking gp210. POM121 (the other integral NPC protein) and NUP107 (of the NUP107/160 complex) were correctly distributed at the nuclear pores in the absence of gp210. Furthermore, fluorescence recovery after photobleaching experiments showed that POM121 and NUP107 remained stably associated at the NPCs. We conclude that gp210 cannot be required for incorporation of POM121 or NUP107 or be required for maintaining NPC stability.

Place, publisher, year, edition, pages
2004. Vol. 572, no 1-3, 261-265 p.
National Category
Biochemistry and Molecular Biology Cell Biology
Identifiers
URN: urn:nbn:se:sh:diva-15469DOI: 10.1016/j.febslet.2004.07.044ISI: 000223519300047PubMedID: 15304359Scopus ID: 2-s2.0-4143137715OAI: oai:DiVA.org:sh-15469DiVA: diva2:504682
Available from: 2012-02-21 Created: 2012-02-20 Last updated: 2017-02-13Bibliographically approved
In thesis
1. Dynamic protein trafficking of the nuclear membrane and in peroxisomes
Open this publication in new window or tab >>Dynamic protein trafficking of the nuclear membrane and in peroxisomes
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cell nucleus is enclosed by the nuclear envelope (NE), a double lipid membrane separating the nucleoplasm from the cytoplasm. Transport of macromolecules between the nucleus and the cytoplasm takes places through nuclear pore complexes (NPCs) in a selective and energy dependent manner. The inner nuclear membrane (INM) contains transmembrane proteins that interact with the nuclear lamina and chromatin. In addition to being a barrier between the nucleoplasm and cytoplasm, an emerging view is that the NE has an active role in chromatin organization and gene regulation.

In order to study structural and functional organization of the NE in live cells, we have used green fluorescent protein (GFP)-labeled proteins and laser scanning confocal microscopy (LSCM). In order to investigate dynamic properties of specific proteins or protein complexes we have used photobleaching techniques. In order to understand the organization of the NPC it is essential to study components necessary for NPC biogenesis and maintenance. We have investigated the possible alterations in the NPC in cells naturally lacking one of the integral membrane proteins of the NPC, gp210. Despite the lack of gp210, we observed no difference in distribution or density of pores. Neither did cell cycle progression nor generation time differ between cells having or lacking gp210. In addition, targeting or dynamic properties of the NPC proteins POM121, Nup107 or Nup153 were unaltered in the absence of gp210. We conclude that gp210 can not be essential for NPC biogenesis or maintaining stability of the NPC.

The steps involved in onset of nuclear apoptosis are unclear. We studied nuclear alterations during apoptosis. We show that the nucleocytoplasmic barrier is disrupted early in apoptosis at the same time as chromatin collapses against the nuclear periphery but prior to nucleosomal DNA fragmentation. In addition, the disruption of nucleocytoplasmic transport correlates with caspase-3 dependent cleavage of POM121 at aspartate-531.

The INM is estimated to contain ~70 uncharacterized transmembrane proteins. We characterized a novel putative mammalian NE protein that we termed Samp1. We show that Samp1 is an integral membrane protein specifically localized to the inner nuclear membrane during interphase. Interestingly, during mitosis a sub fraction of Samp1 distributed in the polar region of the mitotic spindle, colocalizing with tubulin and a lipid marker. However, another inner nuclear membrane protein, emerin, was excluded from this area. Thus Samp1 appears to define a specific membrane domain associated with the mitotic machinery.

The distribution of peroxisomal fatty acid metabolizing enzymes have been reported to vary in different tissues. We investigated whether photobleaching techniques could be used to study the distribution of peroxisomal matrix proteins. We used GFP-labeled peroxisomal proteins and fluorescence recovery after photobleaching to show that peroxisomal matrix proteins become “trapped” inside peroxisomes after import. Thus we conclude that fluorescence loss in photobleaching can be used to distinguish between a strictly cytoplasmic localization and a dual localization when a protein is present both in the cytoplasm and in peroxisomes. Using this technique we determined that GFP-BAAT (bile acid-CoA:amino acid N-acyltransferase) is exclusively localized to the cytoplasm in HeLa cells.

Place, publisher, year, edition, pages
Stockholm: Karolinska Institutet, 2009. 46 p.
National Category
Biological Sciences
Identifiers
urn:nbn:se:sh:diva-31232 (URN)978-91-7409-334-6 (ISBN)
Public defence
2009-02-20, MB503, Alfred Nobels allé 7, Huddinge, 10:00 (English)
Opponent
Supervisors
Available from: 2016-11-28 Created: 2016-11-28 Last updated: 2016-11-28Bibliographically approved
2. Dynamic aspects of nucleocytoplasmic trafficking
Open this publication in new window or tab >>Dynamic aspects of nucleocytoplasmic trafficking
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cellular structures and compartmentalization is the result of a dynamic steady state exchange between its components. This thesis is focused in investigations of dynamic properties of green fluorescent protein (GFP)-labeled proteins in live cells using confocal laser microscopy in combination with bleaching techniques such as fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP).

Studies of dynamic properties of c-Myc in living cells showed that c-Myc is shuttling between the nucleus and the cytoplasm. c-Myc also enters the nucleoli during certain conditions. Nucleolar c-Myc is dynamically associated to structural component(s) of nucleoli, but can exchange with soluble pools in the nucleoplasm and cytoplasm.

Photobleaching experiments showed that a significant fraction of HIV-1 Vpr is dynamically associated with the NE and rapidly exchanges between the nucleoplasm and the cytoplasm. The yeast two-hybrid system, pull-down experiments and co-immunoprecipitating was used to show that Vpr interacts specifically and directly with a domain in the N-terminal portion of the NPC protein hCG1. The results suggest that the specific interaction of HIV-1 Vpr with the nucleoporin hCG1 results in the dynamic retention of Vpr at the nuclear envelope.

The distribution and dynamic properties of NPC proteins was investigated in NIH/3T3 cells, lacking the pore membrane protein gp210. Confocal laser scanning microscopy and FRAP experiments showed that the absence of gp210 from nuclear pores of NIH/3T3 cells did neither alter the distribution nor dynamic properties of POM121 and NUP107 (two NPC proteins stably integrated in the NPC).

Degradation of the integral nuclear pore membrane protein POM121 during apoptosis was investigated in relation to other apoptotic events. POM121 cleavage, which is the earliest sign of dismantling of the nuclear membrane, is due to caspase-3-dependent cleavage at aspartate-531. Loss of nuclear compartmentalization in live cells undergoing apoptosis was monitored as appearance of GFP-NLS in the cytoplasm. The time of appearance of cytoplasmic GFP-NLS correlated with caspase-3-dependent cleavage of POM121. Both events occured concomitantly with collapsing of chromatin against the nuclear periphery, but preceded the onset of nucleosomal DNA fragmentation.

Translocation ability of the cell-penetrating peptide, transportan, into living cells was investigated. Recombinantly expressed GFP was purified and conjugated to chemically synthesized transportan via a disulfide bond and added to tissues culture cells. Transportan was able to internalize a 27 kDa protein such as GFP in a native folded state into living cells.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, Stockholms universitet, 2004. 41 p.
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:sh:diva-32045 (URN)91-7265-803-7 (ISBN)
Public defence
2004-05-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2017-02-13 Created: 2017-02-13 Last updated: 2017-02-13Bibliographically approved

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