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  • 1. Daste, Frederic
    et al.
    Walrant, Astrid
    Holst, Mikkel R.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Gadsby, Jonathan R.
    Mason, Julia
    Lee, Ji-Eun
    Brook, Daniel
    Mettlen, Marcel
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Lee, Steven F.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Gallop, Jennifer L.
    Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature2017Ingår i: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 216, nr 11, s. 3745-3765Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The conditional use of actin during clathrin-mediated endocytosis in mammalian cells suggests that the cell controls whether and how actin is used. Using a combination of biochemical reconstitution and mammalian cell culture, we elucidate a mechanism by which the coincidence of PI(4,5)P-2 and PI(3)P in a curved vesicle triggers actin polymerization. At clathrin-coated pits, PI(3) P is produced by the INPP4A hydrolysis of PI(3,4)P-2, and this is necessary for actin-driven endocytosis. Both Cdc42.guanosine triphosphate and SNX9 activate N-WASP-WIP-and Arp2/3-mediated actin nucleation. Membrane curvature, PI(4,5)P-2, and PI(3) P signals are needed for SNX9 assembly via its PX-BAR domain, whereas signaling through Cdc42 is activated by PI(4,5)P-2 alone. INPP4A activity is stimulated by high membrane curvature and synergizes with SNX9 BAR domain binding in a process we call curvature cascade amplification. We show that the SNX9-driven actin comets that arise on human disease-associated oculocerebrorenal syndrome of Lowe (OCRL) deficiencies are reduced by inhibiting PI(3) P production, suggesting PI(3) P kinase inhibitors as a therapeutic strategy in Lowe syndrome.

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  • 2. Hoernke, M.
    et al.
    Mohan, J.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Kahra, Dana
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Westenhoff, S.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Schwieger, C.
    Determining membrane bound protein structures by infrared reflection-absorption spectroscopy2017Ingår i: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 46, s. S161-S161Artikel i tidskrift (Övrigt vetenskapligt)
  • 3. Hoernke, M
    et al.
    Mohan, Jagan
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Blomberg, J
    Kahra, Dana
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Daumke, O
    Westenhof, S
    Schweiger, C
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    TP driven stabilization of a membrane bound open confirmation of the ATPase EHD2 restrains caveolae dynamicsManuskript (preprint) (Övrigt vetenskapligt)
  • 4. Hoernke, Maria
    et al.
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mohan, Jagan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Blomberg, Jeanette
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Westenhoff, Sebastian
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Schwieger, Christian
    Structural Mechanism in a Membrane Remodelling ATP-ASE2016Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, nr 3, s. 578A-578AArtikel i tidskrift (Övrigt vetenskapligt)
  • 5. Hoernke, Maria
    et al.
    Mohan, Jagan
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Blomberg, Jeanette
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Kahra, Dana
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Westenhoff, Sebastian
    Schwieger, Christian
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation2017Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, nr 22, s. E4360-E4369Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The EH-domain-containing protein 2 (EHD2) is a dynamin-related ATPase that confines caveolae to the cell surface by restricting the scission and subsequent endocytosis of these membrane pits. For this, EHD2 is thought to first bind to the membrane, then to oligomerize, and finally to detach, in a stringently regulated mechanistic cycle. It is still unclear how ATP is used in this process and whether membrane binding is coupled to conformational changes in the protein. Here, we show that the regulatory N-terminal residues and the EH domain keep the EHD2 dimer in an autoinhibited conformation in solution. By significantly advancing the use of infrared reflection-absorption spectroscopy, we demonstrate that EHD2 adopts an open conformation by tilting the helical domains upon membrane binding. We show that ATP binding enables partial insertion of EHD2 into the membrane, where G-domain-mediated oligomerization occurs. ATP hydrolysis is related to detachment of EHD2 from the membrane. Finally, we demonstrate that the regulation of EHD2 oligomerization in a membrane-bound state is crucial to restrict caveolae dynamics in cells.

  • 6.
    Holst, Mikkel Roland
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Vidal-Quadras, Maite
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Song, Jie
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Hubert, Madlen
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Blomberg, Jeanette
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Lundborg, Magnus
    Landström, Maréne
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Clathrin-Independent Endocytosis Suppresses Cancer Cell Blebbing and Invasion2017Ingår i: Cell Reports, E-ISSN 2211-1247, Vol. 20, nr 8, s. 1893-1905Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cellular blebbing, caused by local alterations in cellsurface tension, has been shown to increase the invasiveness of cancer cells. However, the regulatory mechanisms balancing cell-surface dynamics and bleb formation remain elusive. Here, we show that an acute reduction in cell volume activates clathrinindependent endocytosis. Hence, a decrease in surface tension is buffered by the internalization of the plasma membrane (PM) lipid bilayer. Membrane invagination and endocytosis are driven by the tension- mediated recruitment of the membrane sculpting and GTPase-activating protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) to the PM. Disruption of this regulation by depleting cells of GRAF1 or mutating key phosphatidylinositol- interacting amino acids in the protein results in increased cellular blebbing and promotes the 3D motility of cancer cells. Our data support a role for clathrin-independent endocytic machinery in balancing membrane tension, which clarifies the previously reported role of GRAF1 as a tumor suppressor.

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  • 7.
    Hubert, Madlen
    et al.
    Department of Pharmacy, Uppsala University, Uppsala, Sweden.
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Liu, Kang Cheng
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Caveolae biogenesis and lipid sorting at the plasma membrane2022Ingår i: Plasma membrane shaping / [ed] Shiro Suetsugu, Academic Press, 2022, s. 219-228Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    The plasma membrane of many cell types, in particular, endothelia, smooth muscle cells, and adipocytes, contains numerous small invaginations termed caveolae. In nonmuscle cells, caveolae are formed by lipid-driven assembly of the integral membrane protein caveolin 1 (Cav1) and the peripherally attached protein cavin1. Accessory proteins such as Eps15 homology domain-containing 2 (EHD2) control the cell surface association of caveolae, together providing a unique invaginated membrane structure with distinct dynamics and protein and lipid compositions. These features enable caveolae to survey the plasma membrane integrity and to adjust membrane tension, and sort lipids according to the cellular requirements. Currently, characteristics of the protein and lipid interface of caveola are being unraveled, and this chapter is focused on the present knowledge of caveolae biogenesis and dynamics and describes methods that are being used to study the role of caveolae in lipid flux and lipid composition at the cell surface.

  • 8.
    Hubert, Madlen
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Caveolae dynamics is strongly influenced by the lipid composition of the plasma membrane2017Ingår i: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 46, s. S121-S121Artikel i tidskrift (Övrigt vetenskapligt)
  • 9.
    Hubert, Madlen
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Department of Pharmacy, Uppsala University, BMC P.O. Box 580, SE-751 23 Uppsala, Sweden.
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Keeping in touch with the membrane; protein- and lipid-mediated confinement of caveolae to the cell surface2020Ingår i: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 48, s. 155-163Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Caveolae are small Omega-shaped invaginations of the plasma membrane that play important roles in mechanosensing, lipid homeostasis and signaling. Their typical morphology is characterized by a membrane funnel connecting a spherical bulb to the membrane. Membrane funnels (commonly known as necks and pores) are frequently observed as transient states during fusion and fission of membrane vesicles in cells. However, caveolae display atypical dynamics where the membrane funnel can be stabilized over an extended period of time, resulting in cell surface constrained caveolae. In addition, caveolae are also known to undergo flattening as well as short-range cycles of fission and fusion with the membrane, requiring that the membrane funnel closes or opens up, respectively. This mini-review considers the transition between these different states and highlights the role of the protein and lipid components that have been identified to control the balance between surface association and release of caveolae.

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  • 10.
    Hubert, Madlen
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Vegesna, Naga Venkata Gayathri
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Ahnlund, Maria
    Johansson, Annika I.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Moodie, Lindon W. K.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Lipid accumulation controls the balance between surface connection and scission of caveolae2020Ingår i: eLIFE, E-ISSN 2050-084X, Vol. 9, artikel-id e55038Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Caveolae are bulb-shaped invaginations of the plasma membrane (PM) that undergo scission and fusion at the cell surface and are enriched in specific lipids. However, the influence of lipid composition on caveolae surface stability is not well described or understood. Accordingly, we inserted specific lipids into the cell PM via membrane fusion and studied their acute effects on caveolae dynamics. We demonstrate that sphingomyelin stabilizes caveolae to the cell surface, whereas cholesterol and glycosphingolipids drive caveolae scission from the PM. Although all three lipids accumulated specifically in caveolae, cholesterol and sphingomyelin were actively sequestered, whereas glycosphingolipids diffused freely. The ATPase EHD2 restricts lipid diffusion and counteracts lipid-induced scission. We propose that specific lipid accumulation in caveolae generates an intrinsically unstable domain prone to scission if not restrained by EHD2 at the caveolae neck. This work provides a mechanistic link between caveolae and their ability to sense the PM lipid composition.

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  • 11.
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik.
    The use of monogenic disease to study basal and disease associated mechanisms with focus on NGF dependent pain insensitivity and ISCU myopathy2012Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Monogenic diseases make excellent models for the study of gene functions and basal cellular mechanisms in humans. The aim of this thesis was to elucidate how genetic mutations affect the basal cellular mechanisms in the monogenic diseases Nerve growth factor (NGF) dependent pain insensitivity and Iron-Sulphur cluster assembly protein U (ISCU) myopathy.

    NGF dependent pain insensitivity is a rare genetic disorder with clinical manifestations that include insensitivity to deep pain, development of Charcot joints, and impaired temperature sensation but with no effect on mental abilities. The disease is caused by a missense mutation in the NGFβ gene causing a drastic amino acid substitution (R221W) in a well-conserved region of the protein. NGF is secreted in limited amounts by its target tissues and is important for the development and maintenance of the cholinergic forebrain neurons as well as the sensory and sympathetic neurons. To reveal the underlying mechanisms of disease we performed functional studies of the mutant NGF protein. We could show that mutant NGF was unable to induce differentiation of PC12 cells as a consequence of impaired secretion. Furthermore, mutant NGF had different intracellular localisation compared to normal NGF and resided mostly in its unprocessed form proNGF. Mature NGF and proNGF have different binding properties to the receptors TrkA and p75. Individuals with mutations in TRKA are, aside from pain insensitive mentally affected; therefore it has been proposed that the R221W mutation mainly affects the interaction with p75. In agreement with this, we could show that R221W NGF was able to bind and activate TrkA whereas the interaction with p75 was impaired as compared to normal NGF.

    ISCU myopathy is a monogenic disease where the affected patients suffer from severe exercise intolerance resulting in muscle cramps and sometimes severe lactic acidosis. The disease is caused by a point mutation in the last intron of the Iron sulphur cluster assembly gene, ISCU, resulting in the inclusion of a part of the intron in the mRNA. ISCU functions as a scaffold protein in the assembly of iron-sulphur (Fe-S) clusters important for electron transport in Kreb’s cycle and the respiratory chain. We have shown that ISCU is vital in mammals since complete knock-down of Iscu in mice results in early embryonic death. The deletion of ISCU homologous in lower organisms has also been shown fatal. In spite this central role in energy metabolism the disease is restricted to the patient’s skeletal muscles while other energy demanding organs seem unaffected. To address this contradiction we examined if tissue-specific differences in the splicing of mutant ISCU could explain the muscle-specific phenotype. We could show that the splicing pattern did, indeed, differ with more incorrectly spliced ISCU in muscle compared to other tissues. This was accompanied by a decrease in Fe-S containing proteins in muscle, while no decrease was observed in other tissues. Alternative splicing is more common then previously thought and may depend upon interacting factors and/or differences in the surrounding milieu. To reveal plausible mechanisms involved in the tissue-specific splicing we identified nuclear factors that interacted with the region where the mutation was located. Five interacting factors were identified, out of which three affected the splicing of ISCU. PTBP1 was shown to repress the incorrect splicing while IGF2BP1 and RBM39 repressed the formation of normal transcript and could also counteract the effect of PTBP1. IGF2BP1 was the only factor that showed higher affinity to the mutant sequence making it a possible key factor in the incorrect splicing of the mutant ISCU gene.

    Together, these results offer important insights into the cellular mechanisms causing these diseases. We found impaired secretion and inaccurate sorting of NGF to be cellular mechanisms contributing to NGF dependent pain insensitivity while tissue-specific splicing of ISCU was found to be the event contributing to the phenotype of ISCU myopathy.

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    The Use of Monogenic Disease to Study Basal and Disease associated Mechanisms with Focus on NGF Dependent Pain Insensitivity and ISCU Myopathy
  • 12.
    Larsson, Elin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap.
    Fahey, Mark S.
    Watson, Judy J.
    Holmberg, Monica
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap.
    Dawbarn, David
    Allen, Shelley J.
    Purification and Characterization of the Nerve Growth Factor R221W mutant causing Insensitivity to PainManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

     

    We have previously identified a homozygous missense (R221W) mutation in the NGFβ gene which causes insensitivity to pain in patients. The mutation impairs the secretion of NGF and the majority of the protein accumulates as proNGF. NGF mediates its function by binding and activating the TrkA and p75 receptors and is important for the survival of the sensory and sympathetic neurons as well as the cholinergic neurons of the basal forebrain. However, the R221W mutation seems to discriminate between these types of neurons as it is the sensory neurons that are mainly affected in the patients. A second human NGFβ mutation causes a more severe form of pain insensitivity with additional anhidrosis and cognitive dysfunctions in affected patients which is also seen in patients with mutations in the gene encoding the TrkA receptor. Because R221W NGF cause a less severe phenotype we hypothesised that the mutation mainly affects the p75 interaction which is also strengthened by the fact that the substitution is located in a region known to interact with p75. In this report, we show that R221W NGF is able to bind and activate TrkA at a level comparable to wild-type NGF in cells stably expressing TrkA while the activation of the downstream target ERK1/2 is impaired in cells that co-express TrkA and p75. We also describe the effects of the mutation in terms of expression and purification properties from E.coli which indicate the likelihood that eukaryotic folding machinery is needed for correct folding of R221W NGF.

  • 13.
    Larsson, Elin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Hubert, Madlen
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Analysis of protein and lipid interactions using liposome co-sedimentation assays2020Ingår i: Caveolae: methods and protocols / [ed] Cedric M. Blouin, Humana Press, 2020, , s. 9s. 119-127Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    The dynamic assembly of proteins at the membrane interphase is key to many cell biological processes such as the generation and stabilization of caveolae at the cell surface via coat proteins. The liposome co-sedimentation assay has been widely used for studies of protein and lipid interactions and has provided important information about binding mechanisms, lipid-binding specificity, and curvature preference of proteins. Here, we describe this technique in detail and how it can be used as a tool to address the membrane-binding ability and lipid specificity of caveolae-associated proteins.

  • 14.
    Larsson, Elin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik.
    Kuma, Regina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik.
    Norberg, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik.
    Minde, Jan
    Department of Orthopedics, Gällivare Hospital, Gällivare, Sweden.
    Holmberg, Monica
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik.
    Nerve growth factor R221W responsible for insensitivity to pain is defectively processed and accumulates as proNGF2009Ingår i: Neurobiology of Disease, ISSN 0969-9961, E-ISSN 1095-953X, Vol. 33, nr 2, s. 221-228Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have previously identified a homozygous missense (R221W) mutation in the NGFB gene in patients with loss of deep pain perception. NGF is important not only for the survival of sensory neurons but also for the sympathetic neurons and cholinergic neurons of the basal forebrain; however, it is the sensory neurons that are mainly affected in patients with mutant NGFB. In this report, we describe the effects of the mutation on the function of NGF protein and the molecular mechanisms that may underlie the pain insensitivity phenotype in these patients. We show that the mutant NGF has lost its ability to mediate differentiation of PC12 cells into a neuron-like phenotype. We also show that the inability of PC12 cells to differentiate is due to a markedly reduced secretion of mature R221W NGF. The R221W NGF is found mainly as proNGF, in contrast to wild-type NGF which is predominantly in the mature form in both undifferentiated and differentiated PC12 cells. The reduction in numbers of sensory fibers observed in the patients is therefore probably due to loss of trophic support as a result of drastically reduced secretion of NGF from the target organs. Taken together, these data show a clear decrease in the availability of mutant mature NGF and also an accumulation of proNGF in both neuronal and non-neuronal cells. The differential loss of NGF-dependent neurons in these patients, mainly affecting sensory neurons, may depend on differences in the roles of mature NGF and proNGF in different cells and tissues.

  • 15.
    Larsson, Elin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Morén, Björn
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    McMahon, Kerrie-Ann
    Institute for Molecular Bioscience, The University of Queensland, QLD, Brisbane, Australia.
    Parton, Robert G.
    Institute for Molecular Bioscience, The University of Queensland, QLD, Brisbane, Australia; Centre for Microscopy and Microanalysis, The University of Queensland, QLD, Brisbane, Australia.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Dynamin2 functions as an accessory protein to reduce the rate of caveola internalization2023Ingår i: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 222, nr 4, artikel-id e202205122Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Caveolae are small membrane invaginations that generally are stably attached to the plasma membrane. Their release is believed to depend on the GTPase dynamin 2 (Dyn2), in analogy with its role in fission of clathrin-coated vesicles. The mechanistic understanding of caveola fission is, however, sparse. Here, we used microscopy-based tracking of individual caveolae in living cells to determine the role of Dyn2 in caveola dynamics. We report that Dyn2 stably associated with the bulb of a subset of caveolae, but was not required for formation or fission of caveolae. Dyn2-positive caveolae displayed longer plasma membrane duration times, whereas depletion of Dyn2 resulted in shorter duration times and increased caveola fission. The stabilizing role of Dyn2 was independent of its GTPase activity and the caveola stabilizing protein EHD2. Thus, we propose that, in contrast to the current view, Dyn2 is not a core component of the caveolae machinery, but rather functions as an accessory protein that restrains caveola internalization.

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  • 16.
    Larsson, Elin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Morén, Björn
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Shah, Claudio
    Max-Delbrück-Center for Molecular Medicine.
    Daumke, Oliver
    Max-Delbrück-Center for Molecular Medicine.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    An EH-domain switching mechanism regulates stable membrane association of EHD2Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    EHD2 is a dimeric ATPase known to stabilise the surface connection of the characteristic small invaginations of the cell surface termed caveolae. EHD2 oligomerises into rings around lipid membranes thereby controlling their shape. Here, we have analysed the domain interactions and mechanism that control the stable membrane association of EHD2 at caveolae. We have found that the N-terminus of EHD2, which is buried in the core protein and obstruct assembly, has to be relieved by an EH domain dependent mechanism. The binding between the EH domain and a loop in the GTPase domain of EHD2 was required for stable membrane association, but the loop in itself was not sufficient for specific recruitment to caveolae. A positively charged stretch in the EH domain is proposed to bind to lipids and thereby influence the exchange rate of EHD2. Taken together, we propose a stringent regulatory mechanism for the assembly of EHD2 involving switching of the EH domain position to release the N-terminus and facilitate oligomerisation.

  • 17.
    Liu, Kang-Cheng
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Pace, Hudson
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Wallenberg centrum för molekylär medicin vid Umeå universitet (WCMM).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik. Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Hossain, Shakhawath
    Department of Pharmacy, Uppsala Biomedical Center, Uppsala University, Uppsala, Sweden.
    Kabedev, Aleksei
    Department of Pharmacy, Uppsala Biomedical Center, Uppsala University, Uppsala, Sweden.
    Shukla, Ankita
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Jerschabek, Vanessa
    Institute of Physical Chemistry, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
    Mohan, Jagan
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Bergström, Christel A.S.
    Department of Pharmacy, Uppsala Biomedical Center, Uppsala University, Uppsala, Sweden.
    Bally, Marta
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Wallenberg centrum för molekylär medicin vid Umeå universitet (WCMM). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Schwieger, Christian
    Institute of Physical Chemistry, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
    Hubert, Madlen
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Department of Pharmacy, Uppsala Biomedical Center, Uppsala University, Uppsala, Sweden.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Membrane insertion mechanism of the caveola coat protein Cavin12022Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 119, nr 25, artikel-id 2202295119Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P2]-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.

  • 18. Matthaeus, Claudia
    et al.
    Lahmann, Ines
    Kunz, Severine
    Jonas, Wenke
    Melo, Arthur Alves
    Lehmann, Martin
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Kern, Matthias
    Blueher, Matthias
    Olschowski, Hannah
    Kompa, Julian
    Bruegger, Britta
    Mueller, Dominik N.
    Haucke, Volker
    Schuermann, Annette
    Birchmeier, Carmen
    Daumke, Oliver
    EHD2-mediated restriction of caveolar dynamics regulates cellular fatty acid uptake2020Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 117, nr 13, s. 7471-7481Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Eps15-homology domain containing protein 2 (EHD2) is a dynamin-related ATPase located at the neck of caveolae, but its physiological function has remained unclear. Here, we found that global genetic ablation of EHD2 in mice leads to increased lipid droplet size in fat tissue. This organismic phenotype was paralleled at the cellular level by increased fatty acid uptake via a caveolae- and CD36-dependent pathway that also involves dynamin. Concomitantly, elevated numbers of detached caveolae were found in brown and white adipose tissue lacking EHD2, and increased caveolar mobility in mouse embryonic fibroblasts. EHD2 expression itself was down-regulated in the visceral fat of two obese mouse models and obese patients. Our data suggest that EHD2 controls a cell-autonomous, caveolae-dependent fatty acid uptake pathway and imply that low EHD2 expression levels are linked to obesity.

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  • 19. Melo, Arthur Alves
    et al.
    Hegde, Balachandra G.
    Shah, Claudio
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Isas, J. Mario
    Kunz, Severine
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Langen, Ralf
    Daumke, Oliver
    Structural insights into the activation mechanism of dynamin-like EHD ATPases2017Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, nr 22, s. 5629-5634Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Eps15 (epidermal growth factor receptor pathway substrate 15)homology domain containing proteins (EHDs) comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking. Previous studies have revealed the structure of the EHD2 dimer, but the molecular mechanisms of membrane recruitment and assembly have remained obscure. Here, we determined the crystal structure of an amino-terminally truncated EHD4 dimer. Compared with the EHD2 structure, the helical domains are 50 degrees rotated relative to the GTPase domain. Using electron paramagnetic spin resonance (EPR), we show that this rotation aligns the two membrane-binding regions in the helical domain toward the lipid bilayer, allowing membrane interaction. A loop rearrangement in GTPase domain creates a new interface for oligomer formation. Our results suggest that the EHD4 structure represents the active EHD conformation, whereas the EHD2 structure is autoinhibited, and reveal a complex series of domain rearrangements accompanying activation. A comparison with other peripheral membrane proteins elucidates common and specific features of this activation mechanism.

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  • 20.
    Mohan, Jagan
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Hubert, Madlen
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Schweiger, C
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Structural arrangement of membrane-bound cavinManuskript (preprint) (Övrigt vetenskapligt)
  • 21.
    Mohan, Jagan
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Morén, Björn
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Holst, Mikkel
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Cavin3 interacts with cavin1 and caveolin1 to increase surface dynamics of caveolae2015Ingår i: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 128, nr 5, s. 979-991Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Caveolae are invaginations of the cell surface thought to regulate membrane tension, signalling, adhesion and lipid homeostasis due to their dynamic behaviour ranging from stable surface association to dynamic rounds of fission and fusion with the plasma membrane. The caveolae coat is generated by oligomerisation of the membrane protein caveolin and the family of cavin proteins. Here, we show that cavin3 is targeted to caveolae by cavin1 where it interacts with the scaffolding domain of caveolin1 and promote caveolae dynamics. We found that the N-terminal region of cavin3 binds a trimer of the cavin1 N-terminus in competition with a homologous cavin2 region, showing that the cavins form distinct subcomplexes via their N-terminal regions. Our data shows that cavin3 is enriched at deeply invaginated caveolae and that loss of cavin3 in cells results in an increase of stable caveolae and a decrease of caveolae with short duration time at the membrane. We propose that cavin3 is recruited to the caveolae coat by cavin1 to interact with caveolin1 and regulate the duration time of caveolae at the plasma membrane.

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  • 22.
    Nordin, Angelica
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik.
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik.
    Holmberg, Monica
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik.
    The defective splicing caused by the ISCU intron mutation in patients with myopathy with lactic acidosis is repressed by PTBP1 but can be de-repressed by IGF2BP12012Ingår i: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 33, nr 3, s. 467-470Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Hereditary myopathy with lactic acidosis (HML) is caused by an intron mutation in the iron-sulfur cluster assembly gene ISCU which leads to the activation of cryptic splice sites and the retention of part of intron 4. This incorrect splicing is more pronounced in muscle than in other tissues, resulting in a muscle-specific phenotype. In this study, we identified five nuclear factors that interact with the sequence harboring the mutation and analyzed their effect on the splicing of the ISCU gene. The identification revealed three splicing factors, SFRS14, RBM39 and PTBP1, and two additional RNA binding factors, matrin 3 (MATR3) and IGF2BP1. IGF2BP1 showed a preference for the mutant sequence, whereas the other factors showed similar affinity for both sequences. PTBP1 was found to repress the defective splicing of ISCU, resulting in a drastic loss of mutant transcripts. In contrast, IGF2BP1 and RBM39 shifted the splicing ratio toward the incorrect splice form.

  • 23.
    Nordin, Angelica
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik.
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik.
    Thornell, Lars-Eric
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB), Anatomi.
    Holmberg, Monica
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik.
    Tissue-specific splicing of ISCU results in a skeletal muscle phenotype in myopathy with lactic acidosis, while complete loss of ISCU results in early embryonic death in mice2011Ingår i: Human Genetics, ISSN 0340-6717, E-ISSN 1432-1203, Vol. 129, nr 4, s. 371-378Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Hereditary myopathy with lactic acidosis (HML) is caused by an intron mutation in the iron-sulphur cluster assembly gene (ISCU) leading to incorporation of intron sequence into the mRNA. This results in a deficiency of Fe-S cluster proteins, affecting the TCA cycle and the respiratory chain. The proteins involved in the Fe-S machinery are evolutionary conserved and shown to be fundamental in all organisms examined. ISCU is expressed at high levels in numerous tissues in mammals, including high metabolic tissues like the heart, suggesting that a drastic mutation in the ISCU gene would be damaging to all energy-demanding organs. In spite of this, the symptoms in patients with HML are restricted to skeletal muscle, and it has been proposed that splicing events may contribute to the muscle specificity. In this study we confirm that a striking difference in the splicing pattern of mutant ISCU exists between different tissues. The highest level of incorrectly spliced ISCU mRNA was found in skeletal muscle, while the normal splice form predominated in patient heart. The splicing differences were also reflected at a functional level, where loss of Fe-S cluster carrying enzymes and accumulation of iron were present in muscle, but absent in other tissues. We also show that complete loss of ISCU in mice results in early embryonic death. The mice data confirm a fundamental role for ISCU in mammals and further support tissue-specific splicing as the major mechanism limiting the phenotype to skeletal muscle in HML.

  • 24.
    Nygård Skalman, Lars
    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).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Plasma membrane damage caused by listeriolysin O is not repaired through endocytosis of the membrane pore2018Ingår i: Biology Open, ISSN 2046-6390, Vol. 7, nr 10Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Endocytic mechanisms have been suggested to be important for plasma membrane repair in response to pore-forming toxins such as listeriolysin O (LLO), which form membrane pores that disrupt cellular homeostasis. Yet, little is known about the specific role of distinct endocytic machineries in this process. Here, we have addressed the importance of key endocytic pathways and developed reporter systems for real-time imaging of the endocytic response to LLO pore formation. We found that loss of clathrin-independent endocytic pathways negatively influenced the efficiency of membrane repair. However, we did not detect any increased activity of these pathways, or co-localisation with the toxin or markers of membrane repair, suggesting that they were not directly involved in removal of LLO pores from the plasma membrane. In fact, markers of clathrin-independent carriers (CLICs) were rapidly disassembled in the acute phase of membrane damage due to Ca2+ influx, followed by a reassembly about 2 min after pore formation. We propose that these endocytic mechanisms might influence membrane repair by regulating the plasma membrane composition and tension, but not via direct internalisation of LLO pores.

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  • 25. Rodrigues, Leticia
    et al.
    Schneider, Fabian
    Zhang, Xiaohan
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Moodie, Lindon W. K.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Dietz, Hendrik
    Papadakis, Christine M.
    Winter, Gerhard
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Hubert, Madlen
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Cellular uptake of self-assembled phytantriol-based hexosomes is independent of major endocytic machineries2019Ingår i: Journal of Colloid and Interface Science, ISSN 0021-9797, E-ISSN 1095-7103, Vol. 553, s. 820-833Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Despite increasing interests in non-lamellar liquid crystalline dispersions, such as hexosomes, for drug delivery, little is known about their interactions with cells and mechanism of cell entry. Here we examine the cellular uptake of hexosomes based on phytantriol and mannide monooleate by HeLa cells using live cell microscopy in comparison to conventional liposomes. To investigate the importance of specific endocytosis pathways upon particle internalization, we silenced regulatory proteins of major endocytosis pathways using short interfering RNA. While endocytosis plays a significant role in liposome internalization, hexosomes are not taken up via endocytosis but through a mechanism that is dependent on cell membrane tension. Biophysical studies using biomembrane models highlighted that hexosomes have a high affinity for membranes and an ability to disrupt lipid layers. Our data suggest that direct biomechanical interactions of hexosomes with membrane lipids play a crucial role and that the unique morphology of hexosomes is vital for their membrane activity. Based on these results, we propose a mechanism, where hexosomes destabilize the bilayer, allowing them to "phase through" the membrane. Understanding parameters that influence the uptake of hexosomes is critical to establish them as carrier systems that can potentially deliver therapeutics efficiently to intracellular sites of action.

  • 26.
    Vidal-Quadras, Maite
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Holst, Mikkel R.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Francis, Monika K.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Hachimi, Mariam
    Yau, Wai-Lok
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Peranen, Johan
    Martin-Belmonte, Fernando
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Endocytic turnover of Rab8 controls cell polarization2017Ingår i: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 130, nr 6, s. 1147-1157Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Adaptation of cell shape and polarization through the formation and retraction of cellular protrusions requires balancing of endocytosis and exocytosis combined with fine-tuning of the local activity of small GTPases like Rab8. Here, we show that endocytic turnover of the plasma membrane at protrusions is directly coupled to surface removal and inactivation of Rab8. Removal is induced by reduced membrane tension and mediated by the GTPase regulator associated with focal adhesion kinase-1 (GRAF1, also known as ARHGAP26), a regulator of clathrin-independent endocytosis. GRAF1-depleted cells were deficient in multi-directional spreading and displayed elevated levels of GTP-loaded Rab8, which was accumulated at the tips of static protrusions. Furthermore, GRAF1 depletion impaired lumen formation and spindle orientation in a 3D cell culture system, indicating that GRAF1 activity regulates polarity establishment. Our data suggest that GRAF1-mediated removal of Rab8 from the cell surface restricts its activity during protrusion formation, thereby facilitating dynamic adjustment of the polarity axis.

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  • 27.
    Yau, Wai-Lok
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Nguyen-Dinh, Van
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Lindquist, Richard
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Avdelningen för virologi.
    Överby, Anna K.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Avdelningen för virologi.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Model System for the Formation of Tick-Borne Encephalitis Virus Replication Compartments without Viral RNA Replication2019Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 93, nr 18, artikel-id e00292-19Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Flavivirus is a positive-sense, single-stranded RNA viral genus, with members causing severe diseases in humans such as tick-borne encephalitis, yellow fever, and dengue fever. Flaviviruses are known to cause remodeling of intracellular membranes into small cavities, where replication of the viral RNA takes place. Nonstructural (NS) proteins are not part of the virus coat and are thought to participate in the formation of these viral replication compartments (RCs). Here, we used tick-borne encephalitis virus (TBEV) as a model for the flaviviruses and developed a stable human cell line in which the expression of NS proteins can be induced without viral RNA replication. The model system described provides a novel and benign tool for studies of the viral components under controlled expression levels. We show that the expression of six NS proteins is sufficient to induce infection-like dilation of the endoplasmic reticulum (ER) and the formation of RC-like membrane invaginations. The NS proteins form a membrane-associated complex in the ER, and electron tomography reveals that the dilated areas of the ER are closely associated with lipid droplets and mitochondria. We propose that the NS proteins drive the remodeling of ER membranes and that viral RNA, RNA replication, viral polymerase, and TBEV structural proteins are not required. IMPORTANCE TBEV infection causes a broad spectrum of symptoms, ranging from mild fever to severe encephalitis. Similar to other flaviviruses, TBEV exploits intracellular membranes to build RCs for viral replication. The viral NS proteins have been suggested to be involved in this process; however, the mechanism of RC formation and the roles of individual NS proteins remain unclear. To study how TBEV induces membrane remodeling, we developed an inducible stable cell system expressing the TBEV NS polyprotein in the absence of viral RNA replication. Using this system, we were able to reproduce RC-like vesicles that resembled the RCs formed in flavivirus-infected cells, in terms of morphology and size. This cell system is a robust tool to facilitate studies of flavivirus RC formation and is an ideal model for the screening of antiviral agents at a lower biosafety level.

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