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Dynamic aspects of nucleocytoplasmic trafficking
Stockholm University, Faculty of Science, Department of Neurochemistry.
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 , 2004. , 41 p.
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-140ISBN: 91-7265-803-7 (print)OAI: oai:DiVA.org:su-140DiVA: diva2:190019
Public defence
2004-05-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2004-05-06 Created: 2004-05-06Bibliographically approved
List of papers
1. Accumulation of c-Myc and proteasomes at the nucleoli of cells containing elevated c-Myc protein levels
Open this publication in new window or tab >>Accumulation of c-Myc and proteasomes at the nucleoli of cells containing elevated c-Myc protein levels
2003 In: Journal of Cell Science, ISSN 0021-9533, Vol. 116, no 9, 1707-1717 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-23060 (URN)
Note
Part of urn:nbn:se:su:diva-140Available from: 2004-05-06 Created: 2004-05-06Bibliographically approved
2. Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1
Open this publication in new window or tab >>Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1
Show others...
2002 In: J. Biol. Chem., ISSN 0021-9258, Vol. 277, no 47, 45091-45098 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-23061 (URN)
Note
Part of urn:nbn:se:su:diva-140Available from: 2004-05-06 Created: 2004-05-06Bibliographically approved
3. Dynamic properties of pore complex proteins in gp210 deficient cells.
Open this publication in new window or tab >>Dynamic properties of pore complex proteins in gp210 deficient cells.
Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-23062 (URN)
Note
Part of urn:nbn:se:su:diva-140Available from: 2004-05-06 Created: 2004-05-06 Last updated: 2010-01-13Bibliographically approved
4. Correlation between nucleocytoplasmic transport and caspase–3-dependent dismantling of nuclear pores during apoptosis
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, 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.

Keyword
apoptosis, nucleocytoplasmic transport, nuclear membrane, nuclear pores
National Category
Cancer and Oncology Cell Biology
Identifiers
urn:nbn:se:su:diva-23063 (URN)10.1016/j.yexcr.2003.10.019 (DOI)000188462700017 ()
Available from: 2004-05-06 Created: 2004-05-06 Last updated: 2017-12-13Bibliographically approved
5. Cellular translocation of proteins by transportan
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 6, 1451-1453 p.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.

Keyword
peptide-mediated transport, cell-penetrating peptides, drug delivery, transport vectors, protein transduction
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-23064 (URN)10.1096/fj.00-0780fje (DOI)000173705800015 ()
Available from: 2004-05-06 Created: 2004-05-06 Last updated: 2017-12-13Bibliographically approved

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