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  • 1.
    Behm, Mikaela
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Wahlstedt, Helene
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Widmark, Albin
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Eriksson, Maria
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Öhman, Marie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Accumulation of nuclear ADAR2 regulates A-to-I RNA editing during neuronal development2017In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 130, p. 745-753Article in journal (Refereed)
    Abstract [en]

    Adenosine to inosine (A-to-I) RNA editing is important for a functional brain, and most known sites that are subject to selective RNA editing have been found to result in diversified protein isoforms that are involved in neurotransmission. In the absence of the active editing enzymes ADAR1 or ADAR2 (also known as ADAR and ADARB1, respectively), mice fail to survive until adulthood. Nuclear A-to-I editing of neuronal transcripts is regulated during brain development, with low levels of editing in the embryo and a dramatic increase after birth. Yet, little is known about the mechanisms that regulate editing during development. Here, we demonstrate lower levels of ADAR2 in the nucleus of immature neurons than in mature neurons. We show that importin-a4 (encoded by Kpna3), which increases during neuronal maturation, interacts with ADAR2 and contributes to the editing efficiency by bringing it into the nucleus. Moreover, we detect an increased number of interactions between ADAR2 and the nuclear isomerase Pin1 as neurons mature, which contribute to ADAR2 protein stability. Together, these findings explain how the nuclear editing of substrates that are important for neuronal function can increase as the brain develops. 

  • 2. Bembenek, Joshua N.
    et al.
    Meshik, Xenia
    Tsarouhas, Vasilios
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Meeting report - Cellular dynamics: membrane-cytoskeleton interface2017In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 130, no 17, p. 2775-2779Article in journal (Refereed)
    Abstract [en]

    The first ever 'Cellular Dynamics' meeting on the membrane-cytoskeleton interface took place in Southbridge, MA on May 21-24, 2017 and was co-organized by Michael Way, Elizabeth Chen, Margaret Gardel and Jennifer Lippincott-Schwarz. Investigators from around the world studying a broad range of related topics shared their insights into the function and regulation of the cytoskeleton and membrane compartments. This provided great opportunities to learn about key questions in various cellular processes, from the basic organization and operation of the cell to higher-order interactions in adhesion, migration, metastasis, division and immune cell interactions in different model organisms. This unique and diverse mix of research interests created a stimulating and educational meeting that will hopefully continue to be a successful meeting for years to come.

  • 3.
    Bidla, Gawa
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Dushay, Mitchell S.
    Theopold, Ulrich
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Crystal cell rupture after injury in Drosophila requires the JNK pathway, small GTPases and the TNF homolog Eiger2007In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 120, no 7, p. 1209-15Article in journal (Refereed)
    Abstract [en]

    The prophenoloxidase-activating cascade is a key component of arthropod immunity. Drosophila prophenoloxidase is stored in crystal cells, a specialized class of blood cells from which it is released through cell rupture. Within minutes after bleeding, prophenoloxidase is activated leading to visible melanization of the clot matrix. Using crystal cell rupture and melanization as readouts to screen mutants in signal transduction pathways, we show that prophenoloxidase release requires Jun N-terminal kinase, small Rho GTPases and Eiger, the Drosophila homolog of tumor necrosis factor. We also provide evidence that in addition to microbial products, endogenous signals from dying hemocytes contribute to triggering and/or assembly of the prophenoloxidase-activating cascade, and that this process can be inhibited in vitro and in vivo using the viral apoptotic inhibitor p35. Our results provide a more comprehensive view of immune signal transduction pathways, with implications for immune reactions where cell death is used as a terminal mode of cell activation.

  • 4. Boban, Mirta
    et al.
    Pantazopoulou, Marina
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Schick, Anna
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Foisner, Roland
    A nuclear ubiquitin-proteasome pathway targets the inner nuclear membrane protein Asi2 for degradation2014In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 127, no 16, p. 3603-3613Article in journal (Refereed)
    Abstract [en]

    The nuclear envelope consists of inner and outer nuclear membranes. Whereas the outer membrane is an extension of the endoplasmic reticulum, the inner nuclear membrane (INM) represents a unique membranous environment containing specific proteins. The mechanisms of integral INM protein degradation are unknown. Here, we investigated the turnover of Asi2, an integral INM protein in Saccharomyces cerevisiae. We report that Asi2 is degraded by the proteasome independently of the vacuole and that it exhibited a half-life of similar to 45 min. Asi2 exhibits enhanced stability in mutants lacking the E2 ubiquitin conjugating enzymes Ubc6 or Ubc7, or the E3 ubiquitin ligase Doa10. Consistent with these data, Asi2 is post-translationally modified by poly-ubiquitylation in a Ubc7- and Doa10-dependent manner. Importantly Asi2 degradation is significantly reduced in a sts1-2 mutant that fails to accumulate proteasomes in the nucleus, indicating that Asi2 is degraded in the nucleus. Our results reveal a molecular pathway that affects the stability of integral proteins of the inner nuclear membrane and indicate that Asi2 is subject to protein quality control in the nucleus.

  • 5. Buch, Charlotta
    et al.
    Lindberg, Robert
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Södertörn University, Sweden.
    Figueroa, Ricardo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Södertörn University, Sweden.
    Gudise, Santhosh
    Onischenko, Evgeny
    Hallberg, Einar
    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.

  • 6. Busayavalasa, Kiran
    et al.
    Chen, Xin
    Östlund Farrants, Ann-Kristin
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Wagner, Nicole
    Sabri, Nafiseh
    The nup155 mediated organisation of inner nuclear membrane proteins is independent of nup155 anchoring to the metazoan nuclear pore complex2012In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 125, no 18, p. 4214-4218Article in journal (Refereed)
    Abstract [en]

    The nuclear envelope (NE), an important barrier between the nucleus and the cytoplasm, is composed of three structures: the outer nuclear membrane, which is continuous with the ER, the inner nuclear membrane (INM), which interfaces with chromatin, and nuclear pore complexes (NPCs), which are essential for the exchange of macromolecules between the two compartments. The NPC protein Nup155 has an evolutionarily conserved role in the metazoan NE formation; but the in vivo analysis of Nup155 has been severely hampered by the essential function of this protein in cell viability. Here, we take advantage of the hypomorphicity of RNAi systems and use a combination of protein binding and rescue assays to map the interaction sites of two neighbouring NPC proteins Nup93 and Nup53 on Nup155, and to define the requirements of these interactions in INM protein organization. We show that different parts of Drosophila Nup155 have distinct functions: the Nup155 beta-propeller anchors the protein to the NPC, whereas the alpha-solenoid part of Nup155 is essential for the correct localisation of INM proteins lamin-B receptor (LBR) and otefin. Using chromatin extracts from semisynchronized cells, we also provide evidence that the Nup155 alpha-solenoid has a chromatin-binding activity that is stronger at the end of mitosis. Our results argue that the role of Nup155 in INM protein localisation is not mediated through the NPC anchoring activity of the protein and suggest that regions other than Nup155 beta-propeller are necessary for the targeting of proteins to the INM.

  • 7.
    Dinic, Jelena
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Parmryd, Ingela
    Uppsala universitet, Institutionen för medicinsk cellbiologi.
    Actin filaments at the plasma membrane in live cells cause the formation of ordered lipid domains via phosphatidylinositol 4,5-bisphosphateIn: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137Article in journal (Refereed)
    Abstract [en]

    The relationship between ordered plasma membrane nanodomains, known as lipid rafts, and actin filaments is the focus of this study. Plasma membrane order was followed in live cells at 37°C using laurdan and di-4-ANEPPDHQ to report on lipid packing. Disrupting actin polymerization decreased the fraction of ordered domains, which strongly argue that unstimulated cells have a basal level of ordered domains. Stabilising actin filaments had the opposite effect and increased the proportion of ordered domains. Decreasing the plasma membrane level of phosphatidylinositol 4,5-bisphosphate lowers the number of attachment points for actin filaments and reduced the proportion of ordered domains. Aggregation of plasma membrane molecules, both lipid raft and non-lipid raft markers, leads to the formation of ordered domains that is correlated with an increase in cell peripheral actin filaments. In membrane blebs, which are detached from the underlying actin filaments the fraction of ordered domains was low and GM1 could not be patched to form ordered domains. We conclude that ordered domains form where actin filaments attach to the plasma membrane via phosphatidylinositol 4,5-bisphosphate. This downplays lipid-lipid interactions as the main driving force behind the formation of ordered membrane domains in vivo, giving greater prominence to membrane-intracellular filament interactions.

  • 8.
    Gudise, Santhosh
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry. Karolinska Institute (NOVUM), Sweden.
    Figueroa, Ricardo A.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lindberg, Robert
    Stockholm University, Faculty of Science, Department of Neurochemistry. Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Larsson, Veronica
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Samp1 is functionally associated with the LINC complex and A-type lamina networks2011In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 124, p. 2077-2085Article in journal (Refereed)
    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.

  • 9. Guerra, Lina
    et al.
    Guidi, Riccardo
    Slot, Ilse
    Callegari, Simone
    Sompallae, Ramakrishna
    Pickett, Carol L.
    Åström, Stefan
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Eisele, Frederik
    Wolf, Dieter
    Sjögren, Camilla
    Masucci, Maria G.
    Frisan, Teresa
    Bacterial genotoxin triggers FEN1-dependent RhoA activation, cytoskeleton remodeling and cell survival2011In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 124, no 16, p. 2735-2742Article in journal (Refereed)
    Abstract [en]

    The DNA damage response triggered by bacterial cytolethal distending toxins (CDTs) is associated with activation of the actin-regulating protein RhoA and phosphorylation of the downstream-regulated mitogen-activated protein kinase (MAPK) p38, which promotes the survival of intoxicated (i.e. cells exposed to a bacterial toxin) cells. To identify the effectors of this CDT-induced survival response, we screened a library of 4492 Saccharomyces cerevisiae mutants that carry deletions in nonessential genes for reduced growth following inducible expression of CdtB. We identified 78 genes whose deletion confers hypersensitivity to toxin. Bioinformatics analysis revealed that DNA repair and endocytosis were the two most overrepresented signaling pathways. Among the human orthologs present in our data set, FEN1 and TSG101 regulate DNA repair and endocytosis, respectively, and also share common interacting partners with RhoA. We further demonstrate that FEN1, but not TSG101, regulates cell survival, MAPK p38 phosphorylation, RhoA activation and actin cytoskeleton reorganization in response to DNA damage. Our data reveal a previously unrecognized crosstalk between DNA damage and cytoskeleton dynamics in the regulation of cell survival, and might provide new insights on the role of chronic bacteria infection in carcinogenesis.

  • 10. Imreh, Gabriela
    et al.
    Norberg, Helin Vakifahmetoglu
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Imreh, Stefan
    Zhivotovsky, Boris
    Chromosomal breaks during mitotic catastrophe trigger gamma H2AX-ATM-p53-mediated apoptosis2011In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 124, no 17, p. 2951-2963Article in journal (Refereed)
    Abstract [en]

    Although the cause and outcome of mitotic catastrophe (MC) has been thoroughly investigated, precisely how the ensuing lethality is regulated during or following this process and what signals are involved remain unknown. Moreover, the mechanism of the decision of cell death modalities following MC is still not well characterised. We demonstrate here a crucial role of the gamma H2AX-ATM-p53 pathway in the regulation of the apoptotic outcome of MC resulting from cells entering mitosis with damaged DNA. In addition to p53 deficiency, the depletion of ATM (ataxia telangiectasia mutated), but not ATR ( ataxia telangiectasia and Rad3-related protein), protected against apoptosis and shifted cell death towards necrosis. Activation of this pathway is triggered by the augmented chromosomal damage acquired during anaphase in doxorubicin-treated cells lacking 4-3-3 sigma (also known as epithelial cell marker protein-1 or stratifin). Moreover, cells that enter mitosis with damaged DNA encounter segregation problems because of their abnormal chromosomes, leading to defects in mitotic exit, and they therefore accumulate in G1 phase. These multi- or micronucleated cells are prevented from cycling again in a p53- and p21-dependent manner, and subsequently die. Because increased chromosomal damage resulting in extensive H2AX phosphorylation appears to be a direct cause of catastrophic mitosis, our results describe a mechanism that involves generation of additional DNA damage during MC to eliminate chromosomally unstable cells.

  • 11.
    Jin, Shao-Bo
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Zhao, Jian
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Wieslander, Lars
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    CRM1 and Ran are present but a NES-CRM1-RanGTP complex is not required in Balbiani ring mRNP particles from the gene to the cytoplasm2004In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 117, p. 1553-1566Article in journal (Refereed)
    Abstract [en]

    Messenger RNA is formed from precursors known as pre-mRNA. Theseprecursors associate with proteins to form pre-mRNA-protein(pre-mRNP) complexes. Processing machines cap, splice and polyadenylatethe pre-mRNP and in this way build the mRNP. These processingmachines also affect the export of the mRNP complexes from thenucleus to the cytoplasm. Export to the cytoplasm takes placethrough a structure in the nuclear membrane called the nuclearpore complex (NPC). Export involves adapter proteins in themRNP and receptor proteins that bind to the adapter proteinsand to components of the NPC. We show that the export receptorchromosomal region maintenance protein 1 (CRM1), belonging toa family of proteins known as importin-ß-like proteins,binds to gene-specific Balbiani ring (BR) pre-mRNP while transcriptiontakes place. We also show that the GTPase known as Ran bindsto BR pre-mRNP, and that it binds mainly in the interchromatin.However, we also show using leptomycin B treatment that a NES-CRM1-RanGTPcomplex is not essential for export, even though both CRM1 andRan accompany the BR mRNP through the NPC. Our results thereforesuggest that several export receptors associate with BR mRNPand that these receptors have redundant functions in the nuclearexport of BR mRNP.

  • 12. Jung, Sung-Jun
    et al.
    Kim, Ji Eun Hani
    Reithinger, Johannes H.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kim, Hyun
    The Sec62-Sec63 translocon facilitates translocation of the C-terminus of membrane proteins2014In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 127, no 19, p. 4270-4278Article in journal (Refereed)
    Abstract [en]

    The Sec62-Sec63 complex mediates post-translational translocation of a subset of primarily secretory proteins into the endoplasmic reticulum (ER) in yeast. Therefore, it has been thought that membrane proteins, which are mainly co-translationally targeted into the ER, are not handled by the Sec62-Sec63 translocon. By systematic analysis of single and multi-spanning membrane proteins with broad sequence context [with differing hydrophobicity, flanking charged residues and orientation of transmembrane (TM) segments], we show that mutations in the N-terminal cytosolic domain of yeast Sec62 impair its interaction with Sec63 and lead to defects in membrane insertion and translocation of the C-terminus of membrane proteins. These results suggest that there is an unappreciated function of the Sec62-Sec63 translocon in regulating topogenesis of membrane proteins in the eukaryotic cell.

  • 13.
    Kandasamy, Ganapathi
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Hsp70-Hsp110 chaperones deliver ubiquitin-dependent and -independent substrates to the 26S proteasome for proteolysis in yeast2018In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 131, no 6, article id jcs210948Article in journal (Refereed)
    Abstract [en]

    During protein quality control, proteotoxic misfolded proteins are recognized by molecular chaperones, ubiquitylated by dedicated quality control ligases and delivered to the 26S proteasome for degradation. Proteins belonging to the Hsp70 chaperone and Hsp110 (the Hsp70 nucleotide exchange factor) families function in the degradation of misfolded proteins by the ubiquitin-proteasome system via poorly understood mechanisms. Here, we report that the Saccharomyces cerevisiae Hsp110 proteins (Sse1 and Sse2) function in the degradation of Hsp70-associated ubiquitin conjugates at the post-ubiquitylation step and are also required for ubiquitin-independent proteasomal degradation. Hsp110 associates with the 19S regulatory particle of the 26S proteasome and interacts with Hsp70 to f acilitate the delivery of Hsp70 substrates for proteasomal degradation. By using a highly defined ubiquitin-independent proteasome substrate, we show that the mere introduction of a single Hsp70-binding site renders its degradation dependent on Hsp110. The findings define a dedicated and chaperone-dependent pathway for the efficient shuttling of cellular proteins to the proteasome with profound implications for understanding protein quality control and cellular stress management.

  • 14.
    Larsson, Veronica J.
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jafferali, Mohammed Hakim
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Vijayaraghavan, Balaje
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Figueroa, Ricardo A.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Mitotic spindle assembly and γ-tubulin localisation depend on the integral nuclear membrane protein, Samp12018In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 131, no 8, article id jcs211664Article in journal (Refereed)
    Abstract [en]

    We have investigated a possible role of the inner nuclear membrane protein, Samp1, in the mitotic machinery. Live cell imaging showed that Samp1aYFP distributed as filamentous structures in the mitotic spindle, partially co-localising with ß-tubulin. Samp1 depletion resulted in an increased frequency of cells with signs of chromosomal mis-segregation and prolonged metaphase, indicating problems with spindle assembly and/or chromosomal alignment. Consistently, mitotic spindles in Samp1 depleted cells contained significantly lower levels of ß-tubulin and γ-tubulin, phenotypes which were rescued by overexpression of Samp1aYFP. We found that Samp1 can bind directly to γ-tubulin and that Samp1 co-precipitated with γ-tubulin and HAUS6 of the Augmin complex in live cells. The levels of Haus6, in the mitotic spindle also decreased after Samp1 depletion. We show that Samp1 is involved in the recruitment of Haus6 and γ-tubulin to the mitotic spindle. Samp1 is the first inner nuclear membrane protein shown to have a function in mitotic spindle assembly.

  • 15.
    Marin-Vicente, Consuelo
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Domingo-Prim, Judit
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Eberle, Andrea B.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    RRP6/EXOSC10 is required for the repair of DNA double-strand breaks by homologous recombination2015In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 128, no 6, p. 1097-1107Article in journal (Refereed)
    Abstract [en]

    The exosome acts on different RNA substrates and plays important roles in RNA metabolism. The fact that short non-coding RNAs are involved in the DNA damage response led us to investigate whether the exosome factor RRP6 of Drosophila melanogaster and its human ortholog EXOSC10 play a role in DNA repair. Here, we show that RRP6 and EXOSC10 are recruited to DNA double-strand breaks (DSBs) in S2 cells and HeLa cells, respectively. Depletion of RRP6/ EXOSC10 does not interfere with the phosphorylation of the histone variant H2Av (Drosophila) or H2AX (humans), but impairs the recruitment of the homologous recombination factor RAD51 to the damaged sites, without affecting RAD51 levels. The recruitment of RAD51 to DSBs in S2 cells is also inhibited by overexpression of RRP6-Y361A-V5, a catalytically inactive RRP6 mutant. Furthermore, cells depleted of RRP6 or EXOSC10 are more sensitive to radiation, which is consistent with RRP6/EXOSC10 playing a role in DNA repair. RRP6/EXOSC10 can be co-immunoprecipitated with RAD51, which links RRP6/EXOSC10 to the homologous recombination pathway. Taken together, our results suggest that the ribonucleolytic activity of RRP6/EXOSC10 is required for the recruitment of RAD51 to DSBs.

  • 16.
    Pantazopoulou, Marina
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Boban, Mirta
    Foisner, Roland
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Cdc48 and Ubx1 participate in a pathway associated with the inner nuclear membrane that governs Asi1 degradation2016In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 129, no 20, p. 3770-3780Article in journal (Refereed)
    Abstract [en]

    The nuclear envelope is a barrier comprising outer and inner membranes that separate the cytoplasm from the nucleoplasm. The two membranes have different physical characteristics and protein compositions. The processes governing the stability of inner nuclear membrane (INM) proteins are not well characterized. In Saccharomyces cerevisiae, the INM Asi1-Asi3 complex, principally composed of integral membrane proteins Asi1 and Asi3, is an E3 ubiquitin ligase. In addition to its well-documented function in endoplasmic reticulum (ER)-associated degradation, the Doa10 E3 ubiquitin ligase complex partially localizes to the INM. The Asi1-Asi3 and Doa10 complexes define independent INM-associated degradation (INMAD) pathways that target discrete sets of nuclear substrates for proteasomal degradation. Here, we report that Asi1 is rapidly turned over (t(1/2)<= 30 min). Its turnover depends on ubiquitin-mediated degradation by nucleus-localized proteasomes, exhibiting a clear requirement for the E2 ubiquitin-conjugating enzyme Ubc7, Cue1 and the AAA ATPase Cdc48 and co-factor Ubx1. Asi1 turnover occurs largely independently of the Asi1-Asi3 or Doa10 complexes, indicating that it is subject to quality control at the INM in a manner distinct from that of the characterized INMAD pathways.

  • 17. Peng, Lisheng
    et al.
    Berntsson, Ronnie P.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tepp, William H.
    Pitkin, Rose M.
    Johnson, Eric A.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dong, Min
    Botulinum neurotoxin D-C uses synaptotagmin I and II as receptors, and human synaptotagmin II is not an effective receptor for type B, D-C and G toxins2012In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 125, no 13, p. 3233-3242Article in journal (Refereed)
    Abstract [en]

    Botulinum neurotoxins (BoNTs) are classified into seven types (A-G), but multiple subtype and mosaic toxins exist. These subtype and mosaic toxins share a high sequence identity, and presumably the same receptors and substrates with their parental toxins. Here, we report that a mosaic toxin, type D-C (BoNT/D-C), uses different receptors from its parental toxin BoNT/C. BoNT/D-C, but not BoNT/C, binds directly to the luminal domains of synaptic vesicle proteins synaptotagmin (Syt) I and II, and requires expression of SytI/II to enter neurons. The SytII luminal fragment containing the toxin-binding site can block the entry of BoNT/D-C into neurons and reduce its toxicity in vivo in mice. We also found that gangliosides increase binding of BoNT/D-C to SytI/II and enhance the ability of the SytII luminal fragment to block BoNT/D-C entry into neurons. These data establish SytI/II, in conjunction with gangliosides, as the receptors for BoNT/D-C, and indicate that BoNT/D-C is functionally distinct from BoNT/C. We further found that BoNT/D-C recognizes the same binding site on SytI/II where BoNT/B and G also bind, but utilizes a receptor-binding interface that is distinct from BoNT/B and G. Finally, we also report that human and chimpanzee SytII has diminished binding and function as the receptor for BoNT/B, D-C and G owing to a single residue change from rodent SytII within the toxin binding site, potentially reducing the potency of these BoNTs in humans and chimpanzees.

  • 18.
    Tsarouhas, Vasilios
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Yao, Liqun
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Samakovlis, Christos
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Src kinases and ERK activate distinct responses to Stitcher receptor tyrosine kinase signaling during wound healing in Drosophila2014In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 127, no 8, p. 1829-1839Article in journal (Refereed)
    Abstract [en]

    Metazoans have evolved efficient mechanisms for epidermal repair and survival following injury. Several cellular responses and key signaling molecules that are involved in wound healing have been identified in Drosophila, but the coordination of cytoskeletal rearrangements and the activation of gene expression during barrier repair are poorly understood. The Ret-like receptor tyrosine kinase (RTK) Stitcher (Stit, also known as Cad96Ca) regulates both re-epithelialization and transcriptional activation by Grainy head (Grh) to induce restoration of the extracellular barrier. Here, we describe the immediate downstream effectors of Stit signaling in vivo. Drk (Downstream of receptor kinase) and Src family tyrosine kinases bind to the same docking site in the Stit intracellular domain. Drk is required for the full activation of transcriptional responses but is dispensable for re-epithelialization. By contrast, Src family kinases (SFKs) control both the assembly of a contractile actin ring at the wound periphery and Grh-dependent activation of barrier-repair genes. Our analysis identifies distinct pathways mediating injury responses and reveals an RTK-dependent activation mode for Src kinases and their central functions during epidermal wound healing in vivo.

  • 19.
    Vielfort, Katarina
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Söderholm, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Weyler, Linda
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Vare, Daniel
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Löfmark, Sonja
    Aro, Helena
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Neisseria gonorrhoeae infection causes DNA damage and affects the expression of p21, p27 and p53 in non-tumor epithelial cells2013In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 126, no 1, p. 339-347Article in journal (Refereed)
    Abstract [en]

    The constant shedding and renewal of epithelial cells maintain the protection of epithelial barriers. Interference with the processes of host cell-cycle regulation and barrier integrity permits the bacterial pathogen Neisseria gonorrhoeae to effectively colonize and invade epithelial cells. Here, we show that a gonococcal infection causes DNA damage in human non-tumor vaginal VK2/E6E7 cells with an increase of 700 DNA strand breaks per cell per hour as detected by an alkaline DNA unwinding assay. Infected cells exhibited elevated levels of DNA double-strand breaks, as indicated by a more than 50% increase in cells expressing DNA damage-response protein 53BP1-positive foci that co-localized with phosphorylated histone H2AX (gamma H2AX). Furthermore, infected cells abolished their expression of the tumor protein p53 and induced an increase in the expression of cyclin-dependent kinase inhibitors p21 and p27 to 2.6-fold and 4.2-fold of controls, respectively. As shown by live-cell microscopy, flow cytometry assays, and BrdU incorporation assays, gonococcal infection slowed the host cell-cycle progression mainly by impairing progression through the G2 phase. Our findings show new cellular players that are involved in the control of the human cell cycle during gonococcal infection and the potential of bacteria to cause cellular abnormalities.

  • 20.
    Ólafsson, Einar B.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ross, Emily C.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Varas-Godoy, Manuel
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    TIMP-1 promotes hypermigration of Toxoplasma-infected primary dendritic cells via CD63-ITGB1-FAK signaling2019In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 132, no 3, article id UNSP jcs225193Article in journal (Refereed)
    Abstract [en]

    Tissue inhibitor of metalloproteinases-1 (TIMP-1) exerts pleiotropic effects on cells including conferring metastatic properties to cancer cells. As for metastatic cells, recent paradigms of leukocyte migration attribute important roles to the amoeboid migration mode of dendritic cells (DCs) for rapid locomotion in tissues. However, the role of TIMP-1 in immune cell migration and in the context of infection has not been addressed. We report that, upon challenge with the obligate intracellular parasite Toxoplasma gondii, primary DCs secrete TIMP-1 with implications for their migratory properties. Using a short hairpin RNA (shRNA) gene silencing approach, we demonstrate that secreted TIMP-1 and its ligand CD63 are required for the onset of hypermotility in DCs challenged with T. gondii. Further, gene silencing and antibody blockade of the beta 1-integrin CD29 (ITGB1) inhibited DC hypermotility, indicating that signal transduction occurred via ITGB1. Finally, gene silencing of the ITGB1-associated focal adhesion kinase (FAK, also known as PTK2), as well as pharmacological antagonism of FAK and associated kinases SRC and PI3K, abrogated hypermotility. The present study identifies a TIMP-1-CD63-ITGB1-FAK signaling axis in primary DCs, which T. gondii hijacks to drive high-speed amoeboid migration of the vehicle cells that facilitate its systemic dissemination.

  • 21.
    Öjemalm, Karin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Watson, Helen R.
    Roboti, Peristera
    Cross, Benedict C. S.
    Warwicker, Jim
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    High, Stephen
    Positional editing of transmembrane domains during ion channel assembly2013In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 126, no 2, p. 464-472Article in journal (Refereed)
    Abstract [en]

    The integration of transmembrane (TM)-spanning regions of many channels and ion transporters is potentially compromised by the presence of polar and charged residues required for biological function. Although the two TMs of the ATP-gated ion channel subunit P2X2 each contain charged/polar amino acids, we found that each TM is efficiently membrane inserted when it is analysed in isolation, and uncovered no evidence for cooperativity between these two TMs during P2X2 integration. However, using minimal N-glycosylation distance mapping, we find that the positioning of TM2 in newly synthesized P2X2 monomers is distinct from that seen in subunits of the high-resolution structures of assembled homologous trimers. We conclude that P2X2 monomers are initially synthesised at the endoplasmic reticulum in a distinct conformation, where the extent of the TM-spanning regions is primarily defined by the thermodynamic cost of their membrane integration at the Sec61 translocon. In this model, TM2 of P2X2 subsequently undergoes a process of positional editing within the membrane that correlates with trimerisation of the monomer, a process requiring specific polar/charged residues in both TM1 and TM2. We postulate that the assembly process offsets any energetic cost of relocating TM2, and find evidence that positional editing of TM2 in the acid-sensing ion channel (ASIC1a) is even more pronounced than that observed for P2X2. Taken together, these data further underline the potential complexities involved in accurately predicting TM domains. We propose that the orchestrated repositioning of TM segments during subunit oligomerisation plays an important role in generating the functional architecture of active ion channels, and suggest that the regulation of this underappreciated biosynthetic step may provide an elegant mechanism for maintaining ER homeostasis.

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