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  • 1.
    Arabi, Azadeh
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Rustum, Cecilia
    Södertörn University, Avdelning Naturvetenskap. Stockholm University.
    Hallberg, Einar
    Södertörn University, Avdelning Naturvetenskap.
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Accumulation of c-Myc and proteasomes at the nucleoli of cells containing elevated c-Myc protein levels2003In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 116, no 9, p. 1707-1717Article in journal (Refereed)
    Abstract [en]

    c-Myc is a predominately nuclear transcription factor that is a substrate for rapid turnover by the proteasome system. Cancer-related mutations in c-Myc lead to defects in its degradation and thereby contribute to the increase in its cellular level that is associated with the disease. Little is known about the mechanisms that target c-Myc to the proteasomes. By using a GFP fusion protein and live analysis we show that c-Myc shuttles between the nucleus and cytoplasm and thus it could be degraded in either compartment. Strikingly, at elevated levels of expression c-Myc accumulates at nucleoli in some cells, consistent with saturation of a nucleolus-associated degradation system in these cells. This idea is further supported by the observation that proteasome inhibitor treatment causes accumulation of c-Myc at the nucleoli of essentially all cells. Under these conditions c-Myc is relatively stably associated with the nucleolus, as would be expected if the nucleolus functions as a sequestration/degradation site for excess c-Myc. Furthermore, during elevated c-Myc expression or proteasome inhibition, nucleoli that are associated with c-Myc also accumulate proteasomes. c-Myc and proteasomes co-localise in intranucleolar regions distinct from the dense fibrillar component of the nucleolus. Based on these results we propose a model for c-Myc downregulation where c-Myc is sequestered at the nucleoli. Sequestration of c-Myc is accompanied by recruitment of proteasomes and may lead to subsequent degradation.

  • 2. Beckman, M
    et al.
    Kihlmark, Madeleine
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science.
    Iverfeldt, K
    Hallberg, Einar
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science.
    Degradation of GFP-labelled POM121, a non-invasive sensor of nuclear apoptosis, precedes clustering of nuclear pores and externalisation of phosphatidylserine2004In: Apoptosis (London), ISSN 1360-8185, E-ISSN 1573-675X, Vol. 9, no 3, p. 363-368Article in journal (Refereed)
    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.

  • 3.
    Buch, Charlotta
    et al.
    Södertörn University, School of Life Sciences.
    Hunt, Mary C.
    Alexson, Stefan E. H.
    Hallberg, Einar
    Södertörn University, School of Life Sciences.
    Localization of peroxisomal matrix proteins by photobleaching2009In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 388, no 2, p. 355-359Article in journal (Refereed)
    Abstract [en]

    The distribution of some enzymes between peroxisomes and cytosol, or a dual localization in both these compartments, can be difficult to reconcile. We have used photobleaching in live cells expressing green fluorescent protein (GFP)-fusion proteins to show that imported bona fide peroxisomal matrix proteins are retained in the peroxisome. The high mobility of the GFP-fusion proteins in the cytosol and absence of peroxisomal escape makes it possible to eliminate the cytosolic fluorescence by photobleaching, to distinguish between exclusively cytosolic proteins and proteins that are also present at low levels in peroxisomes. Using this technique we found that GFP tagged bile acid-CoA:amino acid N-acyltransferase (BAAT) was exclusively localized in the cytosol in HeLa cells. We conclude that the cytosolic localization was due to its carboxyterminal non-consensus peroxisomal targeting signal (-SQL) since mutation of the -SQL to -SKL resulted in BAAT being efficiently imported into peroxisomes.

  • 4.
    Buch, Charlotta
    et al.
    Södertörn University, School of Life Sciences.
    Lindberg, Robert
    Södertörn University, School of Life Sciences.
    Figueroa, Ricardo
    Södertörn University, School of Life Sciences.
    Gudise, Santhosh
    Södertörn University, School of Life Sciences.
    Onischenko, Evgeny
    Hallberg, Einar
    Södertörn University, School of Life Sciences.
    An integral protein of the inner nuclear membrane localizes to the mitotic spindle in mammalian cells2009In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 122, no 12, p. 2100-2107Article in journal (Refereed)
    Abstract [en]

    Here, we characterize a transmembrane protein of the nuclear envelope that we name spindle-associated membrane protein 1 (Samp1). The protein is conserved in metazoa and fission yeast and is homologous to Net5 in rat and Ima1 in Schizosaccharomyces pombe. We show that, in human cells, the protein is a membrane-spanning polypeptide with an apparent molecular mass of 43 kDa. This is consistent with a predicted polypeptide of 392 amino acids that has five transmembrane segments and its C-terminus exposed to the nucleoplasm. During interphase, Samp1 was specifically distributed in the inner nuclear membrane. Post-transcriptional silencing of Samp1 expression resulted in separation of centrosomes from the nuclear envelope, indicating that it is functionally connected to the cytoskeleton. At the onset of mitosis, most of the protein dispersed out into the ER, as expected. However, during mitosis, a significant fraction of the protein specifically localized to the polar regions of the mitotic spindle. We demonstrate for the first time, in human cells, the existence of a membranous structure overlapping with the mitotic spindle. Interestingly, another integral inner nuclear membrane protein, emerin, was absent from the spindle-associated membranes. Thus, Samp1 defines a specific membrane domain associated with the mitotic spindle.

  • 5.
    Daigle, N
    et al.
    European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
    Beaudouin, J
    European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
    Hartnell, L
    NIH Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD), National Institutes of Health (NIH) - USA.
    Imreh, Gabriela
    Södertörn University, Avdelning Naturvetenskap.
    Hallberg, Einar
    Södertörn University, Avdelning Naturvetenskap.
    Lippincott-Schwartz, J
    NIH Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD), National Institutes of Health (NIH) - USA.
    Ellenberg, J
    European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
    Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells2001In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 154, no 1, p. 71-84Article in journal (Refereed)
    Abstract [en]

    The nuclear pore complex (NPC) and its relationship to the nuclear envelope (NE) was characterized in living cells using POM121-green fluorescent protein (GFP) and GFP-Nup153, and GFP-lamin B1. No independent movement of single pore complexes was found within the plane of the NE in interphase. Only large arrays of NPCs moved slowly and synchronously during global changes in nuclear shape, strongly suggesting mechanical connections which form an NPC network. The nuclear lamina exhibited identical movements. NPC turnover measured by fluorescence recovery after photobleaching of POM121 was less than once per cell cycle. Nup153 association with NPCs was dynamic and turnover of this nucleoporin was three orders of magnitude faster. Overexpression of both nucleoporins induced the formation of annulate lamellae (AL) in the endoplasmic reticulum (ER). Turnover of AL pore complexes was much higher than in the NE (once every 2.5 min). During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase. Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

  • 6.
    Eriksson, Charlotta
    et al.
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institute.
    Rustum, Cecilia
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Stockholm University.
    Hallberg, Einar
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science.
    Dynamic properties of nuclear pore complex proteins in gp210 deficient cells2004In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 572, no 1-3, p. 261-265Article in journal (Refereed)
    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.

  • 7.
    Figueroa, Ricardo
    et al.
    Södertörn University, School of Life Sciences, Molecular biology. Karloinska institutet.
    Gudise, Santhosh
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska institutet.
    Larsson, Veronica
    Karloinska institutet.
    Hallberg, Einar
    Södertörn University, School of Life Sciences, Molecular biology.
    A transmembrane inner nuclear membrane protein in the mitotic spindle2010In: Nucleus, ISSN 1949-1042, Vol. 1, no 3, p. 249-253Article in journal (Refereed)
    Abstract [en]

    We have recently characterized a novel transmembrane protein of the inner nuclear membrane of mammalian cells. The protein has two very interesting features. First, despite being an integral membrane protein it is able to concentrate in the membranes colocalizing with the mitotic spindle in metaphase and anaphase. Hence, the protein was named Samp1, Spindle associated membrane protein 1. Secondly, it displays a functional connection to centrosomes. This article discusses various aspects of Samp1 in relation to possible cellular function(s).

  • 8. Fisher, Linda
    et al.
    Samuelsson, Malin
    Jiang, Yang
    Ramberg, Veronica
    Figueroa, Ricardo
    Södertörn University, School of Life Sciences.
    Hallberg, Einar
    Södertörn University, School of Life Sciences.
    Langel, Ulo
    Iverfeldt, Kerstin
    Targeting cytokine expression in glial cells by cellular delivery of an NF-kappa B decoy2007In: Journal of Molecular Neuroscience, ISSN 0895-8696, E-ISSN 1559-1166, Vol. 31, no 3, p. 209-219Article in journal (Refereed)
    Abstract [en]

    Inhibition of nuclear factor (NF)-kappa B has emerged as an important strategy for design of anti-inflammatory therapies. In neurodegenerative disorders like Alzheimer's disease, inflammatory reactions mediated by glial cells are believed to promote disease progression. Here, we report that uptake of a double-stranded oligonucleotide NF-kappa B decoy in rat primary glial cells is clearly facilitated by noncovalent binding to a cell-penetrating peptide, transportan 10, via a complementary peptide nucleic acid (PNA) sequence. Fluorescently labeled oligonucleotide decoy was detected in the cells within 1 h only when cells were incubated with the decoy in the presence of cell-penetrating peptide. Cellular delivery of the decoy also inhibited effects induced by a neurotoxic fragment of the Alzheimer beta-amyloid peptide in the presence of the inflammatory cytokine interleukin (IL)-1 beta. Pretreatment of the cells with the complex formed by the decoy and the cell-penetrating peptide-PNA resulted in 80% and 50% inhibition of the NF-kappa B binding activity and IL-6 mRNA expression, respectively.

  • 9.
    Gatsinzi, Tom
    et al.
    Stockholm University.
    Ramberg, Veronica
    Stockholm University.
    Figueroa, Ricardo
    Södertörn University, School of Life Sciences, Molecular biology.
    Iverfeldt, Kerstin
    Stockholm University.
    Hallberg, Einar
    Södertörn University, School of Life Sciences, Molecular biology.
    Localized caspase sensors for live cell imaging of amyloid-β induced apoptosis2010In: Alzheimer's & Dementia, ISSN 1552-5260, E-ISSN 1552-5279, Vol. 6, no 4, Supplement, p. S259-S260Article in journal (Other academic)
    Abstract [en]

    Background: Apoptosis is an evolutionary conserved cellular process important for normal development, maintenance of tissue homeostasis and an effective immune system. Cysteine-aspartic proteases, or caspases, are the major mediators of apoptosis, triggering processes which lead to cellular disruption. Dysregulation of apoptotic signaling has been shown to be involved in several pathological conditions, like cancer and degenerative disorders. Alzheimer's disease (AD) is the most common form of dementia involving massive cell death of neurons. However, the cause of AD at the present time is still unknown, although, amyloid-β (Aβ) peptide has been suggested to be the triggering factor. 

    Methods:In order to detect localized caspase activation in live cells we designed sensors for caspase-3, -6 and -9 utilizing fluorescence resonance energy transfer (FRET). The FRET-ing sensor molecules, consisting of CFP and YFP separated by a linker containing a specific caspase cleavage motif, were designed to signal caspase cleavage by the loss of FRET. Differentiated SH-SY5Y cells were used as a model system for neurodegeneration. The cells were treated with oligomeric Aβ42 or staurosporine as a positive control of apoptosis. The cleavage of the sensors during induced apoptosis was verified by western blot analysis. Time-lapse FRET microscopy was used to monitor caspase activity in different parts of the cells. 

    Results: In our study, when the cells were exposed to staurosporine we were able to detect local activity of caspase-6 initially in the soma of the cells, whereas caspase-6 activity in the neurites was delayed. Furthermore, our study shows that oligomeric Aβ42 is able to activate caspase-3, -6 and -9. In contrast to staurosporine, in Aβ42 treated cells loss of FRET occurred globally indicating that caspase was activated simultaneously in soma and axons. 

    Conclusions: In conclusion, we show that our caspase-sensors are able to detect local caspase activity in vitro. We also show that exposure to oligomeric Aβ42 results in global activation of caspases in differentiated SH-SY5Y cells.

  • 10.
    Imreh, Gabriela
    et al.
    Stockhlms univeristet.
    Beckman, M.
    Stockholms universitet.
    Iverfeldt, K.
    Stockholms universitet.
    Hallberg, Einar
    Stockholms universitet.
    Noninvasive monitoring of apoptosis versus necrosis in a neuroblastoma cell line expressing a nuclear pore protein tagged with the green fluorescent protein1998In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 238, no 2, p. 371-376Article in journal (Refereed)
    Abstract [en]

    A fusion chimera between the integral nuclear pore membrane protein POM121 and GFP (green fluorescent protein) has been shown to correctly target to the nuclear pores when transiently expressed in a number of mammalian cell types. POM121-GFP is therefore an excellent marker for the noninvasive studies of the nuclear pores in living cells using fluorescence microscopy. We have established a line of neuroblastoma cells stably expressing the POM121-GFP fusion protein. We also monitored the nuclear envelope in living cells after induction of apoptosis or necrosis using 1 μM staurosporine or 100 μM p-benzoquinone, respectively. Interestingly, the POM121-GFP fluorescence was weaker or missing in the apoptotic cells. The disappearance of the nuclear pore marker accompanied apoptotic progression as judged by the degree of chromatin condensation and DNA fragmentation as analyzed by DNA staining and TUNEL assay, respectively. In contrast, the intensity of the nuclear rim fluorescence was unaffected in necrotic cells displaying an abnormal morphology with tilted nuclei. Thus, it was possible to distinguish between apoptotic and necrotic development in living cells using fluorescence microscopy. This cell line provides a fast and convenient model for screening suspected toxic xenobiotics.

  • 11.
    Imreh, Gabriela
    et al.
    Södertörn University, Avdelning Naturvetenskap. Stockholms unviersitet.
    de Monvel, J. B
    Karolinska universitetssjukhuset.
    Branden, L.
    Karolinska institutet.
    Hallberg, Einar
    Södertörn University, Avdelning Naturvetenskap.
    ER retention may play a role in sorting of the nuclear pore membrane Protein POM121 towards the NPCManuscript (preprint) (Other academic)
  • 12.
    Imreh, Gabriela
    et al.
    Södertörn University, Avdelning Naturvetenskap. Stockholm University.
    Hallberg, Einar
    Södertörn University, Avdelning Naturvetenskap.
    An integral membrane protein from the nuclear pore complex is also present in the annulate lamellae: Implications for annulate lamella formation2000In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 259, no 1, p. 180-190Article in journal (Refereed)
    Abstract [en]

    Annulate lamellae (AL) are cytoplasmic arrays of stacked membrane cisternae containing densely packed pore complexes which are similar in structure to the nuclear pore complexes (NPCs) and thus referred to as annulate lamella pore complexes (ALPCs). We have recently shown that the integral nuclear pore membrane protein POM121 tagged with green fluorescent protein was correctly targeted to the nuclear pores (H. Soderqvist et al., 1997, fur. J. Biochem. 250, 808-813). Here we have investigated if POM121 fused to three tandem molecules of yellow fluorescent protein YFP) (POM121-YFP3,) also was able to distribute in the extensive and well-characterized Al; of RC37 and BMGE cells. Transfected RC37 or BMGE cells displayed YFP fluorescence around the nuclear envelope, as well. as in the cytoplasmic AL structures. The YFP fluorescence colocalized perfectly with immunostaining using antibodies specific for different NPC proteins. The AL of both transfected and untransfected BMGE cells resisted extractions with Tx-100 and 250 mM NaCl, but were completely solubilized at 450 mM NaCl. Loss of YFP fluorescence and immunostaining for other NPC proteins correlated under all extraction conditions tested, suggesting that overexpressed POM121-YFP3, had become an integrated part both of the NPCs and of the ALPCs. Furthermore, we have generated a stable BHK cell line expressing POM121YFP(3,) located exclusively at the nuclear pores. Treatment with vinblastine sulfate, which induces formation of Al; in a variety of cells, resulted in distribution of POM121-YFP3, into cytoplasmic foci colocalizing with immunostaining for peripheral NPC proteins. Taken together, the results show that YFP-tagged POM121 is able to distribute in drug-induced or naturally occurring AL, suggesting that POM121 is a natural constituent of ALPCs. In COS cells, which normally lack or have very little AT-I, YFP-tagged POM121 distributed in the nuclear pores when expressed at low levels. However, at high expression levels the YFP fluorescence also distributed in a number of brightly fluorescing cytoplasmic dots or foci, which were not present in untransfected cells. This was also true for untagged POM121. The cytoplasmic foci varied in size from 0.1 to 2 mu m and were distinctly located in the immediate vicinity of ER cisternae (without colocalizing) and also contained other nuclear pore proteins, indicating that they may represent cytoplasmic AL. This idea is supported by time-lapse studies of postmitotic assembly of these structures. This raises the question of the role of POM121 in ALPC and NPC biogenesis.

  • 13.
    Imreh, Gabriela
    et al.
    Södertörn University, Avdelning Naturvetenskap. Stockholm University.
    Maksel, Danuta
    Södertörn University, Avdelning Naturvetenskap.
    de Monvel, J B
    Branden, L
    Hallberg, Einar
    Södertörn University, Avdelning Naturvetenskap.
    ER retention may play a role in sorting of the nuclear pore membrane protein POM1212003In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 284, no 2, p. 173-184Article in journal (Refereed)
    Abstract [en]

    Integral membrane proteins of the nuclear envelope (NE) are synthesized on the rough endoplasmic reticulum (ER) and following free diffusion in the continuous ER/NE membrane system are targeted to their proper destinations due to interactions of specific domains with other components of the NE. By studying the intracellular distribution and dynamics of a deletion mutant of an integral membrane protein of the nuclear pores, POM121, which lacks the pore-targeting domain, we investigated if ER retention plays a role in sorting of integral membrane proteins to the nuclear envelope. A nascent membrane protein lacking sorting determinants is believed to diffuse laterally in the continuous ER/NE lipid bilayer and expected to follow vesicular traffic to the plasma membrane. The GFP-tagged deletion mutant, POM121(1-129)-GFP, specifically distributed within the ER membrane, but was completely absent from the Golgi compartment and the plasma membrane. Experiments using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) demonstrated that despite having very high mobility within the whole ER network (D = 0.41 +/- 0.11 mum(2)/s) POM121(1-129)-GFP was unable to exit the ER. It was also not detected in post-ER compartments of cells incubated at 15degreesC. Taken together, these experiments show that amino acids 1-129 of POM121 are able to retain GFP in the ER membrane and suggest that this retention occurs by a direct mechanism rather than by a retrieval mechanism. Our data suggest that ER retention might be important for sorting of POM121 to the nuclear pores.

  • 14.
    Imreh, Gabriela
    et al.
    Stockholms unviersitet.
    Söderqvist, H
    Stockholms universitet.
    Kihlmark, Madeleine
    Hallberg, Einar
    Stockholms universitet.
    GFP as a marker for a nuclear pore complex protein1997In: 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 (Other academic)
  • 15.
    Kihlmark, Madeleine
    et al.
    Södertörn University, Avdelning Naturvetenskap.
    Imreh, Gabriela
    Södertörn University, Avdelning Naturvetenskap.
    Hallberg, Einar
    Södertörn University, Avdelning Naturvetenskap.
    Sequential degradation of proteins from the nuclear envelope during apoptosis2001In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 114, no 20, p. 3643-3653Article in journal (Refereed)
    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.

  • 16.
    Kihlmark, Madeleine
    et al.
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Stockholm University.
    Rustum, Cecilia
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Stockholm University.
    Eriksson, Charlotta
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institutet.
    Beckman, M
    Stockholm University.
    Iverfeldt, K
    Stockholm University.
    Hallberg, Einar
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science.
    Correlation between nucleocytoplasmic transport and caspase-3-dependent dismantling of nuclear pores during apoptosis2004In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 293, no 2, p. 346-356Article in journal (Refereed)
    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.

  • 17.
    Kihlmark, Madeleine
    et al.
    Södertörn University, Avdelning Naturvetenskap. Stockholms unviersitet.
    Rustum, Cecilia
    Södertörn University, Avdelning Naturvetenskap. Stockholms universitet.
    Eriksson, Charlotta
    Södertörn University, Avdelning Naturvetenskap. Karolinska institutet.
    Beckman, M
    Stockholms universitet.
    Iverfeldt, Kerstin
    Stockholms universitet.
    Hallberg, Einar
    Södertörn University, Avdelning Naturvetenskap.
    Caspase-3 dependent cleavage of POM121 in relation to nuclear apoptosisManuscript (preprint) (Other academic)
  • 18.
    Le Rouzic, E
    et al.
    Université Paris 5, Paris, France.
    Mousnier, A
    Université Paris VI and Université Paris VII, Paris, France.
    Rustum, Cecilia
    Södertörn University, Avdelning Naturvetenskap. Stockholm University.
    Stutz, F
    Université Paris 5, Paris, France.
    Hallberg, Einar
    Stockholm University.
    Dargemont, C
    Université Paris VI and Université Paris VII, Paris, France.
    Benichou, S
    Université Paris 5, Paris, France.
    Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG12002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 47, p. 45091-45098Article in journal (Refereed)
    Abstract [en]

    The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast two-hybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.

  • 19.
    Olsson, Veronica
    et al.
    Stockholm University.
    Samuelsson, Malin
    Stockholm University.
    Figueroa, Ricardo
    Södertörn University, School of Life Sciences, Molecular biology.
    Zhang, Mu
    Stockholm University.
    Hallberg, Einar
    Södertörn University, School of Life Sciences, Molecular biology.
    Iverfeldt, Kerstin
    Stockholm University.
    Analysis of apoptotic processes in live cells2006In: Alzheimer's & Dementia, ISSN 1552-5260, E-ISSN 1552-5279, Vol. 2, no 3, Supplement, p. S439-S440Article in journal (Other academic)
    Abstract [en]

    Background: Neuronal and synaptic loss can be observed in several neurologic disorders, like Alzheimer’s disease (AD). The mechanism behind cell death in AD has been intensively studied and apoptosis has been proposed to play a central role in death processes, primary affecting cholinergic neurons in the cerebral cortex and the limbic lobe. There are numerous potential death stimuli that may be relevant in AD, including inflammatory responses, growth factor deprivation, oxidative stress and direct effects of the β- amyloid peptide. Objective: In order to get further insights in the initiation of apoptotic processes, we have developed a set of caspase sensors. 

    Methods: We have used fluorescence resonance energy transfer (FRET) technology to, in real time and at single cell level, monitor the crucial event of the activation cysteine aspartate proteases, central in apoptosis. The two chromophores ECFP and EYFP, separated by a caspase cleavage site, have been used to visualize the caspase cleavage event at a chosen subcellular location in different cellular models, including differentiated neuronal cells. Since several apoptotic signalling pathways may be involved, we have designed sensors that can be cleaved by caspase-3, -8 or -9, representing two possible pathways, the death receptor pathway and the mitochondrial pathway. The in vitromodel used initially to characterize the caspase sensors has been HeLa cells, stimulated with staurosporin. The condition of the cells and the different stages of apoptosis were identified by nuclear staining with Hoechst 33258. 

    Results: Our preliminary data indicate that caspase cleavage is an early event in the apoptotic cascade initiated by staurosporin, and that it most likely begins central in the cell body as FRET signals can be detected at later stages only in the cell periphery. Over-expression of the sensors did not result in any detectable toxicity since cells were able to divide successfully and no morphological changes could be revealed. 

    Conclusion: Using this approach, a better temporal and spatial understanding of the apoptotic processes will be achieved. This is necessary in order to identify therapeutic targets to prevent the massive loss of neurons in AD and related disorders.

  • 20.
    Onischenko, Evgeny A.
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Crafoord, Ellinor
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Hallberg, Einar
    Södertörn University, School of Life Sciences.
    Phosphomimetic mutation of the mitotically phosphorylated serine 1880 compromises the interaction of the transmembrane nucleoporin gp210 with the nuclear pore complex2007In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 313, no 12, p. 2744-2751Article in journal (Refereed)
    Abstract [en]

    The nuclear pore complexes (NPCs) reversibly disassemble and reassemble during mitosis. Disassembly of the NPC is accompanied by phosphorylation of many nucleoporins although the function of this is not clear. It was previously shown that in the transmembrane nucleoporin gp210 a single serine residue at position 1880 is specifically phosphorylated during mitosis. Using amino acid substitution combined with live cell imaging, time-lapse microscopy and FRAP, we investigated the role of serine 1880 in binding of gp210 to the NPC in vivo An alanine subtitutions mutant (S1880A) was significantly more dynamic at the NPC compared to the wild-type protein, suggesting that serine 1880 is important for binding of gp210 to the NPC. Moreover a glutamate substitution (S1880E) closely mimicking phosphorylated serine specifically interfered with incorporation of gp210 into the NPC and compromised its post-mitotic recruitment to the nuclear envelope of daughter nuclei. our findings are consistent with the idea that mitotic phosphorylation acts to dissociate gp210 from the structural elements of the NPC.

  • 21.
    Onischenko, Evgeny A
    et al.
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institute.
    Gubanova, N V
    Institute of Cytology and Genetics, Novosibirsk, Russia.
    Kieselbach, T
    Karolinska Institute.
    Kiseleva, E V
    Institute of Cytology and Genetics, Novosibirsk, Russia.
    Hallberg, Einar
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science.
    Annulate lamellae play only a minor role in the storage of excess nucleoporins in Drosophila embryos2004In: Traffic: the International Journal of Intracellular Transport, ISSN 1398-9219, E-ISSN 1600-0854, Vol. 5, no 3, p. 152-164Article in journal (Refereed)
    Abstract [en]

    The nuclear pore complexes (NPCs), multiprotein assemblies embedded in the nuclear envelope, conduct nucleo-cytoplasmic traffic of macromolecules. Mimics of NPCs, called annulate lamellae pore complexes (ALPCs), are usually found in cytoplasmic membranous stacks in oocytes and early embryonic cells. They are believed to constitute storage compartments for excess premade nucleoporins. To evaluate the extent to which ALPCs store nucleoporins in early embryonic cells we took advantage of syncytial Drosophila embryos, containing both AL and rapidly proliferating nuclei in the common cytoplasm. Electron microscopic morphometric analysis showed that the number of ALPCs did not decrease to compensate for the growing number of NPCs during syncytial development. We performed Western blot analysis to quantify seven different nucleoporins and analyzed their intraembryonal distribution by confocal microscopy and subcellular fractionation. Syncytial embryos contained a large maternally contributed stockpile of nucleoporins. However, even during interphases, only a small fraction of the excess nucleoporins was assembled into ALPCs, whereas the major fraction was soluble and contained at least one phosphorylated nucleoporin. We conclude that in Drosophila embryos ALPCs play only a minor role in storing the excess maternally contributed nucleoporins. Factors that may prevent nucleoporins from assembly into ALPCs are discussed.

  • 22.
    Onischenko, Evgeny A
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Gubanova, N V
    Institute of Cytology and Genetics, Novosibirsk, Russia.
    Kiseleva, E V
    Institute of Cytology and Genetics, Novosibirsk, Russia.
    Hallberg, Einar
    Södertörn University, School of Life Sciences.
    Cdk1 and okadaic acid-sensitive phosphatases control assembly of nuclear pore complexes in Drosophila embryos2005In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 16, no 11, p. 5152-5162Article in journal (Refereed)
    Abstract [en]

    Disassembly and reassembly of the nuclear pore complexes (NPCs) is one of the major events during open mitosis in higher eukaryotes. However, how this process is controlled by the mitotic machinery is not clear. To investigate this we developed a novel in vivo model system based on syncytial Drosophila embryos. We microinjected different mitotic effectors into the embryonic cytoplasm and monitored the dynamics of disassembly/reassembly of NPCs in live embryos using fluorescently labeled wheat germ agglutinin (WGA) or in fixed embryos using electron microscopy and immunostaining techniques. We found that in live embryos Cdk1 activity was necessary and sufficient to induce disassembly of NPCs as well as their cytoplasmic mimics: annulate lamellae pore complexes (ALPCs). Cdk1 activity was also required for keeping NPCs and ALPCs disassembled during mitosis. In Agreement recombinant Cdk1/cyclin B was able to induce phosphorylation and dissociation of nucleoporins from the NPCs in vitro. Conversely, reassembly of NPCs and ALPCs was dependent on the activity of protein phosphatases, sensitive to okadaic acid (OA). Our findings suggest a model where mitotic disassembly/reassembly of the NPCs is regulated by a dynamic equilibrium of Cdk1 and OA-sensitive phosphatase activities and provide evidence that mitotic phosphorylation mediates disassembly of the NPC.

  • 23.
    Onischenko, Evgeny A.
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Gubanova, N. V.
    Institute of Cytology and Genetics, Novosibirsk, Russia.
    Kiseleva, E. V.
    Institute of Cytology and Genetics, Novosibirsk, Russia.
    Hallberg, Einar
    Södertörn University, School of Life Sciences.
    Differential requirement of RanGTP production for assembly of pore complexes in the nuclear envelope and annulate lamellaeManuscript (preprint) (Other academic)
  • 24.
    Pooga, M
    et al.
    Stockholms universitet / Estonian Biocentre, Tartu, Estonia.
    Kut, Cecilia
    Södertörn University, Avdelning Naturvetenskap. Stockholms universitet.
    Kihlmark, Madeleine
    Södertörn University, Avdelning Naturvetenskap. Stockholms universitet.
    Hällbrink, M
    Stockholms universitet.
    Fernaeus, S
    Stockholms universitet.
    Raid, R
    Tartu University, Tartu, Estonia.
    Land, T
    Stockholms universitet.
    Hallberg, Einar
    Södertörn University, Avdelning Naturvetenskap.
    Bartfai, T
    Scripps Research Institute, USA.
    Langel, U
    Stockholms universitet / Scripps Research Institute, USA.
    Cellular translocation of proteins by transportan2001In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 15, no 8, p. 1451-Article in journal (Refereed)
    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.

  • 25.
    Samuelsson, Malin
    et al.
    Stockholm University.
    Fisher, Linda
    Stockholm University.
    Jiang, Yang
    Stockholm University.
    Olsson, Veronica
    Stockholm University.
    Figueroa, Ricardo
    Södertörn University, School of Life Sciences, Molecular biology.
    Hallberg, Einar
    Södertörn University, School of Life Sciences, Molecular biology.
    Langel, Ülo
    Stockholm University.
    Iverfeldt, Kerstin
    Stockholm University.
    Transcription factors as targets to block inflammation in neurodegenerative disorder: s2006In: Alzheimer's & Dementia, ISSN 1552-5260, E-ISSN 1552-5279, Vol. 2, no 3, Supplement, p. S457-Article in journal (Other academic)
    Abstract [en]

    Background: Accumulating evidence supports the importance of inflammation in neurodegenerative disorders like Alzheimer’s disease (AD). Epidemiological studies have revealed that patients taking anti-inflammatory drugs for conditions like arthritis have a lower prevalence of Alzheimer’s than others. In addition, there are reports that show that inflammation indeed can cause neurodegeneration in vivo. “The glial loop hypothesis” describes the model where surrounding glial cells are activated and produce neurotoxic products and therefore lead to neuronal death. One of the most important transcription factors involved in the inflammatory signalling cascade is NF-κB (nuclear factor κB). Supporting a role for NF-κB in AD, this transcription factor has been shown to be upregulated in brains from patients suffering from the disease. Another transcription factor family, thought to work together with NF-κB, is CCAAT enhancer binding protein (C/EBP). C/EBPδ has also been shown to be overexpressed in brains from Alzheimer patients. 

    Objective: Our aim was to characterize the activation of transcription factors that may be involved in AD and to investigate the possibilities to block the effects of these transcription factors. 

    Methods: We have used a delivery system that we have previously developed with a decoy non-covalently bound to a cell-penetrating peptide (CPP). In our studies primary mixed glial cultures from rat (5-10% microglia and 90-95% astrocytes) were used as a modelsystem. Transcription factor activation and cytokine mRNA expression were analyzed by electrophoretic mobility shift assay and RT-PCR, respectively. Cellular uptake studies were performed using confocal laser scanning microscopy. 

    Results: Our studies show that a β-amyloid peptide alone or in combination with the inflammatory cytokine interleukin-1β upregulates NF-κB binding activity as well as the mRNA expression of its downstream target gene interleukin-6 (IL-6). Using our delivery system with an NF-κB decoy resulted in inhibition of upregulated NF-κB binding activity by approx. 80% and IL-6 mRNA expression by approx. 50%. We observed a clear uptake of the CPP-coupled decoy. In parallel, we have also investigated the possibility to use C/EBP as a therapeutic target. 

    Conclusion: Facilitated uptake of transcription factor decoys may be a promising strategy to target the inflammation in neurodegenerative disorders like AD.

  • 26.
    Söderqvist, H
    et al.
    Stockhlms univeristet.
    Imreh, Gabriela
    Stockholms universitet.
    Kihlmark, Madeleine
    Stockholms universitet.
    Linnman, C
    Karolinska institutet.
    Ringertz, N
    Karolinska institutet.
    Hallberg, Einar
    Karolinska institutet.
    Intracellular distribution of an integral nuclear pore membrane protein fused to green fluorescent protein - Localization of a targeting domain1997In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 250, no 3, p. 808-813Article in journal (Refereed)
    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.

1 - 26 of 26
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