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  • 1.
    Carlsson, Stina K.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Effects of adenosine and acetylcholine on the lacrimal gland2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    A balanced tear film is essential for a healthy ocular surface. Insufficient tear production may result in dry eye, a common disorder in the elderly population. Dry eye causes significant discomfort in the patients and may lead to visual impairment and ocular infections. The lacrimal gland secretes water, proteins and electrolytes to the aqueous layer of the tear film. Lacrimal gland secretion is tightly regulated by e.g. neuronally released acetylcholine. The effect of acetylcholine on lacrimal gland secretion was recently found to be potentiated by adenosine. Adenosine is an important signaling molecule acting upon the adenosine receptors: A1, A2A, A2B and A3.

    The aim of this thesis was to study effects of adenosine and acetylcholine on intracellular signaling pathways and lacrimal gland secretion. Cholinergic stimulation of secretion was shown to be regulated by the mitogen activated protein kinase p38, a protein previously not known to be involved in exocrine secretion. p38 was activated in response to cholinergic stimulation and inhibition of p38 significantly diminished cholinergic secretion.

    When investigating adenosine effects, potentiation of cholinergic secretion was observed by activation of the A2B receptor in addition to the previously studied A1 receptor. An A2 receptor agonist increased cholinergic rabbit lacrimal gland protein secretion at several concentrations. The increase was inhibited by antagonism of the A2B receptor, but not the A2A receptor. When investigating the intracellular signaling pathways following adenosine and acetylcholine receptor activation, adenosine was shown to increase of cAMP levels. An additional increase in cAMP levels was observed after parallel adenosine and cholinergic receptor activation. Inhibition of Ca2+ release from the endoplasmic reticulum had inhibitory effects of cholinergic stimulation of secretion. In addition, the expression of adenosine receptors in a mouse model of autoimmune dry eye was investigated. The results showed a lymphocyte dependent upregulation of A2A receptors in diseased mice compared to controls.

    In conclusion, the results in this thesis provide significant contributions in the search of dry eye therapeutics through studies of adenosine and acetylcholine receptor activation.

  • 2.
    Carlsson, Stina K.
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Gierow, Peter
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    p38 mitogen-activated protein kinase modulates exocrine secretion in rabbit lacrimal gland2012In: Cellular & Molecular Biology Letters (Druk), ISSN 1425-8153, E-ISSN 1689-1392, Vol. 17, no 1, p. 1-10Article in journal (Refereed)
    Abstract [en]

    The lacrimal gland (LG) is an exocrine gland important for secretion of the tear film. The kinase p38 has important signal transduction functions, e.g. in gene transcription, but has previously not been known to modulate exocrine secretion. The aim of the current study was to investigate the role of p38 in carbachol (Cch)-induced LG secretion in LG acinar cells in vitro. Western blotting was used to determine the phosphorylation status of p38 and p42/44 and determine expression of p38 isoforms. To determine the effect of p38 inhibition on LG secretion, PD 169316, a general p38 inhibitor, and SB 239063, an inhibitor of p38α and β, were added to the cells prior to secretion measurements. The results revealed activation of p38 mediated by Cch stimulation and inhibition of Cch-induced secretion as a result of p38 inhibition. The inhibition was observed with PD 169316 isoforms, but not with SB 239063. The p38δ isoform was shown to have robust expression both by Western blotting of acinar cells and immunofluorescence of the whole gland. In conclusion, p38 activation mediates secretion in cholinergic stimulation of rabbit LG cells.

  • 3.
    Christerson, Ulrika
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Keita, Åsa, V
    Linköping University, Sweden.
    Winberg, Martin E.
    Linköping University, Sweden.
    Söderholm, Johan D.
    Linköping University, Sweden;County Council Östergötland, Sweden.
    Gustafson-Svärd, Christina
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Possible Involvement of Intracellular Calcium-Independent Phospholipase A(2) in the Release of Secretory Phospholipases from Mast Cells-Increased Expression in Ileal Mast Cells of Crohn's Disease2019In: Cells, E-ISSN 2073-4409, Vol. 8, no 7, p. 1-15, article id 672Article in journal (Refereed)
    Abstract [en]

    Increased activity of secretory phospholipases A(2) (sPLA(2)) type-II was previously observed in ileum of Crohn's disease (CD). Our aims were to explore the involvement of calcium-independent (i)PLA(2 beta) in the release of sPLA(2)s from the human mast cell (MC) line (HMC-1) and investigate expressions of cytosolic (c)PLA(2) alpha, iPLA(2)beta, sPLA(2)-IIA and sPLA(2)-V in MCs of CD ileum. The release of sPLA(2) was investigated in HMC-1 by immunocytochemistry and ELISA. The expression intensities of PLA(2)s in mucosal MCs, and the proportion of PLA(2)-positive MCs, were investigated in normal ileum and in ileum from patients with CD by immunohistochemistry. The calcium ionophore-stimulated release of sPLA(2)-IIA and sPLA(2)-V from HMC-1 was reduced by the iPLA(2)-inhibitor bromoenol lactone. All four PLA(2)s were detectable in mucosal MCs, both in normal ileum and in CD, but the proportion of iPLA(2)beta-containing mucosal MCs and the expression intensity of sPLA(2)-IIA was increased in CD. Results indicate that iPLA(2)beta is involved in the secretion of sPLA(2)s from HMC-1, and suggest that iPLA(2)beta-mediated release of sPLA(2) from intestinal MCs may contribute to CD pathophysiology. Ex vivo studies on isolated mucosal mast cells are however needed to clarify the precise role of MC PLA(2)s in the inflammatory processes of CD.

  • 4. Dashevskaya, S
    et al.
    Kopito, R B
    Friedman, Ran
    Tel Aviv University, Israel.
    Elbaum, M
    Epel, B L
    Diffusion of anionic and neutral GFP derivatives through plasmodesmata in epidermal cells of Nicotiana benthamiana2008In: ProtoplasmaArticle in journal (Refereed)
  • 5.
    Evertsson, Kim
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Fjällström, Ann-Kristin
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Norrby, Marlene
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Tågerud, Sven
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    p38 mitogen-activated protein kinase and mitogen-activated protein kinase-activated protein kinase 2 (MK2) signaling in atrophic and hypertrophic denervated mouse skeletal muscle2014In: Journal of Molecular Signaling, ISSN 1750-2187, E-ISSN 1750-2187, Vol. 9, no 2Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: p38 mitogen-activated protein kinase has been implicated in both skeletal muscle atrophy and hypertrophy. T317 phosphorylation of the p38 substrate mitogen-activated protein kinase-activated protein kinase 2 (MK2) correlates with muscle weight in atrophic and hypertrophic denervated muscle and may influence the nuclear and cytoplasmic distribution of p38 and/or MK2. The present study investigates expression and phosphorylation of p38, MK2 and related proteins in cytosolic and nuclear fractions from atrophic and hypertrophic 6-days denervated skeletal muscles compared to innervated controls.

    METHODS: Expression and phosphorylation of p38, MK2, Hsp25 (heat shock protein25rodent/27human, Hsp25/27) and Hsp70 protein expression were studied semi-quantitatively using Western blots with separated nuclear and cytosolic fractions from innervated and denervated hypertrophic hemidiaphragm and atrophic anterior tibial muscles. Unfractionated innervated and denervated atrophic pooled gastrocnemius and soleus muscles were also studied.

    RESULTS: No support was obtained for a differential nuclear/cytosolic localization of p38 or MK2 in denervated hypertrophic and atrophic muscle. The differential effect of denervation on T317 phosphorylation of MK2 in denervated hypertrophic and atrophic muscle was not reflected in p38 phosphorylation nor in the phosphorylation of the MK2 substrate Hsp25. Hsp25 phosphorylation increased 3-30-fold in all denervated muscles studied. The expression of Hsp70 increased 3-5-fold only in denervated hypertrophic muscles.

    CONCLUSIONS: The study confirms a differential response of MK2 T317 phosphorylation in denervated hypertrophic and atrophic muscles and suggests that Hsp70 may be important for this. Increased Hsp25 phosphorylation in all denervated muscles studied indicates a role for factors other than MK2 in the regulation of this phosphorylation.

  • 6.
    Henn, Arnon
    et al.
    Technion, Israel.
    Shneyer, Boris
    Technion, Israel.
    Ušaj, Marko
    Technion, Israel.
    Myosin 19 is an Outer Mitochondrial Membrane Motor and Effector of Starvation Induced Filopodia with Unique Kinetic Features2016In: Biophysical Journal Supplement 1, 2016, Vol. 110, p. 615a-616a, article id 3040-PosConference paper (Refereed)
    Abstract [en]

    The interaction between the actin cytoskeleton, myosin motors and their function in mitochondria dynamics, morphology and cellular localization is now beginning to emerge. A novel function for actin-based motors as regulators of cellular adaptations to stress, linking actin cytoskeleton remodelling to mitochondria dynamics. We reveal a novel function for myosin 19 in mitochondrial dynamics and localization during cellular response to glucose starvation. Ectopically expressed myosin 19 localizes with mitochondria at the tips of starvation-induced filopodia. Corollary to this, RNAi mediated knockdown of myosin 19 diminished their formation without evident effects on the mitochondrial network. We analyzed myosin 19 mitochondria interaction and demonstrated that it is uniquely anchored to the outer mitochondrial membrane (OMM) via a 30-residue motif, indicating that myosin 19 is a stably attached OMM molecular motor. To this end, we have purified myosin 19-3IQ motor domain construct. Myosin 19-3IQ featured characteristic actin-activated ATPase activity with moderate to slow turnover (kcat) and relatively tight KATPase. Our transient kinetics and steady state equilibrium binding experiments revealed that myosin 19-3IQ binds ATP and ADP with tight affinity that, to the best of our knowledge, have not yet been exhibited by any other myosins. We suspect that this feature allows myosin 19 to operate in a unique cellular environment that may be related to cellular stress conditions as we showed in our previous studies. The detailed knowledge of myosin 19 enzymatic adaptation will provide us with a quantitative working model of myosin 19, and will assist us to understand its cellular function. Our work reveals a novel function for myosin 19 in mitochondrial positioning during homeostasis and under stress conditions and broadens our understanding of the actin cytoskeleton- myosin -mitochondria interplay.

  • 7.
    Johansson, Ulrika
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. KTH Royal Instute of Technology, Sweden;Uppsala University, Sweden.
    Widhe, Mona
    KTH Royal Instute of Technology, Sweden.
    Shalaly, Nancy Dekki
    KTH Royal Instute of Technology, Sweden.
    Arregui, Irene Linares
    KTH Royal Instute of Technology, Sweden.
    Nileback, Linnea
    KTH Royal Instute of Technology, Sweden.
    Tasiopoulos, Christos Panagiotis
    KTH Royal Instute of Technology, Sweden.
    Åstrand, Carolina
    KTH Royal Instute of Technology, Sweden.
    Berggren, Per-Olof
    Karolinska Institutet, Sweden;Karolinska University Hospital, Sweden.
    Gasser, Christian
    KTH Royal Instute of Technology, Sweden.
    Hedhammar, My
    KTH Royal Instute of Technology, Sweden.
    Assembly of functionalized silk together with cells to obtain proliferative 3D cultures integrated in a network of ECM-like microfibers2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, p. 1-13, article id 6291Article in journal (Refereed)
    Abstract [en]

    Tissues are built of cells integrated in an extracellular matrix (ECM) which provides a three-dimensional (3D) microfiber network with specific sites for cell anchorage. By genetic engineering, motifs from the ECM can be functionally fused to recombinant silk proteins. Such a silk protein, FN-silk, which harbours a motif from fibronectin, has the ability to self-assemble into networks of microfibers under physiological-like conditions. Herein we describe a method by which mammalian cells are added to the silk solution before assembly, and thereby get uniformly integrated between the formed microfibers. In the resulting 3D scaffold, the cells are highly proliferative and spread out more efficiently than when encapsulated in a hydrogel. Elongated cells containing filamentous actin and defined focal adhesion points confirm proper cell attachment to the FN-silk. The cells remain viable in culture for at least 90 days. The method is also scalable to macro-sized 3D cultures. Silk microfibers formed in a bundle with integrated cells are both strong and extendable, with mechanical properties similar to that of artery walls. The described method enables differentiation of stem cells in 3D as well as facile co-culture of several different cell types. We show that inclusion of endothelial cells leads to the formation of vessel-like structures throughout the tissue constructs. Hence, silk-assembly in presence of cells constitutes a viable option for 3D culture of cells integrated in a ECM-like network, with potential as base for engineering of functional tissue.

  • 8.
    Kandušer, Maša
    et al.
    University of Ljubljana, Slovenia.
    Kokalj Imsirovic, Mojca
    University of Ljubljana, Slovenia.
    Ušaj, Marko
    University of Ljubljana, Slovenia.
    The Effect of Lipid Antioxidant α-Tocopherol on Cell Viability and Electrofusion Yield of B16-F1 Cells In Vitro2019In: The Journal of Membrane Biology, ISSN 0022-2631, Vol. 252, no 1, p. 105-114Article in journal (Refereed)
    Abstract [en]

    Induced cell fusion is a powerful method for production of hybridoma in biotechnology and cell vaccines in medical applications. Among different alternatives, physical methods have an advantage, as they do not require any additives. Among them electrofusion, an electroporation-based cell fusion method holds a great promise. Electric pulses cause cell membrane permeabilization and due to pore formation bring cell membrane into the fusogenic state. At the same time, however, they compromise cell viability. We used a train of 8 × 100 µs electric pulses, delivered at 1 Hz with strengths ranging from 400 to 1600 V/cm. We evaluated electrofusion efficiency by dual color microscopy. We determined cell viability, because during electroporation reactive oxygen species are generated affecting cell survival. The novelty of our study is evaluation of the effect of lipid antioxidant α-tocopherol on cell fusion yield and cell viability on mouse B16-F1 cells. Pretreatment with α-tocopherol slowed down dynamic of cell fusion shortly after electroporation. Twenty-four hours later, fusion yields between α-tocopherol treated and untreated cells were comparable. The viability of α-tocopherol pretreated cells was drastically improved. Pretreatment of cells with α-tocopherol improved whole electrofusion process by more than 60%. We believe that α-tocopherol holds great promise to become an important agent to improve cell electrofusion method.

  • 9.
    Masson, Patrick
    et al.
    Swiss Institute of Bioinformatics, Switzerland.
    Lundin, Daniel
    Stockholm University.
    Söderbom, Fredrik
    Swedish University of Agricultural Sciences (SLU).
    Young, Patrick
    Stockholm University.
    Characterization of a REG/PA28 Proteasome Activator Homolog in Dictyostelium discoideum Indicates that the Ubiquitin- and ATP-Independent REGγ Proteasome Is an Ancient Nuclear Protease2009In: Eukaryotic Cell, ISSN 1535-9778, E-ISSN 1535-9786, Vol. 8, no 6, p. 844-851Article in journal (Refereed)
    Abstract [en]

    The nuclear proteasome activator REGγ/PA28γ is an ATP- and ubiquitin-independent activator of the 20S proteasome and has been proposed to degrade and thereby regulate both a key human oncogene, encoding the coactivator SRC-3/AIB1, and the cyclin-dependent kinase inhibitor p21 (Waf/Cip1). We report the identification and characterization of a PA28/REG homolog in Dictyostelium. Association of a recombinant Dictyostelium REG with the purified Dictyostelium 20S proteasome led to the preferential stimulation of the trypsin-like proteasome peptidase activity. Immunolocalization studies demonstrated that the proteasome activator is localized to the nucleus and is present in growing as well as starving Dictyostelium cells. Our results indicate that the Dictyostelium PA28/REG activator can stimulate both the trypsin-like and chymotrypsin-like activities of the 20S proteasome and supports the idea that the REGγ-20S proteasome represents an early unique nuclear degradation pathway for eukaryotic cells.

  • 10.
    Månsson, Alf
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Norrby, Marlene
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Tågerud, Sven
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Molecular Motors and Self-Healing at the Neuromuscular Synapse2011In: Self-Healing at the Nanoscale: Mechanisms and Key Concepts of Natural and Artificial Systems / [ed] Vincenzo Amendola & Moreno Meneghetti, CRC Press, 2011, 1, p. 357-394Chapter in book (Other academic)
  • 11.
    Norrby, Marlene
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Evertsson, Kim
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Fjällström, Ann-Kristin
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Svensson, Anna
    Tågerud, Sven
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Akt (protein kinase B) isoform phosphorylation and signaling downstream of mTOR (mammalian target of rapamycin) in denervated atrophic and hypertrophic mouse skeletal muscle.2012In: Journal of Molecular Signaling, ISSN 1750-2187, E-ISSN 1750-2187, Vol. 7, no June, p. Article ID: 7-Article in journal (Refereed)
    Abstract [en]

    ABSTRACT: BACKGROUND: The present study examines the hypothesis that Akt (protein kinase B)/mTOR (mammalian target of rapamycin) signaling is increased in hypertrophic and decreased in atrophic denervated muscle. Protein expression and phosphorylation of Akt1, Akt2, glycogen synthase kinase-3beta (GSK-3beta), eukaryotic initiation factor 4E binding protein 1 (4EBP1), 70 kD ribosomal protein S6 kinase (p70S6K1) and ribosomal protein S6 (rpS6) were examined in six-days denervated mouse anterior tibial (atrophic) and hemidiaphragm (hypertrophic) muscles. RESULTS: In denervated hypertrophic muscle expression of total Akt1, Akt2, GSK-3beta, p70S6K1 and rpS6 proteins increased 2-10 fold whereas total 4EBP1 protein remained unaltered. In denervated atrophic muscle Akt1 and Akt2 total protein increased 2-16 fold. A small increase in expression of total rpS6 protein was also observed with no apparent changes in levels of total GSK-3beta, 4EBP1 or p70S6K1 proteins. The level of phosphorylated proteins increased 3-13 fold for all the proteins in hypertrophic denervated muscle. No significant changes in phosphorylated Akt1 or GSK-3beta were detected in atrophic denervated muscle. The phosphorylation levels of Akt2, 4EBP1, p70S6K1 and rpS6 were increased 2-18 fold in atrophic denervated muscle. CONCLUSIONS: The results are consistent with increased Akt/mTOR signaling in hypertrophic skeletal muscle. Decreased levels of phosphorylated Akt (S473/S474) were not observed in denervated atrophic muscle and results downstream of mTOR indicate increased protein synthesis in denervated atrophic anterior tibial muscle as well as in denervated hypertrophic hemidiaphragm muscle. Increased protein degradation, rather than decreased protein synthesis, is likely to be responsible for the loss of muscle mass in denervated atrophic muscles.

  • 12.
    Rahman, Mohammad A.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Salhotra, Aseem
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Comparative analysis of widely used methods to remove nonfunctional myosin heads for the in vitro motility assay2018In: Journal of Muscle Research and Cell Motility, ISSN 0142-4319, E-ISSN 1573-2657, Vol. 39, no 5-6, p. 175-187Article in journal (Refereed)
    Abstract [en]

    The in vitro motility assay allows studies of muscle contraction through observation of actin filament propulsion by surface-adsorbed myosin motors or motor fragments isolated from muscle. A possible problem is that motility may be compromised by nonfunctional, "dead", motors, obtained in the isolation process. Here we investigate the effects on motile function of two approaches designed to eliminate the effects of these dead motors. We first tested the removal of heavy meromyosin (HMM) molecules with ATP-insensitive "dead" heads by pelleting them with actin filaments, using ultracentrifugation in the presence of 1 mM MgATP ("affinity purification"). Alternatively we incubated motility assay flow cells, after HMM surface adsorption, with non-fluorescent "blocking actin" (1 µM) to block the dead heads. Both affinity purification and use of blocking actin increased the fraction of motile filaments compared to control conditions. However, affinity purification significantly reduced the actin sliding speed in five out of seven experiments on silanized surfaces and in one out of four experiments on nitrocellulose surfaces. Similar effects on velocity were not observed with the use of blocking actin. However, a reduced speed was also seen (without affinity purification) if HMM or myosin subfragment 1 was mixed with 1 mM MgATP before and during surface adsorption. We conclude that affinity purification can produce unexpected effects that may complicate the interpretation of in vitro motility assays and other experiments with surface adsorbed HMM, e.g. single molecule mechanics experiments. The presence of MgATP during incubation with myosin motor fragments is critical for the complicating effects.

  • 13.
    Shneyer, Boris I.
    et al.
    Technion, Israel.
    Ušaj, Marko
    Technion, Israel.
    Henn, Arnon
    Technion, Israel.
    Myo19 is an outer mitochondrial membrane motor and effector of starvation-induced filopodia2016In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 129, no 3, p. 543-556Article in journal (Refereed)
    Abstract [en]

    Mitochondria respond to environmental cues and stress conditions. Additionally, the disruption of the mitochondrial network dynamics and its distribution is implicated in a variety of neurodegenerative diseases. Here, we reveal a new function for Myo19 in mitochondrial dynamics and localization during the cellular response to glucose starvation. Ectopically expressed Myo19 localized with mitochondria to the tips of starvation-induced filopodia. Corollary to this, RNA interference (RNAi)-mediated knockdown of Myo19 diminished filopodia formation without evident effects on the mitochondrial network. We analyzed the Myo19–mitochondria interaction, and demonstrated that Myo19 is uniquely anchored to the outer mitochondrial membrane (OMM) through a 30–45-residue motif, indicating that Myo19 is a stably attached OMM molecular motor. Our work reveals a new function for Myo19 in mitochondrial positioning under stress.

  • 14.
    Shneyer, Boris I.
    et al.
    Technion, Israel.
    Ušaj, Marko
    Technion, Israel.
    Wiesel-Motiuk, Naama
    Technion, Israel.
    Regev, Ronit
    Technion, Israel.
    Henn, Arnon
    Technion, Israel.
    ROS induced distribution of mitochondria to filopodia by Myo19 depends on a class specific tryptophan in the motor domain2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, no 1, article id 11577Article in journal (Refereed)
    Abstract [en]

    The role of the actin cytoskeleton in relation to mitochondria function and dynamics is only recently beginning to be recognized. Myo19 is an actin-based motor that is bound to the outer mitochondrial membrane and promotes the localization of mitochondria to filopodia in response to glucose starvation. However, how glucose starvation induces mitochondria localization to filopodia, what are the dynamics of this process and which enzymatic adaptation allows the translocation of mitochondria to filopodia are not known. Here we show that reactive oxygen species (ROS) mimic and mediate the glucose starvation induced phenotype. In addition, time-lapse fluorescent microscopy reveals that ROS-induced Myo19 motility is a highly dynamic process which is coupled to filopodia elongation and retraction. Interestingly, Myo19 motility is inhibited by back-to-consensus-mutation of a unique residue of class XIX myosins in the motor domain. Kinetic analysis of the purified mutant Myo19 motor domain reveals that the duty ratio (time spent strongly bound to actin) is highly compromised in comparison to that of the WT motor domain, indicating that Myo19 unique motor properties are necessary to propel mitochondria to filopodia tips. In summary, our study demonstrates the contribution of actin-based motility to the mitochondrial localization to filopodia by specific cellular cues.

  • 15.
    Shneyer, Boris
    et al.
    Technion, Israel.
    Ušaj, Marko
    Technion, Israel.
    Henn, Arnon
    Technion, Israel.
    Myosin 19 is Anchored to the Mitochondria, Affecting its Localization and Morphology2015In: Biophysical Journal supplement 1, 2015, Vol. 108, p. 303a-, article id 1517-PosConference paper (Refereed)
    Abstract [en]

    Mitochondria undergo continuous cycles of fission and fusion creating a highly dynamic network, which is essential for its proper functions in apoptosis, ATP generation and calcium homeostasis. Mitochondria long-range motility relies on the microtubule motors kinesin and dynein. Recently, actin and myosin 19 have been implicated in mitochondrial motility in vertebrates. However, the interaction of endogenous myosin 19 with the mitochondria remains unknown. Here, we show using multiple complementary approaches that endogenous myosin 19 is anchored directly to the outer mitochondrial membrane (OMM) in a monotopic fashion. We have identified a region of 30 residues at the tail domain of myosin 19, which is both essential and sufficient for myosin 19-OMM interaction. Furthermore, we have purified to near homogeneity a 45 long peptide comprised of this region to study its biochemical and biophysical properties. We performed in-vitro binding assay by fluorescence anisotropy of this specific purified peptide to vesicles with different phospholipid compositions. Our results revealed that that this peptide binds to vesicles mimicking the OMM with the highest affinity. To relate this tight binding to the mitochondria to myosin 19 ATPase activity, we have purified myosin 19-3IQ construct and measured its actin-dependent steady state ATPase activity. Interestingly, we found that it is completely inhibited by very low calcium concentration, suggesting that myosin 19 activity may be regulated by local calcium concentration. The interaction between a motor protein and an organelle, and the calcium dependence implicates that myosin 19 plays a role in mitochondria network dynamics.

  • 16.
    Silberberg, Gilad
    et al.
    Stockholm University.
    Lundin, Daniel
    Stockholm University.
    Navon, Ruth
    Tel Aviv University, Israel.
    Öhman, Marie
    Stockholm University.
    Deregulation of the A-to-I RNA editing mechanism in psychiatric disorders2012In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 21, no 2, p. 311-321Article in journal (Refereed)
    Abstract [en]

    Schizophrenia and bipolar disorder (BPD) are common neurodevelopmental disorders, characterized by various life-crippling symptoms and high suicide rates. Multiple studies support a strong genetic involvement in the etiology of these disorders, although patterns of inheritance are variable and complex. Adenosine-to-inosine RNA editing is a cellular mechanism, which has been implicated in mental disorders and suicide. To examine the involvement of altered RNA editing in these disorders, we: (i) quantified the mRNA levels of the adenosine deaminase acting on RNA (ADAR) editing enzymes by real-time quantitative polymerase chain reaction, and (ii) measured the editing levels in transcripts of several neuroreceptors using 454 high-throughput sequencing, in dorsolateral-prefrontal cortices of schizophrenics, BPD patients and controls. Increased expression of specific ADAR2 variants with diminished catalytic activity was observed in schizophrenia. Our results also indicate that the I/V editing site in the glutamate receptor, ionotropic kainate 2 (GRIK2) transcript is under-edited in BPD (type I) patients (45.8 versus 53.9%, P= 0.023). GRIK2 has been implicated in mood disorders, and editing of its I/V site can modulate Ca(+2) permeability of the channel, consistent with numerous observations of elevated intracellular Ca(+2) levels in BPD patients. Our findings may therefore, at least partly, explain a molecular mechanism underlying the disorder. In addition, an intriguing correlation was found between editing events on separate exons of GRIK2. Finally, multiple novel editing sites were detected near previously known sites, albeit most with very low editing rates. This supports the hypothesis raised previously regarding the existence of wide-spread low-level 'background' editing as a mechanism that enhances adaptation and evolvability.

  • 17. Sköld, H. N.
    et al.
    Amundsen, T.
    Svensson, P. Andreas
    Mayer, I.
    Bjelvenmark, J.
    Forsgren, E.
    Hormonal regulation of female nuptial coloration in a fish2008In: Hormones and Behavior, ISSN 0018-506X, E-ISSN 1095-6867, Vol. 54, no 4, p. 549-556Article in journal (Refereed)
    Abstract [en]

    Physiological color change in camouflage and mating is widespread among fishes, but little is known about the regulation of such temporal changes in nuptial coloration and particularly concerning female coloration. To better understand regulation of nuptial coloration we investigated physiological color change in female two-spotted gobies (Gobiusculus flavescens). Females of this species develop an orange belly that acts as an ornament. The orange color is caused by the color of the gonads combined with the chromathophore based pigmentation and transparency of the skin. Often during courtship and female-female competition, a rapid increase in orange coloration, in combination with lighter sides and back that increases skin and body transparency, gives the belly an intense 'glowing' appearance. To understand how this increased orange coloration can be regulated we analysed chromatic and transparency effects of neurohumoral agents on abdominal skin biopsies in vitro. We found prolactin and alpha-melanocyte stimulating hormone (MSH) to increase orange coloration of the skin. By contrast, melatonin and noradrenaline increased skin transparency, but had a negative effect on orange coloration. However, mixtures of melatonin and MSH, or melatonin and prolactin, increased both orange coloration and transparency. This effect mimics the chromatic 'glow' effect that commonly takes place during courtship and intra sexual aggression. Notably, not only epidermal chromatophores but also internal chromatophores lining the peritoneum responded to hormone treatments. There were no chromatic effects of the sex steroids 17 beta-estradiol, testosterone or 11-ketotestosterone. We hypothesize that similar modulation of nuptial coloration by multiple hormones may be widespread in nature. (C) 2008 Elsevier Inc. All rights reserved.

  • 18.
    Svensson, P. Andreas
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Nilsson-Sköld, Helen
    University of Gothenburg.
    Skin biopsies as tools to measure fish coloration and colour change2011In: Skin Biopsy - Perspectives / [ed] Uday Khopkar, InTech, 2011Chapter in book (Other academic)
  • 19.
    Ušaj, Marko
    et al.
    Technion - Israel Institute of Technology, Israel.
    Zattelman, Lilach
    Technion - Israel Institute of Technology, Israel.
    Regev, Ronit
    Technion - Israel Institute of Technology, Israel.
    Shneyer, Boris I.
    Technion - Israel Institute of Technology, Israel.
    Wiesel-Motiuk, Naama
    Technion - Israel Institute of Technology, Israel.
    Henn, Arnon
    Technion - Israel Institute of Technology, Israel.
    Overexpression and purification of human myosins from transiently and stably transfected suspension adapted HEK293SF-3F6 cells2018In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 558, no 1, p. 19-27Article in journal (Refereed)
    Abstract [en]

    The myosin family of motor proteins is an attractive target of therapeutic small-molecule protein inhibitors and modulators. Milligrams of protein quantities are required to conduct proper biophysical and biochemical studies to understand myosin functions. Myosin protein expression and purification represent a critical starting point towards this goal. Established utilization of Dictyostelium discoideum, Drosophila melanogaster, insect and mouse cells for myosin expression and purification is limited, cost, labor and time inefficient particularly for (full-length) human myosins. Here we are presenting detailed protocols for production of several difficult-to-purify recombinant human myosins in efficient quantities up to 1 mg of protein per liter of cell culture. This is the first time that myosins have been purified in large scales from suspension adapted transiently and stably expressing human cells. The method is also useful for expressing other human proteins in quantities sufficient to perform extensive biochemical and biophysical characterization.

1 - 19 of 19
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