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  • 1. Addario, Barbara
    et al.
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Persson, Karina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Backman, Lars
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Characterisation of Schizosaccharomyces pombe alpha-actinin2016Ingår i: PeerJ, ISSN 2167-8359, E-ISSN 2167-8359, Vol. 4, artikel-id e1858Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The actin cytoskeleton plays a fundamental role in eukaryotic cells. Its reorganization is regulated by a plethora of actin-modulating proteins, such as a-actinin. In higher organisms, alpha-actinin is characterized by the presence of three distinct structural domains: an N-terminal actin-binding domain and a C-terminal region with EF-hand motif separated by a central rod domain with four spectrin repeats. Sequence analysis has revealed that the central rod domain of alpha-actinin from the fission yeast Schizosaccharomyces pombe consists of only two spectrin repeats. To obtain a firmer understanding of the structure and function of this unconventional alpha-actinin, we have cloned and characterized each structural domain. Our results show that this alpha-actinin isoform is capable of forming dimers and that the rod domain is required for this. However, its actin-binding and cross-linking activity appears less efficient compared to conventional alpha-actinins. The solved crystal structure of the actin-binding domain indicates that the closed state is stabilised by hydrogen bonds and a salt bridge not present in other a-actinins, which may reduce the affinity for actin.

  • 2. Bieling, Peter
    et al.
    Laan, Liedewij
    Schek, Henry
    Munteanu, E Laura
    Sandblad, Linda
    European Mol Biol Lab, Cell Biol & Biophys Unit, D-69117 Heidelberg, Germany .
    Dogterom, Marileen
    Brunner, Damian
    Surrey, Thomas
    Reconstitution of a microtubule plus-end tracking system in vitro2007Ingår i: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 450, nr 7172, s. 1100-1105Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The microtubule cytoskeleton is essential to cell morphogenesis. Growing microtubule plus ends have emerged as dynamic regulatory sites in which specialized proteins, called plus-end-binding proteins (+TIPs), bind and regulate the proper functioning of microtubules. However, the molecular mechanism of plus-end association by +TIPs and their ability to track the growing end are not well understood. Here we report the in vitro reconstitution of a minimal plus-end tracking system consisting of the three fission yeast proteins Mal3, Tip1 and the kinesin Tea2. Using time-lapse total internal reflection fluorescence microscopy, we show that the EB1 homologue Mal3 has an enhanced affinity for growing microtubule end structures as opposed to the microtubule lattice. This allows it to track growing microtubule ends autonomously by an end recognition mechanism. In addition, Mal3 acts as a factor that mediates loading of the processive motor Tea2 and its cargo, the Clip170 homologue Tip1, onto the microtubule lattice. The interaction of all three proteins is required for the selective tracking of growing microtubule plus ends by both Tea2 and Tip1. Our results dissect the collective interactions of the constituents of this plus-end tracking system and show how these interactions lead to the emergence of its dynamic behaviour. We expect that such in vitro reconstitutions will also be essential for the mechanistic dissection of other plus-end tracking systems.

  • 3.
    Bonde, Mari
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Olofsson, Annelie
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Frost, Mikaela
    Jegerschöld, Caroline
    Karolinska Institutet, Sweden.
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Structural analysis of the B. burgdorferi integral outer membrane protein, P13, in lipid bilayer NanodiscsManuskript (preprint) (Övrig (populärvetenskap, debatt, mm))
  • 4.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gharibyan, Anna L.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Islam, Tohidul
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Iakovleva, Irina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Nilsson, Lina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lee, Cheng Choo
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Pamrén, Annelie
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Scanning electron microscopy as a tool for evaluating morphology of amyloid structures formed on surface plasmon resonance chips2018Ingår i: Data in Brief, E-ISSN 2352-3409, Vol. 19, s. 1166-1170Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We demonstrate the use of Scanning Electron microscopy (SEM) in combination with Surface Plasmon Resonance (SPR) to probe and verify the formation of amyloid and its morphology on an SPR chip. SPR is a technique that measures changes in the immobilized weight on the chip surface and is frequently used to probe the formation and biophysical properties of amyloid structures. In this context it is of interest to also monitor the morphology of the formed structures. The SPR chip surface is made of a layer of gold, which represent a suitable material for direct analysis of the surface using SEM. The standard SPR chip used here (CM5-chip, GE Healthcare, Uppsala, Sweden) can easily be disassembled and directly analyzed by SEM. In order to verify the formation of amyloid fibrils in our experimental conditions we analyzed also in-solution produced structures by using Transmission Electron Microscopy (TEM). For further details and experimental findings, please refer to the article published in Journal of Molecular Biology, (Brännström K. et al., 2018) [1].

  • 5.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Islam, Tohidul
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gharibyan, Anna L.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Iakovleva, Irina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Nilsson, Lina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lee, Cheng Choo
    Sandblad, Linda
    Pamrén, Annelie
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The Properties of Amyloid-β Fibrils Are Determined by their Path of Formation2018Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, nr 13, s. 1940-1949Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Fibril formation of the amyloid-β peptide (Aβ) follows a nucleation-dependent polymerization process and is associated with Alzheimer's disease. Several different lengths of Aβ are observed in vivo, but Aβ1-40 and Aβ1-42 are the dominant forms. The fibril architectures of Aβ1-40 and Aβ1-42 differ and Aβ1-42 assemblies are generally considered more pathogenic. We show here that monomeric Aβ1-42 can be cross-templated and incorporated into the ends of Aβ1-40 fibrils, while incorporation of Aβ1-40 monomers into Aβ1-42 fibrils is very poor. We also show that via cross-templating incorporated Aβ monomers acquire the properties of the parental fibrils. The suppressed ability of Aβ1-40 to incorporate into the ends of Aβ1-42 fibrils and the capacity of Aβ1-42 monomers to adopt the properties of Aβ1-40 fibrils may thus represent two mechanisms reducing the total load of fibrils having the intrinsic, and possibly pathogenic, features of Aβ1-42 fibrils in vivo. We also show that the transfer of fibrillar properties is restricted to fibril-end templating and does not apply to cross-nucleation via the recently described path of surface-catalyzed secondary nucleation, which instead generates similar structures to those acquired via de novo primary nucleation in the absence of catalyzing seeds. Taken together these results uncover an intrinsic barrier that prevents Aβ1-40 from adopting the fibrillar properties of Aβ1-42 and exposes that the transfer of properties between amyloid-β fibrils are determined by their path of formation.

  • 6.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Islam, Tohidul
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The role of histidines in amyloid β fibril assembly2017Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 591, nr 8, s. 1167-1175Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Low pH has a strong stabilising effect on the fibrillar assembly of amyloid β, which is associated with Alzheimer's disease. The stabilising effect is already pronounced at pH 6.0, suggesting that protonation of histidines might mediate this effect. Through the systematic substitution of the three native histidines in Aβ for alanines, we have evaluated their role in fibril stability. Using surface plasmon resonance, we show that at neutral pH the fibrillar forms of all His-Ala variants are destabilised by a factor of 4-12 compared to wild-type Aβ. However, none of the His-Ala Aβ variants impair the stabilising effect of the fibril at low pH.

  • 7.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Öhman, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Nilsson, Lina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Pihl, Mathias
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The N-terminal Region of Amyloid β Controls the Aggregation Rate and Fibril Stability at Low pH Through a Gain of Function Mechanism2014Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 136, nr 31, s. 10956-10964Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Alzheimer's disease is linked to a pathological polymerization of the endogenous amyloid β-peptide (Aβ) that ultimately forms amyloid plaques within the human brain. We used surface plasmon resonance (SPR) to measure the kinetic properties of Aβ fibril formation under different conditions during the polymerization process. For all polymerization processes, a critical concentration of free monomers, as defined by the dissociation equilibrium constant (KD), is required for the buildup of the polymer, for example, amyloid fibrils. At concentrations below the KD, polymerization cannot occur. However, the KD for Aβ has previously been shown to be several orders of magnitude higher than the concentrations found in the cerebrospinal and interstitial fluids of the human brain, and the mechanism by which Aβ amyloid forms in vivo has been a matter of debate. Using SPR, we found that the KD of Aβ dramatically decreases as a result of lowering the pH. Importantly, this effect enables Aβ to polymerize within a picomolar concentration range that is close to the physiological Aβ concentration within the human brain. The stabilizing effect is dynamic, fully reversible, and notably pronounced within the pH range found within the endosomal and lysosomal pathways. Through sequential truncation, we show that the N-terminal region of Aβ contributes to the enhanced fibrillar stability due to a gain of function mechanism at low pH. Our results present a possible route for amyloid formation at very low Aβ concentrations and raise the question of whether amyloid formation in vivo is restricted to a low pH environment. These results have general implications for the development of therapeutic interventions.

  • 8. Fleury, Christophe
    et al.
    Su, Yu-Ching
    Hallstroem, Teresia
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Zipfel, Peter F.
    Riesbeck, Kristian
    Identification of a Haemophilus influenzae Factor H-Binding Lipoprotein Involved in Serum Resistance2014Ingår i: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 192, nr 12, s. 5913-5923Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Haemophilus influenzae is a Gram-negative human pathogen that resides in the upper respiratory tract. Encapsulated H. influenzae type b (Hib) and type f (Hif) are the most common serotypes associated with invasive disease. H. influenzae displays various strategies to circumvent the host innate immune response, including the bactericidal effect of the complement system. In this study, we identified an H. influenzae lipoprotein having the ability to bind factor H (FH), the major regulator of the alternative pathway of complement activation. This protein, named protein H (PH), was surface exposed and was found in all clinical Hib and Hif isolates tested. Deletion of the gene encoding for PH (lph) in Hib and Hif significantly reduced the interaction between bacteria and FH. When Hib and Hif PH variants were separately expressed in nontypeable ( unencapsulated) H. influenzae, which did not bind FH, an increased FH affinity was observed. We recombinantly expressed the two PH variants in Escherichia coli, and despite sharing only 56% identical amino acids, both FH-binding Haemophilus proteins similarly interacted with the complement regulator FH short consensus repeats 7 and 18-20. Importantly, Hib and Hif resistance against the bactericidal effect of human serum was significantly reduced when bacterial mutants devoid of PH were tested. In conclusion, we have characterized a hitherto unknown bacterial protein that is crucial for mediating an interaction between the human pathogen H. influenzae and FH. This novel interaction is important for H. influenzae resistance against complement activation and will consequently promote bacterial pathogenesis.

  • 9.
    Francis, Monika K.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Holst, Mikkel R.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Vidal-Quadras, Maite
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Henriksson, Sara
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Santarella-Mellwig, Rachel
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Endocytic membrane turnover at the leading edge is driven by a transient interaction between Cdc42 and GRAF12015Ingår i: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 128, nr 22, s. 4183-4195Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Changes in cell morphology require coordination of plasma membrane turnover and cytoskeleton dynamics, processes that are regulated by Rho GTPases. Here, we describe how a direct interaction between the Rho GTPase Cdc42 and the GTPase activating protein (GAP) GRAF1, facilitate rapid cell surface turnover at the leading edge. Both Cdc42 and GRAF1 were required for fluid phase uptake and regulated the generation of transient GRAF1-coated endocytic carriers, distinct from clathrin coated vesicles. GRAF1 was found to transiently assemble at discrete Cdc42-enriched punctae at the plasma membrane resulting in a corresponding decrease in Cdc42 microdomain association. However, Cdc42 captured in its active state was, via a GAP domain mediated interaction, localised together with GRAF1 on accumulated internal structures derived from the cell surface. Correlative fluorescence and electron tomography microscopy revealed that these structures were clusters of small membrane carriers affected in their endosomal processing. We conclude that a transient interaction between Cdc42 and GRAF1 drives endocytic turnover and controls the transition essential for endosomal maturation of plasma membrane internalised by this mechanism.

  • 10. Fuchino, Katsuya
    et al.
    Bagchi, Sonchita
    Cantlay, Stuart
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wu, Di
    Bergman, Jessica
    Kamali-Moghaddam, Masood
    Flardh, Klas
    Ausmees, Nora
    Dynamic gradients of an intermediate filament-like cytoskeleton are recruited by a polarity landmark during apical growth2013Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, nr 21, s. E1889-E1897Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Intermediate filament (IF)-like cytoskeleton emerges as a versatile tool for cellular organization in all kingdoms of life, underscoring the importance of mechanistically understanding its diverse manifestations. We showed previously that, in Streptomyces (a bacterium with a mycelial lifestyle similar to that of filamentous fungi, including extreme cell and growth polarity), the IF protein FilP confers rigidity to the hyphae by an unknown mechanism. Here, we provide a possible explanation for the IF-like function of FilP by demonstrating its ability to self-assemble into a cis-interconnected regular network in vitro and its localization into structures consistent with a cytoskeletal network in vivo. Furthermore, we reveal that a spatially restricted interaction between FilP and DivIVA, the main component of the Streptomyces polarisome complex, leads to formation of apical gradients of FilP in hyphae undergoing active tip extension. We propose that the coupling between the mechanism driving polar growth and the assembly of an IF cytoskeleton provides each new hypha with an additional stress-bearing structure at its tip, where the nascent cell wall is inevitably more flexible and compliant while it is being assembled and matured. Our data suggest that recruitment of cytoskeleton around a cell polarity landmark is a broadly conserved strategy in tip-growing cells.

  • 11. Höög, Johanna L
    et al.
    Huisman, Stephen M
    Sebö-Lemke, Zsofia
    Sandblad, Linda
    European Mol Biol Labs, Cell Biol & Biophys Program, D-69117 Heidelberg, Germany .
    McIntosh, J Richard
    Antony, Claude
    Brunner, Damian
    Electron tomography reveals a flared morphology on growing microtubule ends2011Ingår i: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 124, nr Pt 5, s. 693-698Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Microtubules (MTs) exhibit dynamic instability, alternating between phases of growth and shortening, mostly at their uncapped plus ends. Based on results from cryo-electron microscopy it was proposed that growing MTs display mainly curved sheets and blunt ends; during depolymerisation curled 'ramshorns' predominate. Observations of MTs in mitotic cells have suggested that the situation in vivo differs from that in vitro, but so far, a clear comparison between in vivo and in vitro results has not been possible because MT end structures could not be correlated directly with the dynamic state of that particular MT. Here we combine light microscopy and electron tomography (ET) to show that growing MT plus ends in the fission yeast Schizosaccharomyces pombe display predominantly a flared morphology. This indicates that MT polymerisation in vivo and in vitro can follow different paths.

  • 12.
    Javadi, Ala
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Söderholm, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Olofsson, Annelie
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Flärdh, Klas
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Assembly mechanisms of the bacterial cytoskeletal protein FilP2019Ingår i: Life Science Alliance, ISSN 2575-1077, Vol. 2, nr 3, artikel-id e201800290Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Despite low-sequence homology, the intermediate filament (IF)–like protein FilP from Streptomyces coelicolor displays structural and biochemical similarities to the metazoan nuclear IF lamin. FilP, like IF proteins, is composed of central coiled-coil domains interrupted by short linkers and flanked by head and tail domains. FilP polymerizes into repetitive filament bundles with paracrystalline properties. However, the cations Na+ and K+ are found to induce the formation of a FilP hexagonal meshwork with the same 60-nm repetitive unit as the filaments. Studies of polymerization kinetics, in combination with EM techniques, enabled visualization of the basic building block — a transiently soluble rod-shaped FilP molecule—and its assembly into protofilaments and filament bundles. Cryoelectron tomography provided a 3D view of the FilP bundle structure and an original assembly model of an IF-like protein of prokaryotic origin, thereby enabling a comparison with the assembly of metazoan IF.

  • 13. Kuznetsov, Nikolai V
    et al.
    Sandblad, Linda
    Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
    Hase, Manuela E
    Hunziker, Andreas
    Hergt, Michaela
    Cordes, Volker C
    The evolutionarily conserved single-copy gene for murine Tpr encodes one prevalent isoform in somatic cells and lacks paralogs in higher eukaryotes.2002Ingår i: Chromosoma, ISSN 0009-5915, E-ISSN 1432-0886, Vol. 111, nr 4, s. 236-255Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Vertebrate Tpr and its probable homologs in insects and yeast are heptad repeat-dominated nuclear proteins of M(r) 195,000 to M(r) 267,000 the functions of which are still largely unknown. Whereas two homologs exist in Saccharomyces cerevisiae, it has remained uncertain whether metazoans possess different paralogs or isoforms of Tpr that might explain controversial reports on the subcellular localization of this protein. To address these possibilities, we first determined the sequence and structure of the murine tpr gene, revealing a TATA box-less gene of approximately 57 kb and 52 exons. Southern hybridization of genomic DNA and radiation hybrid mapping showed that murine tpr exists as a single-copy gene on chromosome 1; RNA blotting analyses and EST (expressed sequence tag) database mining revealed that its expression results in only one major mRNA in embryonic and most adult tissues. Accordingly, novel antibodies against the N- and C-terminus of Tpr identified the full-length protein as the major translation product in different somatic cell types; reinvestigation of Tpr localization by confocal microscopy corroborated a predominant localization at the nuclear pore complexes in these cells. Antibody specificity and reliability of Tpr localization was demonstrated by post-transcriptional tpr gene silencing using siRNAs that eliminated the Tpr signal at the nuclear periphery but did not affect intranuclear staining of Tpr-unrelated proteins. Finally, we defined several sequence and structural features that characterize Tpr polypeptides in different species and used these as a guideline to search whole-genome sequence databases for putative paralogs of Tpr in higher eukaryotes. This approach resulted in identification of the Tpr orthologs in Arabidopsis thaliana and Caenorhabditis elegans, but also in the realization that no further paralogs of Tpr exist in several metazoan model organisms or in humans. In summary, these results reveal Tpr to be a unique protein localized at the nuclear periphery of the somatic cell in mammals.

  • 14. Mozziconacci, Julien
    et al.
    Sandblad, Linda
    EMBL, Cell Biol & Biophys Unit, Heidelberg, Germany.
    Wachsmuth, Malte
    Brunner, Damian
    Karsenti, Eric
    Tubulin dimers oligomerize before their incorporation into microtubules2008Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 3, nr 11, s. e3821-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In the presence of GTP, purified dimers of alpha- and beta-tubulin will interact longitudinally and laterally to self-assemble into microtubules (MTs). This property provides a powerful in vitro experimental system to describe MT dynamic behavior at the micrometer scale and to study effects and functioning of a large variety of microtubule associated proteins (MAPs). Despite the plethora of such data produced, the molecular mechanisms of MT assembly remain disputed. Electron microscopy (EM) studies suggested that tubulin dimers interact longitudinally to form short oligomers which form a tube by lateral interaction and which contribute to MT elongation. This idea is however challenged: Based on estimated association constants it was proposed that single dimers represent the major fraction of free tubulin. This view was recently supported by measurements suggesting that MTs elongate by addition of single tubulin dimers. To solve this discrepancy, we performed a direct measurement of the longitudinal interaction energy for tubulin dimers. We quantified the size distribution of tubulin oligomers using EM and fluorescence correlation spectroscopy (FCS). From the distribution we derived the longitudinal interaction energy in the presence of GDP and the non-hydrolysable GTP analog GMPCPP. Our data suggest that MT elongation and nucleation involves interactions of short tubulin oligomers rather than dimers. Our approach provides a solid experimental framework to better understand the role of MAPs in MT nucleation and growth.

  • 15.
    Muthukrishnan, Uma
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Natarajan, Balasubramanian
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Mäger, Imre
    Levén May, Hanna
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Jones, Iwan
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Corso, Giulia
    Nordin, Joel Z.
    Wiklander, Oscar
    Johansson, Henrik J.
    Lehtiö, Janne
    Hällbrink, Mattias
    Wood, Matthew J.
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Nagaev, Ivan
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Baranov, Vladimir
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Mincheva-Nilsson, Lucia
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Rome, Sophie
    Pini, Adrian
    Andaloussi, Samir EL
    Gilthorpe, Jonathan D.
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM). Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    The exosome membrane localization of histones is independent of DNA and upregulated in response to stressManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Extracellular histones contribute to many acute and chronic diseases but also populate the secretomes of healthy cells and biofluids. However, a secretory pathway for histones has not been described. Here we report that core and linker histones localize to multivesicular bodies and are secreted via exosomes. Histones are tightly associated with the exosome membrane, with N-terminal domains exposed, in a DNA-independent manner. Furthermore, rapid upregulation of exosomal histones occurs following heat stress, accompanied by enhanced vesicle secretion and a shift towards a population of smaller vesicles. Proteomic analyses identified the downregulation of endosomal sorting complex required for transport (ESCRT) complex as a possible mechanism underlying increased histone secretion.We show for the first time that membrane-associated histones are actively secreted from intact cells via the multivesicular body/exosomal pathway. We demonstrate a novel pathway for extracellular histone release that may have a role in both health and disease.

  • 16.
    Niemiec, Maria Joanna
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    De Samber, B.
    Garrevoet, J.
    Vergucht, E.
    Vekemans, B.
    De Rycke, R.
    Björn, Erik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wellenreuther, G.
    Falkenberg, G.
    Cloetens, P.
    Vincze, L.
    Urban, Constantin F.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Trace element landscape of resting and activated human neutrophils on the sub-micrometer level2015Ingår i: Metallomics, ISSN 1756-591X, Vol. 7, nr 6, s. 996-1010Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Every infection is a battle for trace elements. Neutrophils migrate first to the infection site and accumulate quickly to high numbers. They fight pathogens by phagocytosis and intracellular toxication. Additionally, neutrophils form neutrophil extracellular traps (NETs) to inhibit extracellular microbes. Yet, neutrophil trace element characteristics are largely unexplored. We investigated unstimulated and phorbol myristate acetate-stimulated neutrophils using synchrotron radiation X-ray fluorescence (SR-XRF) on the sub-micron spatial resolution level. PMA activates pinocytosis, cytoskeletal rearrangements and the release of NETs, all mechanisms deployed by neutrophils to combat infection. By analyzing Zn, Fe, Cu, Mn, P, S, and Ca, not only the nucleus but also vesicular granules were identifiable in the elemental maps. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) revealed a neutrophil-specific composition of Zn, Fe, Cu, and Mn in comparison with J774 and HeLa cells, indicating a neutrophil-specific metallome complying with their designated functions. When investigating PMA-activated neutrophils, the SR-XRF analysis depicted typical subcellular morphological changes: the transformation of nucleus and granules and the emergence of void vacuoles. Mature NETs were evenly composed of Fe, P, S, and Ca with occasional hot spots containing Zn, Fe, and Ca. An ICP-MS-based quantification of NET supernatants revealed a NETosis-induced decrease of soluble Zn, whereas Fe, Cu, and Mn concentrations were only slightly affected. In summary, we present a combination of SR-XRF and ICP-MS as a powerful tool to analyze trace elements in human neutrophils. The approach will be applicable and valuable to numerous aspects of nutritional immunity.

  • 17.
    Nilsson, Lina
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Pamrén, Annelie
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Islam, Tohidul
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Golchin, Solmaz A.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Pettersson, Nina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Iakovleva, Irina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Gharibyan, Anna L.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Transthyretin Interferes with Aβ Amyloid Formation by Redirecting Oligomeric Nuclei into Non-Amyloid Aggregates2018Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, nr 17, s. 2722-2733Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The pathological Aβ aggregates associated with Alzheimer's disease follow a nucleation-dependent path of formation. A nucleus represents an oligomeric assembly of Aβ peptides that acts as a template for subsequent incorporation of monomers to form a fibrillar structure. Nuclei can form de novo or via surface-catalyzed secondary nucleation, and the combined rates of elongation and nucleation control the overall rate of fibril formation. Transthyretin (TTR) obstructs Aβ fibril formation in favor of alternative non-fibrillar assemblies, but the mechanism behind this activity is not fully understood. This study shows that TTR does not significantly disturb fibril elongation; rather, it effectively interferes with the formation of oligomeric nuclei. We demonstrate that this interference can be modulated by altering the relative contribution of elongation and nucleation, and we show how TTR's effects can range from being essentially ineffective to almost complete inhibition of fibril formation without changing the concentration of TTR or monomeric Aβ.

  • 18. Paulsson, Magnus
    et al.
    Che, Karlhans F.
    Ahl, Jonas
    Tham, Johan
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Smith, Margaretha E.
    Qvarfordt, Ingemar
    Su, Yu-Ching
    Linden, Anders
    Riesbeck, Kristian
    Bacterial Outer Membrane Vesicles Induce Vitronectin Release Into the Bronchoalveolar Space Conferring Protection From Complement-Mediated Killing2018Ingår i: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, artikel-id 1559Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Pathogens causing pneumonia utilize the complement regulator vitronectin to evade complement-mediated killing. Although vitronectin is associated with several chronic lung diseases, the role of bronchoalveolar vitronectin in pneumonia has not been studied. This study sought to reveal the involvement of vitronectin in the bronchoalveolar space during pneumonia, to assess the effect of outer membrane vesicles and endotoxin on vitronectin release, and to determine whether bacterial pathogens utilize pulmonary vitronectin for evasion. Vitronectin was analyzed in cell-free bronchoalveolar lavage fluid harvested from patients with pneumonia (n = 8) and from healthy volunteers after subsegmental endotoxin instillation (n = 13). Vitronectin binding by Pseudomonas aeruginosa and Haemophilus influenzae was analyzed, and subsequent complement evasion was assessed by serum challenge. The effects of outer membrane vesicles on vitronectin production in mouse lungs and human type II alveolar epithelial cells (A549) were determined. We detected increased vitronectin concentrations in lavage fluid during pneumonia (p = 0.0063) and after bronchial endotoxin challenge (p = 0.016). The capture of vitronectin by bacteria significantly reduced complement-mediated lysis. Following challenge with vesicles, vitronectin was detected in mouse bronchoalveolar space, and mouse alveolar epithelial cells in vivo as well as A549 cells in vitro contained increased levels of vitronectin. Taken together, outer membrane vesicles and endotoxin from Gram-negative bacteria induce vitronectin, which is released into the bronchoalveolar space, and used for evasion of complement-mediated clearance.

  • 19.
    Rajan, Anandi
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Persson, B. David
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Frängsmyr, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Olofsson, Annelie
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Heino, Jyrki
    Department of Biochemistry, University of Turku, Finland.
    Takada, Yoshikazu
    Department of Dermatology, Biochemistry and Molecular Medicine, UC Davis School of Medicine, California, USA.
    Mould, A. Paul
    Biomolecular Analysis Core Facility, Faculty of Biology, Medicine and Health, University of Manchester, United Kingdom.
    Schnapp, Lynn M.
    Division of Pulmonary, Critical Care, Allergy and Sleep Medicine, Medical University of South Carolina, Charleston, USA.
    Gall, Jason
    Vaccine Research Center (VRC), NIAID, NIH, Bethesda, USA.
    Arnberg, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Enteric species F human adenoviruses use laminin-binding integrins as co-receptors for infection of Ht-29 cells2018Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, nr 1, artikel-id 10019Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The enteric species F human adenovirus types 40 and 41 (HAdV-40 and -41) are the third most common cause of infantile gastroenteritis in the world. Knowledge about HAdV-40 and -41 cellular infection is assumed to be fundamentally different from that of other HAdVs since HAdV-40 and -41 penton bases lack the αV-integrin-interacting RGD motif. This motif is used by other HAdVs mainly for internalization and endosomal escape. We hypothesised that the penton bases of HAdV-40 and -41 interact with integrins independently of the RGD motif. HAdV-41 transduction of a library of rodent cells expressing specific human integrin subunits pointed to the use of laminin-binding α2-, α3- and α6-containing integrins as well as other integrins as candidate co-receptors. Specific laminins prevented internalisation and infection, and recombinant, soluble HAdV-41 penton base proteins prevented infection of human intestinal HT-29 cells. Surface plasmon resonance analysis demonstrated that HAdV-40 and -41 penton base proteins bind to α6-containing integrins with an affinity similar to that of previously characterised penton base:integrin interactions. With these results, we propose that laminin-binding integrins are co-receptors for HAdV-40 and -41.

  • 20.
    Sandblad, Linda
    et al.
    Structural and Computational Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
    Busch, Karl Emanuel
    Tittmann, Peter
    Gross, Heinz
    Brunner, Damian
    Hoenger, Andreas
    The Schizosaccharomyces pombe EB1 homolog Mal3p binds and stabilizes the microtubule lattice seam2006Ingår i: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 127, nr 7, s. 1415-1424Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    End binding 1 (EB1) proteins are highly conserved regulators of microtubule dynamics. Using electron microscopy (EM) and high-resolution surface shadowing we have studied the microtubule-binding properties of the fission yeast EB1 homolog Mal3p. This allowed for a direct visualization of Mal3p bound on the surface of microtubules. Mal3p particles usually formed a single line on each microtubule along just one of the multiple grooves that are formed by adjacent protofilaments. We provide structural data showing that the alignment of Mal3p molecules coincides with the microtubule lattice seam as well as data suggesting that Mal3p not only binds but also stabilizes this seam. Accordingly, Mal3p stabilizes microtubules through a specific interaction with what is potentially the weakest part of the microtubule in a way not previously demonstrated. Our findings further suggest that microtubules exhibit two distinct reaction platforms on their surface that can independently interact with target structures such as microtubule-associated proteins, motors, kinetochores, or membranes.

  • 21.
    Sellin, Mikael E.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sandblad, Linda
    Department of Cell and Molecular Biology, Karolinska Institute, S-171 77 Stockholm, Sweden.
    Stenmark, Sonja
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Gullberg, Martin
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Deciphering the rules governing assembly order of mammalian septin complexes2011Ingår i: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 22, nr 17, s. 3152-3164Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Septins are conserved GTP-binding proteins that assemble into lateral diffusion barriers and molecular scaffolds. Vertebrate genomes contain 9-17 septin genes that encode both ubiquitous and tissue-specific septins. Expressed septins may assemble in various combinations through both heterotypic and homotypic G-domain interactions. However, little is known regarding assembly states of mammalian septins and mechanisms directing ordered assembly of individual septins into heteromeric units, which is the focus of this study. Our analysis of the septin system in cells lacking or overexpressing selected septins reveals inter-dependencies coinciding with previously described homology subgroups. Hydrodynamic and single-particle data show that individual septins exist solely in the context of stable six-to eight-subunit core heteromers, all of which contain SEPT2 and SEPT6 subgroup members and SEPT7, while heteromers comprising more than six subunits also contain SEPT9. The combined data suggest a generic model for how the temporal order of septin assembly is homology subgroup-directed, which in turn determines the subunit arrangement of native heteromers. Because mammalian cells normally express multiple members and/or isoforms of some septin subgroups, our data also suggest that only a minor fraction of native heteromers are arranged as perfect palindromes.

  • 22. Sen, Beer Chakra
    et al.
    Wasserstrom, Sebastian
    Findlay, Kim
    Söderholm, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    von Wachenfeldt, Claes
    Flardh, Klas
    Specific amino acid substitutions in β strand S2 of FtsZ cause spiraling septation and impair assembly cooperativity in Streptomyces spp.2019Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 112, nr 1, s. 184-198Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacterial cell division is orchestrated by the Z ring, which is formed by single-stranded treadmilling protofilaments of FtsZ. In Streptomyces, during sporulation, multiple Z rings are assembled and lead to formation of septa that divide a filamentous hyphal cell into tens of prespore compartments. We describe here mutant alleles of ftsZ in Streptomyces coelicolor and Streptomyces venezuelae that perturb cell division in such a way that constriction is initiated along irregular spiral-shaped paths rather than as regular septa perpendicular to the cell length axis. This conspicuous phenotype is caused by amino acid substitutions F37I and F37R in beta strand S2 of FtsZ. The F37I mutation leads, instead of regular Z rings, to formation of relatively stable spiral-shaped FtsZ structures that are capable of initiating cell constriction. Further, we show that the F37 mutations affect the polymerization properties and impair the cooperativity of FtsZ assembly in vitro. The results suggest that specific residues in beta strand S2 of FtsZ affect the conformational switch in FtsZ that underlies assembly cooperativity and enable treadmilling of protofilaments, and that these features are required for formation of regular Z rings. However, the data also indicate FtsZ-directed cell constriction is not dependent on assembly cooperativity.

  • 23.
    Sjöström, Annika E
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Membrane vesicle-mediated release of bacterial RNA2015Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, artikel-id 15329Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the host by delivering virulence factors. Here, we report for the first time that RNA is among the wide variety of bacterial components that are associated with OMVs. To characterize the RNA profiles of bacterial OMVs, we performed RNA deep sequencing analysis using OMV samples isolated from a wild type Vibrio cholerae O1 El Tor strain. The results showed that RNAs originating from intergenic regions were the most abundant. Our findings reveal a hitherto unrecognised feature of OMVs mimicking eukaryotic exosomes and highlight a need to evaluate the potential role of RNA-containing bacterial membrane vesicles in bacteria-host interactions.

  • 24. Svensson, Lennart
    et al.
    Svensson, Stina
    Nyström, Ingela
    Nysjö, Fredrik
    Laloeuf, Aurelie
    den Hollander, Lianne
    Brun, Anders
    Masich, Sergej
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sani, Musa
    Sintorn, Ida-Maria
    ProViz: a tool for explorative 3-D visualization and template matching in electron tomograms2017Ingår i: Computer Methods in Biomechanics and Biomedical Engineering: Imaging & Visualization, ISSN 2168-1163, Vol. 5, nr 6, s. 446-454Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Visual understanding is a key aspect when studying electron tomography data-sets, aside quantitative assessments such as registration of high-resolution structures. We here present the free software tool ProViz (Protein Visualization) for visualisation and template matching in electron tomograms of biological samples. The ProViz software contains methods and tools which we have developed, adapted and computationally optimised for easy and intuitive visualisation and analysis of electron tomograms with low signal-to-noise ratio. ProViz complements existing software in the application field and serves as an easy and convenient tool for a first assessment and screening of the tomograms. It provides enhancements in three areas: (1) improved visualisation that makes connections as well as intensity differences between and within objects or structures easier to see and interpret, (2) interactive transfer function editing with direct visual result feedback using both piecewise linear functions and Gaussian function elements, (3) computationally optimised template matching and tools to visually assess and interactively explore the correlation results. The visualisation capabilities and features of ProViz are demonstrated on various biological volume data-sets: bacterial filament structures in vitro, a desmosome and the transmembrane cadherin connections therein in situ, and liposomes filled with doxorubicin in solution. The explorative template matching is demonstrated on a synthetic IgG data-set.

  • 25.
    Söderholm, Niklas
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Javadi, Ala
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sierra Flores, Isabel
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Flärdh, Klas
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Affinity to cellulose is a shared property among coiled-coil domains of intermediate filaments and prokaryotic intermediate filament-like proteins2018Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, artikel-id 16524Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Coiled-coil domains of intermediate filaments (IF) and prokaryotic IF-like proteins enable oligomerisation and filamentation, and no additional function is ascribed to these coiled-coil domains. However, an IF-like protein from Streptomyces reticuli was reported to display cellulose affinity. We demonstrate that cellulose affinity is an intrinsic property of the IF-like proteins FilP and Scy and the coiled-coil protein DivIVA from the genus Streptomyces. Furthermore, IF-like proteins and DivIVA from other prokaryotic species and metazoan IF display cellulose affinity despite having little sequence homology. Cellulose affinity-based purification is utilised to isolate native FilP protein from the whole cell lysate of Scoelicolor. Moreover, cellulose affinity allowed for the isolation of IF and IF-like protein from the whole cell lysate of Ccrescentus and a mouse macrophage cell line. The binding to cellulose is mediated by certain combinations of coiled-coil domains, as demornstrated for FilP and lamin. Fusions of target proteins to cellulose-binding coiled-coil domains allowed for cellulose-based protein purification. The data presented show that cellulose affinity is a novel function of certain coiled-coil domains of IF and IF-like proteins from evolutionary diverse species.

  • 26.
    Taheri, Nayyer
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Mahmud, A K M Firoj
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Fahlgren, Anna
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Campylobacter jejuni bile exposure influences outer membrane vesicles protein content and bacterial interaction with epithelial cells2018Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, artikel-id 16996Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Campylobacter jejuni is a prevalent human pathogen and a major cause of bacterial gastroenteritis in the world. In humans, C. jejuni colonizes the intestinal tract and its tolerance to bile is crucial for bacteria to survive and establish infection. C. jejuni produces outer membrane vesicles (OMVs) which have been suggested to be involved in virulence. In this study, the proteome composition of C. jejuni OMVs in response to low concentration of bile was investigated. We showed that exposure of C. jejuni to low concentrations of bile, similar to the concentration in cecum, induced significant changes in the protein profile of OMVs released during growth without affecting the protein profile of the bacteria. This suggests that bile influences a selective packing of the OMVs after bacterial exposure to low bile. A low concentration of bile was found to increase bacterial adhesion to intestinal epithelial cells, likely by an enhanced hydrophobicity of the cell membrane following exposure to bile. The increased bacterial adhesiveness was not associated with increased invasion, instead bile exposure decreased C. jejuni invasion. OMVs released from bacteria upon exposure to low bile showed to increase both adhesion and invasion of non-bile-exposed bacteria into intestinal epithelial cells. These findings suggest that C. jejuni in environments with low concentrations of bile produce OMVs that facilitates colonization of the bacteria, and this could potentially contribute to virulence of C. jejuni in the gut.

  • 27. Wasserström, Sebastian
    et al.
    Grantcharova, Nina
    Ubhayasekera, Wimal
    Ausmees, Nora
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Flardh, Klas
    Non-sporulating ftsZ mutants in Streptomyces coelicolor reveal amino acid residues critical for FtsZ polymerization dynamics2013Ingår i: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 159, s. 890-901Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    During sporulation of Streptomyces coelicolor, the cytokinetic protein FtsZ is assembled into dozens of regularly spaced Z rings, which orchestrate the division of aerial hyphae into spores. We have previously found that a missense allele of ftsZ, ftsZ17(Spo), primarily affects sporulation septation rather than formation of cross-walls in vegetative mycelium. To clarify what aspect of FtsZ function is compromised in such non-sporulating mutants, we here use a genetic strategy to identify new ftsZ(Spo) alleles and describe how some of the mutations affect the biochemical properties of FtsZ. We have established a system for purification of recombinant untagged S. coelicolor FtsZ, and shown that it assembles dynamically into single protofilaments, displays a critical concentration indicative of cooperative assembly and has a rate of GTP hydrolysis that is substantially higher than that of the closely related Mycobacterium tuberculosis FtsZ. Of the nine isolated ftsZ(Spo) mutations, four affect the interface between the two main subdomains of FtsZ that is implicated in the assembly-induced conformational changes thought to mediate the GTP/GDP-driven cooperative assembly of FtsZ. We find that all these four mutations affect the polymerization behaviour of FtsZ in vitro. In addition, at least one ftsZ(Spo) mutation at the longitudinal contact surface between subunits in protofilaments strongly affects formation of polymers in vitro. We conclude that the assembly of Z rings during sporulation of S. coelicolor is highly sensitive to disturbances of FtsZ polymerization and therefore constitutes an excellent system for analysis of the elusive properties of FtsZ that mediate its characteristic polymerization dynamics.

  • 28.
    Zhang, Hanqing
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Söderholm, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wiklund, Krister
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Andersson, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    DSeg: a dynamic image segmentation program to extract backbone patterns for filamentous bacteria and hyphae structures2019Ingår i: Microscopy and Microanalysis, ISSN 1431-9276, E-ISSN 1435-8115, Vol. 25, nr 3, s. 711-719Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Analysis of numerous filamentous structures in an image is often limited by the ability of algorithms to accurately segment complex structures or structures within a dense population. It is even more problematic if these structures continuously grow when recording a time-series of images. To overcome these issues we present DSeg; an image analysis program designed to process time-series image data, as well as single images, to segment filamentous structures. The program includes a robust binary level-set algorithm modified to use size constraints, edge intensity, and past information. We verify our algorithms using synthetic data, differential interference contrast images of filamentous prokaryotes, and transmission electron microscopy images of bacterial adhesion fimbriae. DSeg includes automatic segmentation, tools for analysis, and drift correction, and outputs statistical data such as persistence length, growth rate, and growth direction. The program is available at Sourceforge.

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