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Kihlmark, Madeleine
Publications (8 of 8) Show all publications
Kihlmark, M., Rustum, C., Eriksson, C., Beckman, M., Iverfeldt, K. & Hallberg, E. (2004). Correlation between nucleocytoplasmic transport and caspase-3-dependent dismantling of nuclear pores during apoptosis. Experimental Cell Research, 293(2), 346-356
Open this publication in new window or tab >>Correlation between nucleocytoplasmic transport and caspase-3-dependent dismantling of nuclear pores during apoptosis
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2004 (English)In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 293, no 2, p. 346-356Article 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.

National Category
Cell Biology
Identifiers
urn:nbn:se:sh:diva-15492 (URN)10.1016/j.yexcr.2003.10.019 (DOI)000188462700017 ()14729472 (PubMedID)2-s2.0-0347722245 (Scopus ID)
Available from: 2012-02-21 Created: 2012-02-20 Last updated: 2017-12-07Bibliographically approved
Beckman, M., Kihlmark, M., Iverfeldt, K. & Hallberg, E. (2004). Degradation of GFP-labelled POM121, a non-invasive sensor of nuclear apoptosis, precedes clustering of nuclear pores and externalisation of phosphatidylserine. Apoptosis (London), 9(3), 363-368
Open this publication in new window or tab >>Degradation of GFP-labelled POM121, a non-invasive sensor of nuclear apoptosis, precedes clustering of nuclear pores and externalisation of phosphatidylserine
2004 (English)In: Apoptosis (London), ISSN 1360-8185, E-ISSN 1573-675X, Vol. 9, no 3, p. 363-368Article in journal (Refereed) Published
Abstract [en]

The nuclear pore membrane protein POM121 is specifically degraded during apoptosis by a caspase-3-dependent process enabling early detection of apoptosis in living cells expressing POM121-GFP. Here we further investigated temporal aspects of apoptotic degradation of POM121-GFP. We demonstrate that decreased POM121-GFP fluorescence precedes annexin V-labelling of apoptotic cells. This indicates that degradation of the nuclear pore complex starts prior to redistribution of plasma membrane phosphatidylserine, which serves as a signal for phagocytotic elimination of apoptotic cells. Furthermore, a caspase-resistant GFP-labelled mutant of POM121 resisted degradation even in late apoptosis and was detected in clustered nuclear pores. Thus, it can be concluded that loss of POM121-GFP is a specific sensor of the activation of caspase-3-dependent proteolysis at the nuclear pores.

National Category
Biochemistry and Molecular Biology Cell Biology
Identifiers
urn:nbn:se:sh:diva-15480 (URN)10.1023/B:APPT.0000025813.75258.b5 (DOI)000221102700012 ()15258468 (PubMedID)2-s2.0-3242805164 (Scopus ID)
Available from: 2012-02-21 Created: 2012-02-20 Last updated: 2017-12-07Bibliographically approved
Kihlmark, M. (2002). Targeting of a nascent integral membrane protein to the nuclear pores and its degradation during apoptosis. (Doctoral dissertation). Stockholm: Stockholms universitet
Open this publication in new window or tab >>Targeting of a nascent integral membrane protein to the nuclear pores and its degradation during apoptosis
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Place, publisher, year, edition, pages
Stockholm: Stockholms universitet, 2002. p. 46
National Category
Biological Sciences
Identifiers
urn:nbn:se:sh:diva-31521 (URN)91-7265-559-3 (ISBN)
Public defence
2002-12-13, Magnélisalen, Kemiska Övningslaboratoriet, Svante Arrenihusväg 12, Stockholm, 10:00
Opponent
Supervisors
Available from: 2016-12-22 Created: 2016-12-22 Last updated: 2016-12-22Bibliographically approved
Pooga, M., Kut, C., Kihlmark, M., Hällbrink, M., Fernaeus, S., Raid, R., . . . Langel, U. (2001). Cellular translocation of proteins by transportan. The FASEB Journal, 15(8), 1451
Open this publication in new window or tab >>Cellular translocation of proteins by transportan
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2001 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 15, no 8, p. 1451-Article in journal (Refereed) Published
Abstract [en]

Proteins with molecular masses ranging from 30 kDa. (green fluorescent protein, GFP) to 150 kDa (monoclonal and polyclonal antibodies) were coupled to the cellular translocating peptide transportan. We studied the ability of the resulting protein-peptide constructs to penetrate into Bowes melanoma, BRL, and COS-7 cells. After 0.5-3 h incubation with recombinant GFP coupled to transportan, most of the GFP fluorescence was found in intracellular membranes of BRL and COS-7 cells, which suggests that transportan could internalize covalently linked proteins of about 30 kDa in a folded state. Transportan could internalize covalently coupled molecules of even larger size; that is, avidin and antibodies, (up to 150 kDa). The covalent bond between the transport peptide and its cargo is not obligatory because streptavidin was translocated into the cells within 15 min as a noncovalent complex with biotinylated transportan. Inside the cells, the delivered streptavidin was first located mainly in close proximity to the plasma membrane and was later distributed to the perinuclear region. Most of the internalized streptavidin was confined to vesicular structures, but a significant fraction of the protein was distributed in the cytoplasm. Our data suggest that transportan can deliver proteins and other hydrophilic macromolecules into intact mammalian cells, and this finding demonstrates good potential as powerful cellular delivery vector for scientific and therapeutic purposes.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:sh:diva-15859 (URN)10.1096/fj.00-0780fje (DOI)000173705800015 ()
Available from: 2012-03-12 Created: 2012-03-09 Last updated: 2017-12-07Bibliographically approved
Kihlmark, M., Imreh, G. & Hallberg, E. (2001). Sequential degradation of proteins from the nuclear envelope during apoptosis. Journal of Cell Science, 114(20), 3643-3653
Open this publication in new window or tab >>Sequential degradation of proteins from the nuclear envelope during apoptosis
2001 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 114, no 20, p. 3643-3653Article in journal (Refereed) Published
Abstract [en]

We have produced new antibodies specific for the integral pore membrane protein POM121. Using these antibodies we show that during apoptosis POM121 becomes proteolytically degraded in a caspase-dependent manner. The POM121 antibodies and antibodies specific for other proteins of the nuclear envelope were used in a comparative study of nuclear apoptosis in staurosporine-treated buffalo rat liver cells. Nuclei from these cells were classified in three different stages of apoptotic progression: stage I, moderately condensed chromatin surrounded by a smooth nuclear periphery; stage II, compact patches of condensed chromatin collapsing against a smooth nuclear periphery; stage III, round compact chromatin bodies surrounded by grape-shaped nuclear periphery. We have performed double labeling immunofluorescence microscopy of individual apoptotic cells and quantitative immunoblotting analysis of total proteins from apoptotic cell cultures. The results showed that degradation of nuclear envelope marker proteins occurred in a specific order. POM121 degradation occurred surprisingly early and was initiated before nucleosomal DNA degradation could be detected using TUNEL assay and completed before clustering of the nuclear pores. POM121 was eliminated significantly more rapid compared with NUP153 (a peripheral protein located in the nucleoplasmic basket of the nuclear pore complex) and lamin B (a component of the nuclear lamina). Disappearance of NUP153 and lamin B was coincident with onset of DNA fragmentation and clustering of nuclear pores. By contrast, the peripheral NPC protein p62 was degraded much later. The results suggest that degradation of POM121 may be an important early step in propagation of nuclear apoptosis.

National Category
Cell Biology
Identifiers
urn:nbn:se:sh:diva-15844 (URN)000171889300008 ()11707516 (PubMedID)2-s2.0-0034757949 (Scopus ID)
Available from: 2012-03-12 Created: 2012-03-09 Last updated: 2017-12-07Bibliographically approved
Imreh, G., Söderqvist, H., Kihlmark, M. & Hallberg, E. (1997). GFP as a marker for a nuclear pore complex protein. In: J.W. Hastings, L.J. kricka & P.E. Stanley (Ed.), Bioluminescence and chemiluminescence: proceedings of the 9th International Symposium on Bioluminescence and Chemiluminescence at Woods Hole, Massachusetts, October 1996. Paper presented at 9th International Symposium on Bioluminescence and Chemiluminescence at Woods Hole, Massachusetts, October 1996.. Sussex: John Wiley & Sons
Open this publication in new window or tab >>GFP as a marker for a nuclear pore complex protein
1997 (English)In: Bioluminescence and chemiluminescence: proceedings of the 9th International Symposium on Bioluminescence and Chemiluminescence at Woods Hole, Massachusetts, October 1996 / [ed] J.W. Hastings, L.J. kricka & P.E. Stanley, Sussex: John Wiley & Sons, 1997Conference paper, Published paper (Other academic)
Place, publisher, year, edition, pages
Sussex: John Wiley & Sons, 1997
National Category
Biological Sciences
Identifiers
urn:nbn:se:sh:diva-31516 (URN)0-471-97502-8 (ISBN)
Conference
9th International Symposium on Bioluminescence and Chemiluminescence at Woods Hole, Massachusetts, October 1996.
Available from: 2016-12-22 Created: 2016-12-22 Last updated: 2016-12-22Bibliographically approved
Söderqvist, H., Imreh, G., Kihlmark, M., Linnman, C., Ringertz, N. & Hallberg, E. (1997). Intracellular distribution of an integral nuclear pore membrane protein fused to green fluorescent protein - Localization of a targeting domain. European Journal of Biochemistry, 250(3), 808-813
Open this publication in new window or tab >>Intracellular distribution of an integral nuclear pore membrane protein fused to green fluorescent protein - Localization of a targeting domain
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1997 (English)In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 250, no 3, p. 808-813Article in journal (Refereed) Published
Abstract [en]

The 121-kDa pore membrane protein (POM121) is a bitopic integral membrane protein specifically located in the pore membrane domain of the nuclear envelope with its short N-terminal tail exposed on the luminal side and its major C-terminal portion adjoining the nuclear pore complex, In order to locate a signal for targeting of POM121 to the nuclear pores, we overexpressed selected regions of POM121 alone or fused to the green fluorescent protein (GFP) in transiently transfected COS-1 cells or in a stably transfected neuroblastoma cell line. Microscopic analysis of the GFP fluorescence or immunostaining was used to determine the intracellular distribution of the overexpressed proteins. The endofluorescent GFP tag had no effect on the distribution of POM121, since the chimerical POM121-GFP fusion protein was correctly targeted to the nuclear ports of both COS-1 cells and neuroblastoma cells. Based on the differentiated intracellular sorting of the POM121 variants, we conclude that the first 128 amino acids of POM121 contains signals for targeting to the continuous endoplasmic reticulum/nuclear envelope membrane system but not specifically to the nuclear pol es and that a specific nuclear pore targeting signal is located between amino acids 129 and 618 in the endoplasmically exposed portion of POM121.

National Category
Biological Sciences
Identifiers
urn:nbn:se:sh:diva-31518 (URN)10.1111/j.1432-1033.1997.00808.x (DOI)000071370300024 ()9461306 (PubMedID)
Available from: 2016-12-22 Created: 2016-12-22 Last updated: 2017-11-29Bibliographically approved
Kihlmark, M., Rustum, C., Eriksson, C., Beckman, M., Iverfeldt, K. & Hallberg, E.Caspase-3 dependent cleavage of POM121 in relation to nuclear apoptosis.
Open this publication in new window or tab >>Caspase-3 dependent cleavage of POM121 in relation to nuclear apoptosis
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(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:sh:diva-31522 (URN)
Note

Som manuskript i avhandling. As manuscript in dissertation.

Available from: 2016-12-22 Created: 2016-12-22 Last updated: 2016-12-22Bibliographically approved
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