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Correlation between nucleocytoplasmic transport and caspase-3-dependent dismantling of nuclear pores during apoptosis
Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Stockholm University.
Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Stockholm University.
Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institutet.
Stockholm University.
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2004 (English)In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 293, no 2, 346-356 p.Article in journal (Refereed) Published
Abstract [en]

During apoptosis (also called programmed cell death), the chromatin condenses and the DNA is cleaved into oligonucleosomal fragments. Caspases are believed to play a major role in nuclear apoptosis. However, the relation between dismantling of nuclear pores, disruption of the nucleocytoplasmic barrier, and nuclear entry of caspases is unclear. We have analyzed nuclear import of the green fluorescent protein fused to a nuclear localization signal (GFP-NLS) in tissue culture cells undergoing apoptosis. Decreased nuclear accumulation of GFP-NLS could be detected at the onset of nuclear apoptosis manifested as dramatic condensation and redistribution of chromatin toward the nuclear periphery. At this step, dismantling of nuclear pores was already evident as indicated by proteolysis of the nuclear pore membrane protein POM121. Thus, disruption of nuclear compartmentalization correlated with early signs of nuclear pore damage. Both these events clearly preceded massive DNA fragmentation, detected by TUNEL assay. Furthermore, we show that in apoptotic cells, POM121 is specifically cleaved at aspartate-531 in its large C-terminal portion by a caspase-3-dependent mechanism. Cleavage of the C-terminal portion of POM121, which is adjoining the nuclear pore complex, is likely to disrupt interactions with other nuclear pore proteins affecting the stability of the pore complex. A temporal correlation of apoptotic events supports a model where caspase-dependent disassembly of nuclear pores and disruption of the nucleocytoplasmic barrier paves the way for nuclear entry of caspases and subsequent activation of CAD-mediated DNA fragmentation.

Place, publisher, year, edition, pages
2004. Vol. 293, no 2, 346-356 p.
National Category
Cell Biology
URN: urn:nbn:se:sh:diva-15492DOI: 10.1016/j.yexcr.2003.10.019ISI: 000188462700017PubMedID: 14729472ScopusID: 2-s2.0-0347722245OAI: diva2:504561
Available from: 2012-02-21 Created: 2012-02-20 Last updated: 2016-11-28Bibliographically 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
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)
Available from: 2016-11-28 Created: 2016-11-28 Last updated: 2016-11-28Bibliographically approved

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