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The functional organization of nuclear membrane proteins and development of new technology for studies of cell signaling
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Einar Hallberg)ORCID iD: 0000-0003-1476-6675
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The eukaryotic cell is defined by the nucleus, which is delimited by a double membrane structure termed the nuclear envelope (NE). The NE is implicated in a multitude of different processes, for example chromatin organization. During mitosis in higher eukaryotes the nucleus is disassembled to allow the formation of the mitotic spindle, which segregates the duplicated chromosomes between daughter cells. We have characterized a novel transmembrane protein of the inner nuclear membrane. Because of its distribution along spindle microtubule during mitosis, we termed the protein Samp1 (Spindle associated membrane protein 1). Samp1 is the founding member of transmembrane proteins that define a novel membrane domain that we have termed the SE (spindle endomembrane). Furthermore, we have shown that in interphase Samp1 specifically interacts with the centrosome and A-type lamina network proteins. Moreover, Samp1 contains an evolutionary highly conserved N-terminal tail containing two putative zinc fingers.

Recent studies indicate local caspase activity in dendrites or axons during development and in neurodegenerative disorders. Here I present the development of a novel and unique system to monitor protease activity at sub-cellular resolution in live cells. This system relies on a cleavable FRET sensor that is anchored to the cytoskeleton. Using this system we demonstrate local caspase activation of the soma in neuronaly differentiated cells. We also used the anchored FRET sensors to monitor caspase activation after treatment with the Alzheimer’s decease related amyloid-β peptide.

Moreover we have improved a NF-ĸB decoy delivery system. The system consists of a cell penetrating peptide, transportan-10, covalently linked to a peptide nucleic acid sequence that hybridizes with a nonanucleotide sequence in the decoy. We show that this system effectively delivered the decoy and inhibited an inflammatory response in primary rat glial cells.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2011. , 54 p.
National Category
Biochemistry and Molecular Biology Cell Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-63559ISBN: 978-91-7447-386-5 (print)OAI: oai:DiVA.org:su-63559DiVA: diva2:450861
Public defence
2011-11-25, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2011-11-02 Created: 2011-10-23 Last updated: 2015-03-06Bibliographically approved
List of papers
1. An integral protein of the inner nuclear membrane localizes to the mitotic spindle in mammalian cells
Open this publication in new window or tab >>An integral protein of the inner nuclear membrane localizes to the mitotic spindle in mammalian cells
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2009 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 122, no 12, 2100-2107 p.Article in journal (Refereed) Published
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.

Keyword
Inner nuclear membrane, Transmembrane proteins, Mitotic spindle, Mitosis, Cancer
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-60074 (URN)10.1242/jcs.047373 (DOI)000266634800018 ()
Note

authorCount :6

Available from: 2011-08-08 Created: 2011-08-08 Last updated: 2017-12-08Bibliographically approved
2. Samp1 is functionally associated with the LINC complex and A-type lamina networks
Open this publication in new window or tab >>Samp1 is functionally associated with the LINC complex and A-type lamina networks
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2011 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 124, 2077-2085 p.Article in journal (Refereed) Published
Abstract [en]

The transmembrane inner nuclear membrane (INM) protein Samp1 is required for anchoring centrosomes near the nuclei. Using high-resolution fluorescence microscopy we show that Samp1 is distributed in a distinct and characteristic pattern in the nuclear envelope (NE), where it partially colocalizes with the LINC complex protein Sun1. By studying the localization of Samp1 deletion mutants and fusion proteins, we conclude that the cysteine-rich N-terminal half of Samp1 is nucleoplasmically exposed and is responsible for targeting to the INM. It contains four conserved CxxC motifs with the potential to form zinc fingers. The distribution of cysteine-to-alanine substitution mutants, designed to prevent zinc finger formation, showed that NE localization of Samp1 depends on intact CxxC motifs. Overexpression of Samp1 zinc finger mutants produced an abnormal dominant phenotype characterized by disrupted organization of a selective subset NE proteins, including emerin, Sun1, endogenous Samp1 and, in some cases, lamin A/C, but not lamin B, Sun2 or nucleoporins. Silencing of Samp1 expression showed that emerin depends on Samp1 for its correct localization in the NE. Our results demonstrate that Samp1 is functionally associated with the LINC complex protein Sun1 and proteins of the A-type lamina network.

Keyword
Cancer, Centrosome, Laminopathies, Nuclear membrane, Nuclear migration
National Category
Biological Sciences Chemical Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-63558 (URN)10.1242/jcs.078923 (DOI)000291048000014 ()
Available from: 2011-10-23 Created: 2011-10-23 Last updated: 2017-12-08Bibliographically approved
3. Anchored FRET sensors detect local caspase activation prior to neuronal degeneration
Open this publication in new window or tab >>Anchored FRET sensors detect local caspase activation prior to neuronal degeneration
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2011 (English)In: Molecular Neurodegeneration, ISSN 1750-1326, E-ISSN 1750-1326, Vol. 6, 35- p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Recent studies indicate local caspase activation in dendrites or axons during development and in neurodegenerative disorders such as Alzheimer's disease (AD). Emerging evidences point to soluble oligomeric amyloid-beta peptide as a causative agent in AD.

RESULTS: Here we describe the design of fluorescence resonance energy transfer (FRET)-based caspase sensors, fused to the microtubule associated protein tau. Specific caspase sensors preferentially cleaved by caspase-3, -6 or -9 were expressed in differentiated human neuroblastoma SH-SY5Y cells. The anchoring of the sensors resulted in high FRET signals both in extended neurites and soma and made analysis of spatiotemporal signal propagation possible. Caspase activation was detected as loss of FRET after exposure to different stimuli. Interestingly, after staurosporine treatment caspase-6 activation was significantly delayed in neurites compared to cell bodies. In addition, we show that exposure to oligomer-enriched amyloid-beta peptide resulted in loss of FRET in cells expressing sensors for caspase-3 and -6, but not -9, in both soma and neurites before neurite degeneration was observed.

CONCLUSIONS: Taken together, the results show that by using anchored FRET sensors it is possible to detect stimuli-dependent differential activation of caspases and to distinguish local from global caspase activation in live neuronal cells. Furthermore, in these cells oligomer-enriched amyloid-beta peptide induces a global, rather than local activation of caspase-3 and -6, which subsequently leads to neuronal cell death.

Keyword
Amyloid-beta, Caspases, FRET, Live Cell Imaging, Neurite degeneration, Neurodegeneration, Spatiotemporal analysis
National Category
Biological Sciences Chemical Sciences
Research subject
Biochemistry; Neurochemistry and Neurotoxicology; Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-58646 (URN)10.1186/1750-1326-6-35 (DOI)000291991900001 ()21605370 (PubMedID)
Funder
Swedish Research Council, 2010-4481Swedish Research Council, 2010-4505
Available from: 2011-06-07 Created: 2011-06-07 Last updated: 2017-12-11Bibliographically approved
4. Targeting cytokine expression in glial cells by cellular delivery of an NFκB decoy
Open this publication in new window or tab >>Targeting cytokine expression in glial cells by cellular delivery of an NFκB decoy
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2007 (English)In: Journal of Molecular Neuroscience, ISSN 0895-8696, E-ISSN 1559-1166, Vol. 31, no 3, 209-219 p.Article in journal (Refereed) Published
Abstract [en]

Inhibition of nuclear factor (NF)-κB 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-κB 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 β amyloid peptide in the presence of the inflammatory cytokine interleukin (IL) 1β. 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-κB binding activity and IL-6 mRNA expression, respectively.

Keyword
β-amyloid, astrocytes, cell-penetrating peptide, inflammation, nuclear factor-κB, peptide nucleic acid
National Category
Biological Sciences
Research subject
Biochemistry; Neurochemistry and Neurotoxicology
Identifiers
urn:nbn:se:su:diva-25921 (URN)10.1385/JMN:31:03:209 (DOI)000246588800003 ()
Available from: 2006-05-18 Created: 2006-05-18 Last updated: 2017-12-13Bibliographically approved

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