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  • 51.
    Dinic, Jelena
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Adler, Jeremy
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Parmryd, Ingela
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Plasma membrane order in T cell signalling2009Conference paper (Other academic)
    Abstract [en]

    Plasma membrane nanodomains, referred to as lipid rafts, more ordered than the bulk membrane play an important role in T cell signalling by forming signalling platforms in activated T cells. However, the existence of lipid rafts in resting T cells is contentious. Using laurdan, a membrane probe whose peak emission wavelength depends on the lipid environment, evidence is presented for the existence of ordered nanodomains in resting T cells.

    T cell signalling can be initiated by stimulating the T cell receptor (TCR), crosslinking the lipid raft markers GM1 (sphingolipid) or glycosylphosphatidylinositol (GPI) anchored proteins. The aggregation of lipid raft components induces the same response in Jurkat T cells as the ligation of an antigen to the TCR. Changes in membrane order linked with reorganization of the plasma membrane upon Jurkat T cell activation were followed at 37°C. Fluorescent images were analyzed for generalised polarisation values - a measure of the relative abundance of liquid ordered and liquid disordered domains. TCR patching does not increase the overall membrane order suggesting that membrane domains of high order are brought together in the patches. This supports the existence of small ordered membrane domains in resting T cells that aggregate upon activation. Patching of GM1, the GPI-anchored protein CD59 and the non lipid raft marker CD45 significantly increases the overall membrane order. So does general crosslinking of membrane components with Concanavalin A. Remodelling of the actin cytoskeleton is an integral part of TCR signaling and T cell activation. Disrupting actin polymerization using latrunculin B decreases membrane order and stabilizing actin filaments with jasplakinolide increases membrane order. An increase in membrane order appears to be a general effect of plasma membrane component patching and is likely due to a global induction of actin polymerization at the plasma membrane.

  • 52.
    Dinic, Jelena
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute.
    Biverståhl, Henrik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Parmryd, Ingela
    Stockholm University, Faculty of Science, The Wenner-Gren Institute.
    Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing2011In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1808, no 1, p. 298-306Article in journal (Refereed)
    Abstract [en]

    Laurdan and di-4-ANEPPDHQ are used as probes for membrane order, with a blue shift in emission for membranes in liquid-ordered (lo) phase relative to membranes in liquid-disordered (ld) phase. Their use as membrane order probes requires that their spectral shifts are unaffected by membrane proteins, which we have examined by using membrane inserting peptides and large unilamellar vesicles (LUVs). The transmembrane polypeptides, mastoparan and bovine prion protein-derived peptide (bPrPp), were added to LUVs of either lo or ld phase, up to 1:10 peptide/total lipid ratio. The excitation and emission spectra of laurdan and di-4-ANEPPDHQ in both lipid phases were unaltered by peptide addition. The integrity and size distribution of the LUVs upon addition of the polypeptides were determined by dynamic light scattering. The insertion efficiency of the polypeptides into LUVs was determined by measuring their secondary structure by circular dichroism. Mastoparan had an α-helical and bPrPp a β-strand conformation compatible with insertion into the lipid bilayer. Our results suggest that the presence of proteins in biological membranes does not influence the spectra of laurdan and di-4-ANEPPDHQ, supporting that the dyes are appropriate probes for assessing lipid order in cells.

  • 53.
    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.

  • 54.
    Dinic, Jelena
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Parmryd, Ingela
    Uppsala universitet, Institutionen för medicinsk cellbiologi .
    Riehl, Astrid
    Adler, Jeremy
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    The T cell receptor resides in small ordered plasma membrane domains that aggregate upon T cell activationManuscript (preprint) (Other academic)
    Abstract [en]

    T cell signaling emanates from large lipid raft platforms. Whether lipid rafts form upon T cell receptor (TCR) engagement or exist in resting T cells was the focus of this study. Plasma membrane order was followed in live T cells at 37°C using laurdan to report on lipid packing. Patching of the TCR in both Jurkat and human primary CD4+ T cells resulted in higher fractions of ordered plasma domains in the patches but did not increase the overall membrane order. The TCR colocalized with actin filaments in unstimulated Jurkat T cells and this colocalization was most prominent for cells in G1 phase. Moreover, the TCR located to the nuclear envelope, in addition to the plasma membrane, in cells in S and G2/M phase. Our study suggests that the TCR resides in ordered plasma membrane domains/lipid rafts that are linked to actin filament and aggregate upon T cell activation.

  • 55.
    Domingo Prim, Judit
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    The exosome and the maintenance of genome integrity2016Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    The RNA 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 plays a role in DNA repair. We have shown that the exosome catalytic subunit RRP6/EXOSC10 is recruited to DNA double-strand breaks (DSBs) in Drosophila S2 cells and human HeLa cells exposed to either ionizing radiation or I-PpoI endonuclease cleavage. DIS3, the other catalytic subunit of the nuclear exosome, is also recruited to DSBs, whereas the exosome core subunit EXOSC7 is not. Depletion of different exosome subunits does not interfere with the phosphorylation of the histone variants H2Av (Drosophila) or H2AX (humans), but depletion of RRP6/EXOSC10 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 in animal cells. Taken together, our results suggest that a 3’-5’ ribonucleolytic activity is required for efficient DNA repair. 

  • 56. Fergusson, Joannah R.
    et al.
    Smith, Kira E.
    Fleming, Vicki M.
    Rajoriya, Neil
    Newell, Evan W.
    Simmons, Ruth
    Marchi, Emanuele
    Björkander, Sophia
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kang, Yu-Hoi
    Swadling, Leo
    Kurioka, Ayako
    Sahgal, Natasha
    Lockstone, Helen
    Baban, Dilair
    Freeman, Gordon J.
    Sverremark-Ekström, Eva
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Davis, Mark M.
    Davenport, Miles P.
    Venturi, Vanessa
    Ussher, James E.
    Willberg, Christian B.
    Klenerman, Paul
    CD161 Defines a Transcriptional and Functional Phenotype across Distinct Human T Cell Lineages2014In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 9, no 3, p. 1075-1088Article in journal (Refereed)
    Abstract [en]

    The C-type lectin CD161 is expressed by a large proportion of human T lymphocytes of all lineages, including a population known as mucosal-associated invariant T (MAIT) cells. To understand whether different T cell subsets expressing CD161 have similar properties, we examined these populations in parallel using mass cytometry and mRNA microarray approaches. The analysis identified a conserved CD161++/MAIT cell transcriptional signature enriched in CD161+CD8+ T cells, which can be extended to CD161+ CD4+ and CD161+TCR gamma delta+ T cells. Furthermore, this led to the identification of a shared innate-like, TCR-independent response to interleukin (IL)-12 plus IL-18 by different CD161-expressing T cell populations. This response was independent of regulation by CD161, which acted as a costimulatory molecule in the context of T cell receptor stimulation. Expression of CD161 hence identifies a transcriptional and functional phenotype, shared across human T lymphocytes and independent of both T cell receptor (TCR) expression and cell lineage.

  • 57.
    Figueroa, Ricardo A.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The functional organization of nuclear membrane proteins and development of new technology for studies of cell signaling2011Doctoral 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.

  • 58.
    Forsby, Anna
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Norman, Kimberly
    EL Andaloussi-Lilja, Johanna
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lundqvist, Jessica
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Wojcik, Beata
    Walczak, Vincent
    Curren, Rodger
    Martin, Katharine
    Tierney, Neena
    Predicting eye stinging potential of baby shampoos by assessing TRPV1 channel activity2012In: Toxicology Letters, ISSN 0378-4274, E-ISSN 1879-3169, Vol. 211, p. S113-S113Article in journal (Refereed)
    Abstract [en]

    The Transient Receptor Potential Vanilloid type 1 (TRPV1) receptor is one of the most well characterized pain-inducing receptors. The purpose of this study was to predict human eye stinging of 19 baby bath and shampoo formulations by studying TRPV1 activity. The NociOcular test, a novel recombinant neuronal in vitro model with high expression of functional TRPV1 channels was used to test shampoo formulations containing surfactants, preservatives, and fragrances (sodium laureth sulfate, cocoamidopropylbetaine, cocoglucoside, sodium benzoate, quaternium-15, etc.). The increase in intracellular free Ca2+ was analysed by fluorescence during exposure. TRPV1-specific Ca2+ influx was abolished when the TRPV1 channel antagonist capsazepine was applied to the cells prior to shampoo samples. The positive control, i.e. adult shampoo, was the most active sample tested in the NociOcular test and also induced the worst stinging sensation. The negative control, i.e. marketed baby shampoo, was negative in both tests. Seven of the formulations induced stinging in the human test, and of those six were positive in the NociOcular test. Twelve of the formulations were classified as non-stinging in the human test, and of those 10 were negative in the NociOcular test. None of the established in vitro tests for eye irritation were able to correctly predict the human stinging sensation of the baby products. Our data support that the TRPV1 channel is a principle mediator of eye stinging sensation induced by baby bath and shampoo formulations and that the NociOcular test may be a valuable in vitro tool to predict human eye stinging sensation.

  • 59. Gabarayeva, Nina I.
    et al.
    Grigorjeva, Valentina V.
    Rowley, John R.
    Stockholm University, Faculty of Science, Department of Botany.
    Sporoderm development in Acer tataricum (Aceraceae): an interpretation2010In: Protoplasma, ISSN 0033-183X, E-ISSN 1615-6102, Vol. 247, no 1-2, p. 65-81Article in journal (Refereed)
    Abstract [en]

    For the first time, the developmental events in the course of complicated exine structure establishment have been traced in detail with transmission electron microscope in the representative of Acer. A new look at unfolding events is suggested using the knowledge of a boundary field, colloid science. Our purpose was to find out whether the sequence of sporoderm developmental events represents, in essence, the sequence of self-assembling micellar mesophases, initiated by genomically given physicochemical parameters and induced by surfactant glycoproteins at increasing concentration. Indeed, the first units observed in the periplasmic space are globular ones (=spherical micelles) which become arranged into rod-like units (=cylindrical micelles). Then, twisted clusters of rodlets form a layer of procolumellae (middle micellar mesophase). The tectum emerges as an untwisting and merging of distal ends of procolumellae (distal untwist of micelle clusters). In the end of tetrad period, when a hydrophilic-hydrophobic switch occurs in the periplasmic space, the contrast reversal of the columellae corresponds to the change of normal micelles to reverse ones. The initiation of the foot layer and the endexine lamellae, with their typical central "white lines", corresponds to the next-"neat"aEuro"mesophase, with its typical central gaps between layers. Aperture sites during development show all the main micellar mesophases and their transitional forms. The data received have supported our previous hypothesis.

  • 60.
    Garcia-Gonzalez, Claudia
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Morrison, Jamie Ian
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Cardiac regeneration in non-mammalian vertebrates2014In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 321, no 1, p. 58-63Article, review/survey (Refereed)
    Abstract [en]

    The heart is a robust organ, capable of pumping nutrients and transferring oxygen throughout the body via a network of capillaries, veins and arteries, for the entirety of a human's life. However, the fragility of mammalian hearts is also evident when it becomes damaged and parts of the organ fail to function. This is due to the fact that rather than replenishing the damaged areas with functional cellular mass, fibrotic scar tissue is the preferred replacement, resulting in an organ with functional deficiencies. Due to the mammalian hearts incapability to regenerate following damage and the ever-increasing number of people worldwide suffering from heart disease, tireless efforts are being made to discover ways of inducing a regenerative response in this most important organ. One such avenue of investigation involves studying our distantly related non-mammalian vertebrate cousins, which over the last decade has proved to us that cardiac regeneration is possible. This review will highlight these organisms and provide insights into some of the seminal discoveries made in the heart regeneration field using these amazing chordates.

  • 61. Gorelova, O. A.
    et al.
    Baulina, O. I.
    Rasmussen, Ulla
    Stockholm University, Faculty of Science, Department of Botany.
    Koksharova, O. A.
    The pleiotropic effects of ftn2 and ftn6 mutations in cyanobacterium Synechococcus sp PCC 79422013In: Protoplasma, ISSN 0033-183X, E-ISSN 1615-6102, Vol. 250, no 4, p. 931-942Article in journal (Refereed)
    Abstract [en]

    Two cell division mutants (Ftn2 and Ftn6) of the cyanobacterium Synechococcus sp. PCC 7942 were studied using scanning electron microscopy and transmission electron microscopy methods. This included negative staining and ultrathin section analysis. Different morphological and ultrastructural features of mutant cells were identified. Ftn2 and Ftn6 mutants exhibited particularly elongated cells characterized by significantly changed shape in comparison with the wild type. There was irregular bending, curving, spiralization, and bulges as well as cell branching. Elongated mutant cells were able to initiate cytokinesis simultaneously in several division sites which were localized irregularly along the cell. Damaged rigidity of the cell wall was typical of many cells for both mutants. Thylakoids of mutants showed modified arrangement and ultrastructural organization. Carboxysome-like structures without a shell and/or without accurate polyhedral packing protein particles were often detected in the mutants. However, in the case of Ftn2 and Ftn6, the average number of carboxysomes per section was less than in the wild type by a factor of 4 and 2, respectively. These multiple morphological and ultrastructural changes in mutant cells evinced pleiotropic responses which were induced by mutations in cell division genes ftn2 and ftn6. Ultrastructural abnormalities of Ftn2 and Ftn6 mutants were consistent with differences in their proteomes. These results could support the significance of FTN2 and FTN6 proteins for both cyanobacterial cell division and cellular physiology.

  • 62.
    Gowda, Naveen Kumar C.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kaimal, Jayasankar Mohanakrishnan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kityk, Roman
    Daniel, Chammiran
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Liebau, Jobst
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öhman, Marie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Mayer, Matthias P.
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Substrate-mimicking domain of nucleotide-exchange factor Fes1/HspBP1 ensures efficient release of persistent substrates from Hsp70Manuscript (preprint) (Other academic)
  • 63.
    Gowda, Naveen Kumar Chandappa
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Hsp70 nucleotide exchange factor Fes1 is essential for protein quality control in Saccharomyces cerevisiae2014Licentiate thesis, comprehensive summary (Other academic)
  • 64.
    Gowda, Naveen Kumar Chandappa
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Regulation of Hsp70 function by nucleotide-exchange factors2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Protein folding is the process in which polypeptides in their non-native states attain the unique folds of their native states. Adverse environmental conditions and genetic predisposition challenge the folding process and accelerate the production of proteotoxic misfolded proteins. Misfolded proteins are selectively recognized and removed from the cell by processes of protein quality control (PQC). In PQC molecular chaperones of the Heat shock protein 70 kDa (Hsp70) family play important roles by recognizing and facilitating the removal of misfolded proteins. Hsp70 function is dependent on cofactors that regulate the intrinsic ATPase activity of the chaperone. In this thesis I have used yeast genetic, cell biological and biochemical experiments to gain insight into the regulation of Hsp70 function in PQC by nucleotide-exchange factors (NEFs). Study I shows that the NEF Fes1 is a key factor essential for cytosolic PQC. A reverse genetics approach demonstrated that Fes1 NEF activity is required for the degradation of misfolded proteins associated with Hsp70 by the ubiquitin-proteasome system. Specifically, Fes1 association with Hsp70-substrate complexes promotes interaction of the substrate with downstream ubiquitin E3 ligase Ubr1. The consequences of genetic removal of FES1 (fes1Δ) are the failure to degrade misfolded proteins, the accumulation of protein aggregates and constitutive induction of the heat-shock response. Taken the experimental data together, Fes1 targets misfolded proteins for degradation by releasing them from Hsp70. Study II describes an unusual example of alternative splicing of FES1 transcripts that leads to the expression of the two alternative splice isoforms Fes1S and Fes1L. Both isoforms are functional NEFs but localize to different compartments. Fes1S is localized to the cytosol and is required for the efficient degradation of Hsp70-associated misfolded proteins. In contrast, Fes1L is targeted to the nucleus and represents the first identified nuclear NEF in yeast. The identification of distinctly localized Fes1 isoforms have implications for the understanding of the mechanisms underlying nucleo-cytoplasmic PQC. Study III reports on the mechanism that Fes1 employs to regulate Hsp70 function. Specifically Fes1 carries an N-terminal domain (NTD) that is conserved throughout the fungal kingdom. The NTD is flexible, modular and is required for the cellular function of Fes1. Importantly, the NTD forms ATP-sensitive complexes with Hsp70 suggesting that it competes substrates of the chaperone during Fes1-Hsp70 interactions. Study IV reports on methodological development for the efficient assembly of bacterial protein-expression plasmids using yeast homologous recombination cloning and the novel vector pSUMO-YHRC. The findings support the notion that Fes1 plays a key role in determining the fate of Hsp70-associated misfolded substrates and thereby target them for proteasomal degradation. From a broader perspective, the findings provide information essential to develop models that describe how Hsp70 function is regulated by different NEFs to participate in protein folding and degradation.

  • 65.
    Gowda, Naveen Kumar Chandappa
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kaimal, Jayasankar Mohanakrishnan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Masser, Anna E.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kang, Wenjing
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Friedländer, Marc R.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Cytosolic splice isoform of Hsp70 nucleotide exchange factor Fes1 is required for the degradation of misfolded proteins in yeast2016In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 27, no 8, p. 1210-1219Article in journal (Refereed)
    Abstract [en]

    Cells maintain proteostasis by selectively recognizing and targeting misfolded proteins for degradation. In Saccharomyces cerevisiae, the Hsp70 nucleotide exchange factor Fes1 is essential for the degradation of chaperone-associated misfolded proteins by the ubiquitin-proteasome system. Here we show that the FES1 transcript undergoes unique 3' alternative splicing that results in two equally active isoforms with alternative C-termini, Fes1L and Fes1S. Fes1L is actively targeted to the nucleus and represents the first identified nuclear Hsp70 nucleotide exchange factor. In contrast, Fes1S localizes to the cytosol and is essential to maintain proteostasis. In the absence of Fes1S, the heat-shock response is constitutively induced at normally non-stressful conditions. Moreover, cells display severe growth defects when elevated temperatures, amino acid analogues or the ectopic expression of misfolded proteins, induce protein misfolding. Importantly, misfolded proteins are not targeted for degradation by the ubiquitin-proteasome system. These observations support the notion that cytosolic Fes1S maintains proteostasis by supporting the removal of toxic misfolded proteins by proteasomal degradation. This study provides key findings for the understanding of the organization of protein quality control mechanisms in the cytosol and nucleus.

  • 66.
    Gowda, Naveen Kumar Chandappa
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kandasamy, Ganapathi
    Froehlich, Marceli S.
    Dohmen, R. Jürgen
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Hsp70 nucleotide exchange factor Fes1 is essential for ubiquitin-dependent degradation of misfolded cytosolic proteins2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 15, p. 5975-5980Article in journal (Refereed)
    Abstract [en]

    Protein quality control systems protect cells against the accumulation of toxic misfolded proteins by promoting their selective degradation. Malfunctions of quality control systems are linked to aging and neurodegenerative disease. Folding of polypeptides is facilitated by the association of 70 kDa Heat shock protein (Hsp70) molecular chaperones. If folding cannot be achieved, Hsp70 interacts with ubiquitylation enzymes that promote the proteasomal degradation of the misfolded protein. However, the factors that direct Hsp70 substrates toward the degradation machinery have remained unknown. Here, we identify Fes1, an Hsp70 nucleotide exchange factor of hitherto unclear physiological function, as a cytosolic triaging factor that promotes proteasomal degradation of misfolded proteins. Fes1 selectively interacts with misfolded proteins bound by Hsp70 and triggers their release from the chaperone. In the absence of Fes1, misfolded proteins fail to undergo polyubiquitylation, aggregate, and induce a strong heat shock response. Our findings reveal that Hsp70 direct proteins toward either folding or degradation by using distinct nucleotide exchange factors.

  • 67. Grantham, Julie
    et al.
    Lassing, Ingrid
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Karlsson, Roger
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Controlling the cortical actin motor2012In: Protoplasma, ISSN 0033-183X, E-ISSN 1615-6102, Vol. 249, no 4, p. 1001-1015Article, review/survey (Refereed)
    Abstract [en]

    Actin is the essential force-generating component of the microfilament system, which powers numerous motile processes in eukaryotic cells and undergoes dynamic remodeling in response to different internal and external signaling. The ability of actin to polymerize into asymmetric filaments is the inherent property behind the site-directed force-generating capacity that operates during various intracellular movements and in surface protrusions. Not surprisingly, a broad variety of signaling pathways and components are involved in controlling and coordinating the activities of the actin microfilament system in a myriad of different interactions. The characterization of these processes has stimulated cell biologists for decades and has, as a consequence, resulted in a huge body of data. The purpose here is to present a cellular perspective on recent advances in our understanding of the microfilament system with respect to actin polymerization, filament structure and specific folding requirements.

  • 68.
    Grenklo, Staffan
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Cross-linked Profilin:actin - A tool to study actin dynamics in non-muscle cells2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The microfilament system, consisting of actin and a number of auxiliary proteins, is fundamental for cell motility. Its dynamic organization depends on receptor-mediated signals, leading to rapid polymerizations and depolymerizations of actin. Profilin binds to non-filamentous actin, inhibits spontaneous filament formation, and functions as a regulator of actin polymerization. The profilin:actin complex, is thought to be the principal source of actin for filament formation although the role of profilin is not fully elucidated.

    In this thesis, a cross-linked profilin:actin complex (PxA), that retains the properties of ordinary profilin:actin, except for being non-dissociable, has been used to characterize the role of profilin and profilin:actin in non-muscle cells. A rapid screening method, employing PxA and based on the far western technique and mass-spectrometry, was designed to identify cellular components that specifically bind profilin:actin. Microinjection of PxA into cells infected with the bacteria Listeria monocytogenes impaired bacterial motility but a mutant PxA, unable to bind proline-rich sequences had no effect, demonstrating that profilin:actin is vital for the activity of the actin polymer-forming complex that the pathogen recruits to its surface upon infection.

    Fluorescence microscopy using two distinct sets of affinity-purified actin and profilin antibodies generated against PxA enabled localization of monomeric actin in cells. One of the actin and both profilin antibodies resulted in a dotted pattern of fluorescence partially aligning with microtubules whereas the other actin antibody detected filamentous actin. The result demonstrates extensive variability in epitope recognition, and indicates that unpolymerized actin, i.e. profilin:actin and maybe other complex-bound forms of actin, distributes in small packages that might be transported along microtubules. Microinjection of PxA into lamprey axons demonstrated the involvement of actin polymerization during synaptic signaling.

  • 69.
    Grenklo, Staffan
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Geese, Marcus
    Department of Cell Biology, Gesellschaft für Biotechnologische Forschung (GBF).
    Lindberg, Uno
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Department of Cell Biology.
    Wehland, Jürgen
    Department of Cell Biology, Gesellschaft für Biotechnologische Forschung (GBF).
    Karlsson, Roger
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Department of Cell Biology.
    Sechi, Antonio S
    Department of Cell Biology, Gesellschaft für Biotechnologische Forschung (GBF).
    A crucial role for profilin-actin in the intracellular motility of Listeria monocytogenes2003In: EMBO reports, ISSN 1469-221, Vol. 4, no 5, p. 523-529Article in journal (Refereed)
    Abstract [en]

    We have examined the effect of covalently crosslinked profilin–actin (PxA), which closely matches the biochemical properties of ordinary profilin–actin and interferes with actin polymerization in vitro and in vivo, on Listeria monocytogenes motility. PxA caused a marked reduction in bacterial motility, which was accompanied by the detachment of bacterial tails. The effect of PxA was dependent on its binding to proline-rich sequences, as shown by the inability of PH133SxA, which cannot interact with such sequences, to impair Listeria motility. PxA did not alter the motility of a Listeria mutant that is unable to recruit Ena (Enabled)/VASP (vasodilator-stimulated phosphoprotein) proteins and profilin to its surface. Finally, PxA did not block the initiation of actin-tail formation, indicating that profilin–actin is only required for the elongation of actin filaments at the bacterial surface. Our findings provide further evidence that profilin–actin is important for actin-based processes, and show that it has a key function in Listeria motility.

  • 70. Grundberg, Ida
    et al.
    Kiflemariam, Sara
    Mignardi, Marco
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Imgenberg-Kreuz, Juliana
    Edlund, Karolina
    Micke, Patrick
    Sundström, Magnus
    Sjöblom, Tobias
    Botling, Johan
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics2013In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 4, no 12, p. 2407-2418Article in journal (Refereed)
    Abstract [en]

    Current assays for somatic mutation analysis are based on extracts from tissue sections that often contain morphologically heterogeneous neoplastic regions with variable contents of genetically normal stromal and inflammatory cells, obscuring the results of the assays. We have developed an RNA-based in situ mutation assay that targets oncogenic mutations in a multiplex fashion that resolves the heterogeneity of the tissue sample. Activating oncogenic mutations are targets for a new generation of cancer drugs. For anti-EGFR therapy prediction, we demonstrate reliable in situ detection of KRAS mutations in codon 12 and 13 in colon and lung cancers in three different types of routinely processed tissue materials. High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray. Moreover, we show how the patterns of expressed mutated and wild-type alleles can be studied in situ in tumors with complex combinations of mutated EGFR, KRAS and TP53. This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy.

  • 71.
    Gubanova, Evgenia
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Science for Life Laboratory; Division of Translational Medicine and Chemical Biology; Department of Medical Biochemistry and Biophysics; Karolinska Institut; Stockholm, Sweden.
    Issaeva, Natalia
    Djureinovic, Tatjana
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    SMG-1 regulates senescence and suppresses epithelial-mesenchymal transitionManuscript (preprint) (Other academic)
  • 72.
    Gubanova, Evgenia
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Issaeva, Natalia
    Gokturk, Camilla
    Djureinovic, Tatjana
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Helleday, Thomas
    SMG-1 suppresses CDK2 and tumor growth by regulating both the p53 and Cdc25A signaling pathways2013In: Cell Cycle, ISSN 1538-4101, E-ISSN 1551-4005, Vol. 12, no 24, p. 3770-3780Article in journal (Refereed)
    Abstract [en]

    The DNA damage response is coordinated by phosphatidylinositol 3-kinase-related kinases, ATM, ATR, and DNA-PK. SMG-1 is the least studied stress-responsive member of this family. Here, we show that SMG-1 regulates the G 1/S checkpoint through both a p53-dependent, and a p53-independent pathway. We identify Cdc25A as a new SMG-1 substrate, and show that cells depleted of SMG-1 exhibit prolonged Cdc25A stability, failing to inactivate CDK2 in response to radiation. Given an increased tumor growth following depletion of SMG-1, our data demonstrate a novel role for SMG-1 in regulating Cdc25A and suppressing oncogenic CDK2 driven proliferation, confirming SMG-1 as a tumor suppressor.

  • 73.
    Gustafsson, Helena
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Adamson, Lars
    Hedander, Jan
    Walum, Erik
    Forsby, Anna
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Insulin-like growth factor type 1 upregulates uncoupling protein 3.2001In: Biochem Biophys Res Commun, ISSN 0006-291X, Vol. 287, no 5, p. 1105-11Article in journal (Refereed)
    Abstract [en]

    In this study the expression of uncoupling protein 3 (UCP3) and its regulation by insulin-like growth factor 1 (IGF-I) and insulin in human neuroblastoma SH-SY5Y cells were characterized. Reverse transcriptase-PCR, Western blot, and immunofluorescence analysis showed that SH-SY5Y cells express UCP3 natively. IGF-I induced a time- and concentration-dependent induction of UCP3 protein reaching a twofold expression after 72 h with 10 nM IGF-I. Extremely high insulin concentrations (860 nM) and 10 nM trIGF-I, a truncated form of IGF-I with the same affinity for the IGF-I receptor as the full-length IGF-I, but with lower activity on the insulin receptor, also upregulated UCP3. We conclude that SH-SY5Y cells express UCP3 natively and that the expression is regulated by IGF-I via the IGF-I receptor. Copyright 2001 Academic Press.

  • 74. Gustafsson, Nina M. S.
    et al.
    Färnegårdh, Katarina
    Stockholm University, Faculty of Science, Department of Organic Chemistry. Stockholm University, Science for Life Laboratory (SciLifeLab). Kancera AB, Sweden.
    Bonagas, Nadilly
    Ninou, Anna Huguet
    Groth, Petra
    Wiita, Elisee
    Jönsson, Mattias
    Hallberg, Kenth
    Lehto, Jemina
    Pennisi, Rosa
    Martinsson, Jessica
    Norström, Carina
    Hollers, Jessica
    Schultz, Johan
    Andersson, Martin
    Markova, Natalia
    Marttila, Petra
    Kim, Baek
    Norin, Martin
    Olin, Thomas
    Helleday, Thomas
    Targeting PFKFB3 radiosensitizes cancer cells and suppresses homologous recombination2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 3872Article in journal (Refereed)
    Abstract [en]

    The glycolytic PFKFB3 enzyme is widely overexpressed in cancer cells and an emerging anticancer target. Here, we identify PFKFB3 as a critical factor in homologous recombination (HR) repair of DNA double-strand breaks. PFKFB3 rapidly relocates into ionizing radiation (IR)-induced nuclear foci in an MRN-ATM-gamma H2AX-MDC1-dependent manner and co-localizes with DNA damage and HR repair proteins. PFKFB3 relocalization is critical for recruitment of HR proteins, HR activity, and cell survival upon IR. We develop KAN0438757, a small molecule inhibitor that potently targets PFKFB3. Pharmacological PFKFB3 inhibition impairs recruitment of ribonucleotide reductase M2 and deoxynucleotide incorporation upon DNA repair, and reduces dNTP levels. Importantly, KAN0438757 induces radiosensitization in transformed cells while leaving non-transformed cells unaffected. In summary, we identify a key role for PFKFB3 enzymatic activity in HR repair and present KAN0438757, a selective PFKFB3 inhibitor that could potentially be used as a strategy for the treatment of cancer.

  • 75. Hansson Petersen, Camilla A.
    et al.
    Alikhani, Nyosha
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Behbahani, Homira
    Wiehager, Birgitta
    Pavlov, Pavel F
    Alafuzoff, Irina
    Leinonen, Ville
    Ito, Akira
    Winblad, Bengt
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ankarcrona, Maria
    The amyloid beta-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 35, p. 13145-13150Article in journal (Refereed)
    Abstract [en]

    The amyloid beta-peptide (Abeta) has been suggested to exert its toxicity intracellularly. Mitochondrial functions can be negatively affected by Abeta and accumulation of Abeta has been detected in mitochondria. Because Abeta is not likely to be produced locally in mitochondria, we decided to investigate the mechanisms for mitochondrial Abeta uptake. Our results from rat mitochondria show that Abeta is transported into mitochondria via the translocase of the outer membrane (TOM) machinery. The import was insensitive to valinomycin, indicating that it is independent of the mitochondrial membrane potential. Subfractionation studies following the import experiments revealed Abeta association with the inner membrane fraction, and immunoelectron microscopy after import showed localization of Abeta to mitochondrial cristae. A similar distribution pattern of Abeta in mitochondria was shown by immunoelectron microscopy in human cortical brain biopsies obtained from living subjects with normal pressure hydrocephalus. Thus, we present a unique import mechanism for Abeta in mitochondria and demonstrate both in vitro and in vivo that Abeta is located to the mitochondrial cristae. Importantly, we also show that extracellulary applied Abeta can be internalized by human neuroblastoma cells and can colocalize with mitochondrial markers. Together, these results provide further insight into the mitochondrial uptake of Abeta, a peptide considered to be of major significance in Alzheimer's disease.

  • 76.
    Hedengren, Marika
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Borge, Karin
    Umeå Centre for Molecular Pathogenesis, Umeå University.
    Hultmark, Dan
    Umeå Centre for Molecular Pathogenesis, Umeå University.
    Expression and evolution of the Drosophila attacin/diptericin gene family2000In: Biochemical and Biophysical Research Communications, ISSN 0006-291X, Vol. 279, no 2, p. 574-81Article in journal (Refereed)
    Abstract [en]

    We describe the genes for three new glycine-rich antimicrobial peptides in (support the proposal that these glycine-rich antimicrobial peptides evolved from a common ancestor and are probably also related to proline-rich peptides such as drosocin. Drosophila, two attacinsAttC and AttD) and one diptericin (DptB). Their structuresAttC is similar to the nearby  AttA on a different chromosome. Intriguingly, and AttB genes. AttD is more divergent and locatedAttD  may encode an intracellular attacin tandem to the closely related DptB is linked inDiptericin. However, the  DptB and may be processed in an attacin-like fashion. All attacin and diptericin genes are induced after bacterial challenge. This induction is reduced in and unexpectedly also in gene product contains a furin-like cleavage siteimd mutants,Tl2 mutants. The 18w  mutation particularly affects the induction of AttC,  which may be a useful marker for 18w signaling.

  • 77.
    Hedengren, Marika
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Åsling, Bengt
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Dushay, Mitchell
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Ando, Istvan
    Umeå Center for Molecular Pathogenesis, Umeå university.
    Ekengren, Sophia
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Wihlborg, Margareta
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Hultmark, Dan
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Relish, a central factor in the control of humoral but not cellular immunity in Drosophila1999In: Molecular cell, ISSN 1097-2765, Vol. 4, no 5, p. 827-37Article in journal (Refereed)
    Abstract [en]

    The NF-κB-like Relish gene is complex, with four transcripts that are all located within an intron of the Nmdmc gene. Using deletion mutants, we show that Relish is specifically required for the induction of the humoral immune response, including both antibacterial and antifungal peptides. As a result, the Relish mutants are very sensitive to infection. A single cell of E. cloacae is sufficient to kill a mutant fly, and the mutants show increased susceptibility to fungal infection. In contrast, the blood cell population, the hematopoietic organs, and the phagocytic, encapsulation, and melanization responses are normal. Our results illustrate the importance of the humoral response in Drosophila immunity and demonstrate that Relish plays a key role in this response.

  • 78.
    Hedengren-Olcott, Marika
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Olcott, Micael C
    Dept. of Microbiology, Oregon State University, Corvallis.
    Mooney, Duan T
    Ekengren, Sophia
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Geller, Bruce L
    Taylor, Barbara J
    Differential activation of the NF-kappaB-like factors Relish and Dif in Drosophila melanogaster by fungi and gram-positive bacteria2004In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 279, no 20, p. 21121-7Article in journal (Refereed)
    Abstract [en]

    The current model of immune activation in Drosophila melanogaster suggests that fungi and Gram-positive (G+) bacteria activate the Toll/Dif pathway and that Gram-negative (G-) bacteria activate the Imd/Relish pathway. To test this model, we examined the response of Relish and Dif (Dorsal-related immunity factor) mutants to challenge by various fungi and G+ and G- bacteria. In Relish mutants, the Cecropin A gene was induced by the G+ bacteria Micrococcus luteus and Staphylococcus aureus, but not by other G+ or G- bacteria. This Relish-independent Cecropin A induction was blocked in Dif/Relish double mutant flies. Induction of the Cecropin A1 gene by M. luteus required Relish, whereas induction of the Cecropin A2 gene required Dif. Intact peptidoglycan (PG) was necessary for this differential induction of Cecropin A. PG extracted from M. luteus induced Cecropin A in Relish mutants, whereas PGs from the G+ bacteria Bacillus megaterium and Bacillus subtilis did not, suggesting that the Drosophila immune system can distinguish PGs from various G+ bacteria. Various fungi stimulated antimicrobial peptides through at least two different pathways requiring Relish and/or Dif. Induction of Attacin A by Geotrichum candidum required Relish, whereas activation by Beauvaria bassiana required Dif, suggesting that the Drosophila immune system can distinguish between at least these two fungi. We conclude that the Drosophila immune system is more complex than the current model. We propose a new model to account for this immune system complexity, incorporating distinct pattern recognition receptors of the Drosophila immune system, which can distinguish between various fungi and G+ bacteria, thereby leading to selective induction of antimicrobial peptides via differential activation of Relish and Dif.

  • 79.
    Hedman, Rickard
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dynamics of peptide chains during co-translational translocation, membrane integration & domain folding2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The biosynthesis of proteins occurs at the ribosomes, where amino acids are linked together into linear chains. Nascent protein chains may undergo several different processes during their synthesis. Some proteins begin to fold, while others interact with chaperones, targeting factors or processing enzymes. Nascent membrane proteins are targeted to the cell membrane for integration, which involves the translocation of periplasmic domains and the insertion of membrane-embedded parts.

    The aim of this thesis was to gain insights about the dynamics of nascent peptide chains undergoing folding, membrane translocation and integration. To this end, we explored the use of arrest peptides (APs) as force sensors. APs stall ribosomes when translated unless there is tension in the nascent peptide chain: the higher the tension, the more full-length protein can be detected. By using APs, we could show that a transmembrane helix is strongly ‘pulled’ twice on its way into the membrane and that strong electric forces act on negatively charged peptide segments translocating through the membrane. Furthermore, we discovered that APs could be used to detect protein folding and made the surprising discovery that a small protein domain folded well inside the ribosomal tunnel. Finally, we explored the arrest-stability of a large set of AP variants and found two extremely stable APs.

  • 80.
    Hildenbeutel, Markus
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hegg, Eric L.
    Stephan, Katharina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gruschke, Steffi
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Meunier, Brigitte
    Ott, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Assembly factors monitor sequential hemylation of cytochrome b to regulate mitochondria! translation2014In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 205, no 4, p. 511-524Article in journal (Refereed)
    Abstract [en]

    Mitochondrial respiratory chain complexes convert chemical energy into a membrane potential by connecting electron transport with charge separation. Electron transport relies on redox cofactors that occupy strategic positions in the complexes. How these redox cofactors are assembled into the complexes is not known. Cytochrome b, a central catalytic subunit of complex III, contains two henne bs. Here, we unravel the sequence of events in the mitochondrial inner membrane by which cytochrome b is hemylated. Heme incorporation occurs in a strict sequential process that involves interactions of the newly synthesized cytochrome b with assembly factors and structural complex III subunits. These interactions are functionally connected to cofactor acquisition that triggers the progression of cytochrome b through successive assembly intermediates. Failure to hemylate cytochrome b sequesters the Cbp3-Cbp6 complex in early assembly intermediates, thereby causing a reduction in cytochrome b synthesis via a feedback loop that senses hemylation of cytochrome b.

  • 81.
    Hjelm, Anna
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schlegel, Susan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Baumgarten, Thomas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Klepsch, Mirjam
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wickström, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Drew, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Optimizing E. coli-Based Membrane Protein Production Using Lemo21(DE3) and GFP-Fusions2013In: Membrane Biogenesis: Methods and Protocols / [ed] Doron Rapaport, Johannes M. Herrmann, Totowa, USA: Humana Press, 2013, p. 381-400Chapter in book (Refereed)
    Abstract [en]

    Optimizing the conditions for the overexpression of membrane proteins in E. coli and their subsequent purification is usually a laborious and time-consuming process. Combining the Lemo21(DE3) strain, which conveniently allows to identify the optimal expression intensity of a membrane protein using only one strain, and membrane proteins C-terminally fused to Green Fluorescent Protein (GFP) greatly facilitates the production of high-quality membrane protein material for functional and structural studies.

  • 82.
    Hossein, Moradi
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Simoff, Ivailo
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Bartish, Galyna
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Nygård, Odd
    Functional features of the C-terminal region of yeast ribosomal protein L52008In: Molecular Genetics and Genomics, ISSN 1617-4615, E-ISSN 1617-4623, Vol. 280, no 4, p. 337-350Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to analyze the functional importance of the C-terminus of the essential yeast ribosomal protein L5 (YrpL5). Previous studies have indicated that the C-terminal region of YrpL5 forms an α-helix with a positively charged surface that is involved in protein–5S rRNA interaction. Formation of an YrpL5·5S rRNA complex is a prerequisite for nuclear import of YrpL5. Here we have tested the importance of the α-helix and the positively charged surface for YrpL5 function in Saccharomyces cerevisiae using site directed mutagenesis in combination with functional complementation. Alterations in the sequence forming the putative α-helix affected the functional capacity of YrpL5. However, the effect did not correlate with a decreased ability of the protein to bind to 5S rRNA as all rpL5 mutants tested were imported to the nucleus whether or not the α-helix or the positively charged surface were intact. The alterations introduced in the C-terminal sequence affected the growth rate of cells expressing mutant but functional forms of YrpL5. The reduced growth rate was correlated with a reduced ribosomal content per cell indicating that the alterations introduced in the C-terminus interfered with ribosome assembly.

  • 83. Huenchuguala, Sandro
    et al.
    Munoz, Patricia
    Zavala, Patricio
    Villa, Monica
    Cuevas, Carlos
    Ahumada, Ulises
    Graumann, Rebecca
    Nore, Beston F.
    Couve, Eduardo
    Mannervik, Bengt
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Paris, Irmgard
    Segura-Aguilar, Juan
    Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction2014In: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635, Vol. 10, no 4, p. 618-630Article in journal (Refereed)
    Abstract [en]

    U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit H-3-dopamine uptake, which is inhibited by 2 mu M of nomifensine and 15 mu M of estradiol. We generated a stable cell line (U373MGsiGST6) expressing an siRNA against GSTM2 that resulted in low GSTM2 expression (26% of wild-type U373MG cells). A significant increase in cell death was observed when U373MGsiGST6 cells were incubated with 50 mu M purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 mu M aminochrome resulted in the formation of autophagic vacuoles containing undigested cellular components, as determined using transmission electron microscopy. A significant increase in autophagosomes was determined by measuring endogenous LC3-II, a significant decrease in cell death was observed in the presence of bafilomycin A(1), and a significant increase in cell death was observed in the presence of trehalose. A significant increase in LAMP2 immunostaining was observed, a significant decrease in bright red fluorescence of lysosomes with acridine orange was observed, and bafilomycin A(1) pretreatment reduced the loss of lysosome acidity. A significant increase in cell death was observed in the presence of lysosomal protease inhibitors. Aggregation of TUBA/-tubulin (tubulin, ) and SQSTM1 protein accumulation were also observed. Moreover, a significant increase in the number of lipids droplets was observed compared with U373MG cells with normal expression of GSTM2. These results support the notion that GSTM2 is a protective enzyme against aminochrome toxicity in astrocytes and that aminochrome cell death in U373MGsiGST6 cells involves autophagic-lysosomal dysfunction.

  • 84.
    Hugosson, Marie
    Stockholm University, Faculty of Science.
    Intracellular protein transport in plants: targeting, sorting and processing1996Doctoral thesis, comprehensive summary (Other academic)
  • 85.
    Ivanova, Elena V.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Caspase activation in human neuroblastoma cells: mechanisms and spatiotemporal aspects2015Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Apoptosis is one of the modes of programmed cell death, in which several members of the caspase family of proteases play the central role. However, activation of apoptotic caspases does not necessarily lead to cell death. Instead, these caspases may mediate, for instance, differentiation or synaptic plasticity, if their activity is restricted in space and time. Such localized caspase activation has been also implicated in the initial stages of neurodegeneration. In order to assess this kind of events at a subcellular level, our research group has previously constructed tau-anchored FRET-based caspase sensors (tAFSs). Here, we demonstrate that localization of tAFSs to the cytoskeleton results in enrichment of the sensors in neuritic processes and enables increased spatiotemporal resolution for live cell imaging of caspase activation, as compared to soluble FRET sensors. This feature is particularly beneficial for investigation of neurodegeneration-related processes.

    tAFSs were further employed for investigation of caspase activation in neuroblastoma, an extracranial solid pediatric tumor. Tumor necrosis factor-related apoptosis inducing factor (TRAIL) is a promising candidate for cancer treatment due to its ability to selectively trigger apoptosis malignant cells. However, many cancer cells, including neuroblastoma, acquire resistance to TRAIL. Here, we show that in S-type neuroblastoma cell lines, TRAIL resistance is dependent on incomplete activation of apoptotic caspase-3. Sensitization to TRAIL was achieved with protein kinase C (PKC)-inhibiting compounds, suggesting a role for this kinase in blocking the apoptotic response to TRAIL. This effect of PKC could possibly involve stabilization of XIAP, an endogenous caspase inhibitor, as PKC inhibition, in combination with TRAIL treatment, led to downregulation of XIAP.

  • 86.
    Jafari-Mamaghani, Mehrdad
    Stockholm University, Faculty of Science, Department of Mathematics.
    A Treatise on Measuring Wiener-Granger Causality2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Wiener-Granger causality is a well-established concept of causality based on stochasticity and the flow of time, with applications in a broad array of quantitative sciences. The majority of methods used to measure Wiener-Granger causality are based on linear premises and hence insensitive to non-linear signals. Other frameworks based on non-parametric techniques are often computationally expensive and susceptible to overfitting or lack of sensitivity.

    In this thesis, Paper I investigates the application of linear Wiener-Granger causality to migrating cancer cell data obtained using a Systems Microscopy experimental platform. Paper II represents a review of non-parametric measures based on information theory and discusses a number of related bottlenecks and potential routes of circumvention. Paper III studies the properties of a frequently used non-parametric information theoretical measure for a class of non-Gaussian distributions. Paper IV introduces a new efficient scheme for non-parametric analysis of Wiener-Granger causality based on kernel canonical correlations, and studies the connection between this new scheme and the information theoretical approach. Lastly, Paper V draws upon the results in the preceding paper to discuss non-parametric analysis of Wiener-Granger causality in partially observed systems.

    Altogether, the work presented in this thesis constitutes a comprehensive review on measures of Wiener-Granger causality in general, and in particular, features new insights on efficient non-parametric analysis of Wiener-Granger causality in high-dimensional settings.

  • 87.
    Jaffer Ali, Mohammed Hakim
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Multifaceted roles of the transmembrane nuclear envelope protein, Samp12017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The eukaryotic nuclear envelope (NE), separates the nucleoplasm from cytoplasm and is made up of two concentric lipid membranes, the outer and the inner nuclear membranes (ONM and INM), the nuclear pore complexes (NPCs) and an underlying filamentous nuclear lamina. The INM contains hundreds of unique transmembrane proteins of which only a handful have been characterized. In this thesis, I aimed to understand the functional organization of proteins in the nuclear envelope and I focused on investigating the functions of a recently identified INM transmembrane protein, Samp1. We have developed a novel and robust approach, MCLIP, to identify specific protein-protein interactions taking place in live cells. Using MCLIP, we have shown that Samp1 interacts with proteins of the LINC complex, the nuclear lamina and components of the mitotic spindle. Samp1's specific interactions with a variety of binding partners, suggest that Samp1 plays important roles both in interphase and in mitosis.  We have also shown that Samp1 can provide a binding site at the INM for the GTPase Ran, a master regulator of protein interactions in interphase and in mitosis. Furthermore, we have also investigated the role of Samp1 in cell differentiation using two independent model systems. In human iPSCs, ectopic expression of Samp1 promoted differentiation despite pluripotent culture conditions. In C2C12 myoblast, depletion of Samp1 completely blocked differentiation into myotubes. The two studies complement each other and suggest that Samp1 has a strong differentiation promoting activity. Taken together, the findings in this thesis, give insights on the unexpected and unforeseen roles played by a transmembrane protein in different fundamental cellular process.

  • 88. Jemth, Ann-Sofie
    et al.
    Gustafsson, Robert
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bräutigam, Lars
    Henriksson, Linda
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Desroses, Matthieu
    Carreras Puigvert, Jordi
    Homan, Evert
    Warpman Berglund, Ulrika
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Helleday, Thomas
    MutT homologue 1 (MTH1) catalyses the hydrolysis of mutagenic O6-methyl-dGTPManuscript (preprint) (Other academic)
  • 89.
    Jenvert, Rose-Marie
    Stockholm University, Faculty of Science, Wenner-Gren Institute for Experimental Biology.
    The ribosome, stringent factor and the bacterial stringent response2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The stringent response plays a significant role in the survival of bacteria during different environmental conditions. It is activated by the binding of stringent factor (SF) to stalled ribosomes that have an unacylated tRNA in the ribosomal A-site which leads to the synthesis of (p)ppGpp. ppGpp binds to the RNA polymerase, resulting in a rapid down-regulation of rRNA and tRNA transcription and up-regulation of mRNAs coding for enzymes involved in amino acid biosynthesis. The importance of the A-site and unacylated tRNA in the activation of SF was confirmed by chemical modification and subsequent primer extension experiments (footprinting experiments) which showed that binding of SF to ribosomes resulted in the protection of regions in 23S rRNA, the A-loop and helix 89 that are involved in the binding of the A-site tRNA. An in vitro assay showed that the ribosomal protein L11 and its flexible N-terminal part was important in the activation of SF. Interestingly the N-terminal part of L11 was shown to activate SF on its own and this activation was dependent on both ribosomes and an unacylated tRNA in the A-site. The N-terminal part of L11 was suggested to mediate an interaction between ribosome-bound SF and the unacylated tRNA in the A-site or interact with SF and the unacylated tRNA independently of each other. Footprinting experiments showed that SF bound to the ribosome protected bases in the L11 binding domain of the ribosome that were not involved in an interaction with ribosomal protein L11. The sarcin/ricin loop, in close contact with the L11 binding domain on the ribosome and essential for the binding and activation of translation elongation factors was also found to be protected by the binding of SF. Altogether the presented results suggest that SF binds to the factor-binding stalk of the ribosome and that activation of SF is dependent on the flexible N-terminal domain of L11 and an interaction of SF with the unacylated tRNA in the A-site of the 50S subunit.

  • 90.
    Jin, Haining
    Stockholm University.
    Functional studies of mRNA in translation initation and termination in E. coli2002Doctoral thesis, comprehensive summary (Other academic)
  • 91.
    Johansson, Hans Erik
    Stockholm University, Faculty of Science.
    Eukaryotic translation initiation: natural and artificial control1994Doctoral thesis, comprehensive summary (Other academic)
  • 92.
    Johansson, Thomas
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Profilin:actin in cell motility: A search for profilin:actin binding proteins2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The profilin:actin complex is a major source of actin for actin filament growth in vivo. A number of proteins regulating either profilin or actin has been described since profilin:actin was isolated during the 1970s. Since then, profilin and actin and their binding partners have been intensively studied. The ability of profilin:actin to interact with the fast polymerizing end of actin filaments focus the interest to components that regulates this interaction; this is the theme in this thesis.

    A chemically cross-linked and therefore non-dissociable profilin:actin complex, called PxA, was used in these studies which led to development of a rapid screening method to search for proteins that bind to profilin:actin. The method allows a simultaneous detection of proteins that separately interact with profilin, actin and/or profilin:actin. Here the technique was used to screen cell and tissue extracts, before and after gel filtration, for components that showed a unique interaction with the profilin:actin complex. Mass spectrometry was then used for their identification. Furthermore it was demonstrated that profilin:actin binding components are present in RNA containing, large molecular weight complexes.

    Two different PxA immunizations generated two separate populations of affinity purified profilin and actin antibodies. The actin antibodies from these two populations showed significant differences in the staining pattern when used for fluorescence microscopy of tissue cultured cells. One of these appeared to bind monomeric actin while the other bound to filamentous actin. Both of the profilin antibody preparations stained cells in a dotted pattern. The distribution of epitopes recognized by the different actin antibody preparations was determined using a combination of protease digestion, gel electrophoresis and mass spectrometry. The result demonstrated partially different epitope recognition.

    The actin associated protein palladin contains sequence motifs typical for profilin-binding proteins suggesting that profilin may bind palladin. The potential profilin-palladin interaction was studied using a combination of biochemical and histochemical techniques. The interaction was observed in vitro, and the two proteins co-distributed in actin rich regions in tissue cultured cells. These results suggest that palladin recruits profilin and/or profilin:actin to sites of actin dynamics.

  • 93.
    Johnsson, Anna-Karin
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    The Actin Filament System: Its Involvement in Cell Migration and Neurotransmitter Release2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The microfilament system consists of actin filaments as the major component and is regulated by a number of actin binding proteins. It is juxtaposed to the plasma membrane where it forms a dense cortical weave from where it pervades into the cell interior. This filament system has multiple roles and participates in virtually all motile processes where its dynamic activities depend on receptor mediated signaling leading to constant polymerizations and depolymerizations. These activities are now also known to affect gene regulation. This thesis discusses these dynamic reorganizations of the microfilament system and how components are supplied to support these motile processes. The focus is on profilin/profilin:actin, actin polymerization and the localization of the transcripts of these proteins.

    The localization of profilin mRNA was examined in relation to the distribution of β-actin mRNA using fluorescent in situ hybridization. It was concluded that both these mRNAs localize to sites of massive actin polymerization called dorsal ruffles albeit the data obtained suggests that this localization must be dependent on distinct mechanisms. Additionally signal transduction and cell motility was studied after depleting the two profilin isoforms 1 and 2. The activity of the transcription factor SRF is known to be coupled to microfilament system dynamics via the cofactor MAL which binds to actin monomers and is released upon receptor mediated actin polymerization. Depletion of profilin was seen to influence SRF dependent signaling, most likely because the lack of profilin enables more MAL to bind actin monomers thereby preventing SRF dependent transcription. Finally, it was also investigated how the synaptic vesicle protein synaptotagmin 1 which is involved in exocytosis, has a role in actin polymerization. This protein has previously been described to cause filopodia formation when ectopically expressed. A polybasic sequence motif was identified as the effector sequence for this activity and it was established that this sequence interacts with anionic lipids. It is also discussed how this sequence could have a role in neurotransmitter release and actin polymerization in the nerve synapse.

  • 94.
    Johnsson, Anna-Karin
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Karlsson, Roger
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Synaptotagmin 1 causes phosphatidyl inositol lipid-dependent actin remodeling in cultured non-neuronal and neuronal cells2012In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 318, no 2, p. 114-126Article in journal (Refereed)
    Abstract [en]

    Here we demonstrate that a dramatic actin polymerizing activity caused by ectopic expression of the synaptic vesicle protein synaptotagmin 1 that results in extensive filopodia formation is due to the presence of a lysine rich sequence motif immediately at the cytoplasmic side of the transmembrane domain of the protein. This polybasic sequence interacts with anionic phospholipids in vitro, and, consequently, the actin remodeling caused by this sequence is interfered with by expression of a phosphatidyl inositol (4,5)-bisphosphate (PIP2)-targeted phosphatase, suggesting that it intervenes with the function of PIP2-binding actin control proteins. The activity drastically alters the behavior of a range of cultured cells including the neuroblastoma cell line SH-SY5Y and primary cortical mouse neurons, and, since the sequence is conserved also in synaptotagmin 2, it may reflect an important fine-tuning role for these two proteins during synaptic vesicle fusion and neurotransmitter release.

  • 95. 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.

  • 96.
    Kader, Md Abdul
    et al.
    Stockholm University, Faculty of Science, Department of Botany.
    Rasmusson, Allan
    University of Lund.
    Lindberg, Sylvia
    Stockholm University, Faculty of Science, Department of Botany.
    Expressions of HKT members in salt-tolerant and salt-sensitive rice cultivars are differentially regulated under salinity stress2009In: 9th IMPB Congress October 2009: Abiotic stress, Water, salts, minerals, 2009Conference paper (Other academic)
  • 97.
    Kaimal, Jayasankar Mohanakrishnan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Regulation of cellular Hsp70: Proteostasis and aggregate management2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Proteins have to be folded to their native structures to be functionally expressed. Misfolded proteins are proteotoxic and negatively impact on cellular fitness. To maintain the proteome functional proteins are under the constant surveillance of dedicated molecular chaperones that perform protein quality control (PQC). Using the model organism yeast Saccharomyces cerevisiae this thesis investigates the molecular mechanisms that cells employ to maintain protein homeostasis (proteostasis). In Study I the role of the molecular chaperone Hsp110 in the disentanglement and reactivation of aggregated proteins was investigated. We found that Hsp110 is essential for cellular protein disaggregation driven by the molecular chaperones Hsp40, Hsp70 and Hsp104 and characterized its involvement via regulation of Hsp70 ATPase activity as a nucleotide exchange factor. In Study II we found out that Hsp110 undergoes translational frameshifting during its expression resulting in a nuclear targeting. Nuclear Hsp110 interacts with Hsp70 and reprograms the proteostasis system to better deal with stress and to confer longevity. Study III describes regulation of Hsp70 function in PQC by the nucleotide exchange factor Fes1. We found that rare alternative splicing regulates Fes1 subcellular localization in the cytosol and nucleus and that the cytosolic isoform has a key role in PQC. In Study IV we have revealed the molecular mechanism that Fes1 employ in PQC. We show that Fes1 carries a specialized release domain (RD) that ensures the efficient release of protein substrates from Hsp70, explaining how Fes1 maintains the Hsp70-chaperone system clear of persistent misfolded proteins. In Study V we report on the use of a novel bioluminescent reporter (Nanoluc) for use in yeast to measure the gene expression and protein levels. In summary, this thesis contributes to the molecular understanding of chaperone-dependent PQC mechanisms both at the level of individual components as well as how they interact to ensure proteostasis.

  • 98.
    Kaimal, Jayasankar Mohanakrishnan
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Habernig, Lukas
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Büttner, Sabrina
    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.
    Nuclear targeting of Hsp110 modifies the proteostasis system by mobilizing latent Hsp70 chaperonesManuscript (preprint) (Other academic)
  • 99.
    Kalinovich, Anastasia V.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    de Jong, Jasper M. A.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Cannon, Barbara
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nedergaard, Jan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    UCP1 in adipose tissues: two steps to full browning2017In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 134, p. 127-137Article in journal (Refereed)
    Abstract [en]

    The possibility that brown adipose tissue thermogenesis can be recruited in order to combat the development of obesity has led to a high interest in the identification of "browning agents", i.e. agents that increase the amount and activity of UCP1 in brown and brite/beige adipose tissues. However, functional analysis of the browning process yields confusingly different results when the analysis is performed in one of two alternative steps. Thus, in one of the steps, using cold acclimation as a potent model browning agent, we find that if the browning process is followed in mice initially housed at 21 °C (the most common procedure), there is only weak molecular evidence for increases in UCP1 gene expression or UCP1 protein abundance in classical brown adipose tissue; however, in brite/beige adipose depots, there are large increases, apparently associating functional browning with events only in the brite/beige tissues. Contrastingly, in another step, if the process is followed starting with mice initially housed at 30 °C (thermoneutrality for mice, thus similar to normal human conditions), large increases in UCP1 gene expression and UCP1 protein abundance are observed in the classical brown adipose tissue depots; there is then practically no observable UCP1 gene expression in brite/beige tissues. This apparent conundrum can be resolved when it is realized that the classical brown adipose tissue at 21 °C is already essentially fully differentiated and thus expands extensively through proliferation upon further browning induction, rather than by further enhancing cellular differentiation. When the limiting factor for thermogenesis, i.e. the total amount of UCP1 protein per depot, is analyzed, classical brown adipose tissue is by far the predominant site for the browning process, irrespective of which of the two steps is analyzed. There are to date no published data demonstrating that alternative browning agents would selectively promote brite/beige tissues versus classical brown tissue to a higher degree than does cold acclimation. Thus, to restrict investigations to examine adipose tissue depots where only a limited part of the adaptation process occurs (i.e. the brite/beige tissues) and to use initial conditions different from the thermoneutrality normally experienced by adult humans may seriously hamper the identification of therapeutically valid browning agents. The data presented here have therefore important implications for the analysis of the potential of browning agents and the nature of human brown adipose tissue.

  • 100.
    Kanatani, Sachie
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Fuks, Jonas M.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Olafsson, Einar B.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Westermark, Linda
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Chambers, Benedict
    Varas-Godoy, Manuel
    Uhlén, Per
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Voltage-dependent calcium channel signaling mediates GABA(A) receptor-induced migratory activation of dendritic cells infected by Toxoplasma gondii2017In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 13, no 12, article id e1006739Article in journal (Refereed)
    Abstract [en]

    The obligate intracellular parasite Toxoplasma gondii exploits cells of the immune system to disseminate. Upon T. gondii-infection,. Upsilon-aminobutyric acid (GABA)/GABAA receptor signaling triggers a hypermigratory phenotype in dendritic cells (DCs) by unknown signal transduction pathways. Here, we demonstrate that calcium (Ca2+) signaling in DCs is indispensable for T. gondii-induced DC hypermotility and transmigration in vitro. We report that activation of GABAA receptors by GABA induces transient Ca2+ entry in DCs. Murine bone marrow-derived DCs preferentially expressed the L-type voltage-dependent Ca2+ channel (VDCC) subtype Cav1.3. Silencing of Cav1.3 by short hairpin RNA or selective pharmacological antagonism of VDCCs abolished the Toxoplasma-induced hypermigratory phenotype. In a mouse model of toxoplasmosis, VDCC inhibition of adoptively transferred Toxoplasma-infected DCs delayed the appearance of cell-associated parasites in the blood circulation and reduced parasite dissemination to target organs. The present data establish that T. gondii-induced hypermigration of DCs requires signaling via VDCCs and that Ca2+ acts as a second messenger to GABAergic signaling via the VDCC Cav1.3. The findings define a novel motility-related signaling axis in DCs and unveil that interneurons and DCs share common GABAergic motogenic pathways. T. gondii employs GABAergic non-canonical pathways to induce host cell migration and facilitate dissemination.

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