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  • 1. Cortese, Katia
    et al.
    Howes, Mark T.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Tagliatti, Erica
    Bagnato, Paola
    Petrelli, Annalisa
    Bono, Maria
    McMahon, Harvey T.
    Parton, Robert G.
    Tacchetti, Carlo
    The HSP90 inhibitor geldanamycin perturbs endosomal structure and drives recycling ErbB2 and transferrin to modified MVBs/lysosomal compartments2013In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 24, no 2, p. 129-144Article in journal (Refereed)
    Abstract [en]

    The ErbB2 receptor is a clinically validated cancer target whose internalization and trafficking mechanisms remain poorly understood. HSP90 inhibitors, such as geldanamycin (GA), have been developed to target the receptor to degradation or to modulate downstream signaling. Despite intense investigations, the entry route and postendocytic sorting of ErbB2 upon GA stimulation have remained controversial. We report that ErbB2 levels inversely impact cell clathrin-mediated endocytosis (CME) capacity. Indeed, the high levels of the receptor are responsible for its own low internalization rate. GA treatment does not directly modulate ErbB2 CME rate but it affects ErbB2 recycling fate, routing the receptor to modified multivesicular endosomes (MVBs) and lysosomal compartments, by perturbing early/recycling endosome structure and sorting capacity. This activity occurs irrespective of the cargo interaction with HSP90, as both ErbB2 and the constitutively recycled, HSP90-independent, transferrin receptor are found within modified endosomes, and within aberrant, elongated recycling tubules, leading to modified MVBs/lysosomes. We propose that GA, as part of its anticancer activity, perturbs early/recycling endosome sorting, routing recycling cargoes toward mixed endosomal compartments.

  • 2. Daumke, Oliver
    et al.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Vallis, Yvonne
    Martens, Sascha
    Butler, P Jonathan G
    McMahon, Harvey T
    Architectural and mechanistic insights into an EHD ATPase involved in membrane remodelling2007In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 449, no 7164, p. 923-927Article in journal (Refereed)
    Abstract [en]

    The ability to actively remodel membranes in response to nucleotide hydrolysis has largely been attributed to GTPases of the dynamin superfamily, and these have been extensively studied. Eps15 homology (EH)-domain-containing proteins (EHDs/RME-1/pincher) comprise a less-well-characterized class of highly conserved eukaryotic ATPases implicated in clathrin-independent endocytosis, and recycling from endosomes. Here we show that EHDs share many common features with the dynamin superfamily, such as a low affinity for nucleotides, the ability to tubulate liposomes in vitro, oligomerization around lipid tubules in ring-like structures and stimulated nucleotide hydrolysis in response to lipid binding. We present the structure of EHD2, bound to a non-hydrolysable ATP analogue, and provide evidence consistent with a role for EHDs in nucleotide-dependent membrane remodelling in vivo. The nucleotide-binding domain is involved in dimerization, which creates a highly curved membrane-binding region in the dimer. Oligomerization of dimers occurs on another interface of the nucleotide-binding domain, and this allows us to model the EHD oligomer. We discuss the functional implications of the EHD2 structure for understanding membrane deformation.

  • 3. Doherty, Gary J.
    et al.
    Åhlund, Monika K.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Howes, Mark T.
    Moren, Bjorn
    Parton, Robert G.
    McMahon, Harvey T.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The endocytic protein GRAF1 is directed to cell-matrix adhesion sites and regulates cell spreading2011In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 22, no 22, p. 4380-4389Article in journal (Refereed)
    Abstract [en]

    The rho GTPase-activating protein GTPase regulator associated with focal adhesion kinase-1 (GRAF1) remodels membranes into tubulovesicular clathrin-independent carriers (CLICs) mediating lipid-anchored receptor endocytosis. However, the cell biological functions of this highly prevalent endocytic pathway are unclear. In this article, we present biochemical and cell biological evidence that GRAF1 interacted with a network of endocytic and adhesion proteins and was found enriched at podosome-like adhesions and src-induced podosomes. We further demonstrate that these sites comprise microdomains of highly ordered lipid enriched in GRAF1 endocytic cargo. GRAF1 activity was upregulated in spreading cells and uptake via CLICs was concentrated at the leading edge of migrating cells. Depletion of GRAF1, which inhibits CLIC generation, resulted in profound defects in cell spreading and migration. We propose that GRAF1 remodels membrane microdomains at adhesion sites into endocytic carriers, facilitating membrane turnover during cell morphological changes.

  • 4. Eberth, Alexander
    et al.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gremer, Lothar
    Dvorsky, Radovan
    Koessmeier, Katja T
    McMahon, Harvey T
    Ahmadian, Mohammad Reza
    A BAR domain-mediated autoinhibitory mechanism for RhoGAPs of the GRAF family2009In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 417, no 1, p. 371-377Article in journal (Refereed)
    Abstract [en]

    The BAR (Bin/amphiphysin/Rvs) domain defines an emerging superfamily of proteins implicated in fundamental biological processes by sensing and inducing membrane curvature. We identified a novel autoregulatory function for the BAR domain of two related GAPs' (GTPase-activating proteins) of the GRAF (GTPase regulator associated with focal adhesion kinase) subfamily. We demonstrate that the N-terminal fragment of these GAPs including the BAR domain interacts directly with the GAP domain and inhibits its activity. Analysis of various BAR and GAP domains revealed that the BAR domain-mediated inhibition of these GAPs' function is highly specific. These GAPs, in their autoinhibited state, are able to bind and tubulate liposomes in vitro, and to generate lipid tubules in cells. Taken together, we identified BAR domains as cis-acting inhibitory elements that very likely mask the active sites of the GAP domains and thus prevent down-regulation of Rho proteins. Most remarkably, these BAR proteins represent a dual-site system with separate membrane-tubulation and GAP-inhibitory functions that operate simultaneously.

  • 5. Howes, Mark T
    et al.
    Kirkham, Matthew
    Riches, James
    Cortese, Katia
    Walser, Piers J
    Simpson, Fiona
    Hill, Michelle M
    Jones, Alun
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. MRC Laboratory of Molecular Biology, Cambridge, England.
    Lindsay, Margaret R
    Hernandez-Deviez, Delia J
    Hadzic, Gordana
    McCluskey, Adam
    Bashir, Rumasia
    Liu, Libin
    Pilch, Paul
    McMahon, Harvey
    Robinson, Phillip J
    Hancock, John F
    Mayor, Satyajit
    Parton, Robert G
    Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells2010In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 190, no 4, p. 675-691Article in journal (Refereed)
    Abstract [en]

    Although the importance of clathrin- and caveolin-independent endocytic pathways has recently emerged, key aspects of these routes remain unknown. Using quantitative ultrastructural approaches, we show that clathrin-independent carriers (CLICs) account for approximately three times the volume internalized by the clathrin-mediated endocytic pathway, forming the major pathway involved in uptake of fluid and bulk membrane in fibroblasts. Electron tomographic analysis of the 3D morphology of the earliest carriers shows that they are multidomain organelles that form a complex sorting station as they mature. Proteomic analysis provides direct links between CLICs, cellular adhesion turnover, and migration. Consistent with this, CLIC-mediated endocytosis of key cargo proteins, CD44 and Thy-1, is polarized at the leading edge of migrating fibroblasts, while transient ablation of CLICs impairs their ability to migrate. These studies provide the first quantitative ultrastructural analysis and molecular characterization of the major endocytic pathway in fibroblasts, a pathway that provides rapid membrane turnover at the leading edge of migrating cells.

  • 6.
    Håberg, Karin
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Carlsson, Sven
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    SNX18 is an SNX9 paralog that acts as a membrane tubulator in AP-1-positive endosomal trafficking.2008In: Journal of Cell Science, ISSN 0021-9533, Vol. 121, no Pt 9, p. 1495-505Article in journal (Refereed)
  • 7.
    Larsson, Elin
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morén, Björn
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Shah, Claudio
    Max-Delbrück-Center for Molecular Medicine.
    Daumke, Oliver
    Max-Delbrück-Center for Molecular Medicine.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    An EH-domain switching mechanism regulates stable membrane association of EHD2Manuscript (preprint) (Other academic)
    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.

  • 8.
    Lundmark, Richard
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sorting nexin 9 participates in clathrin-mediated endocytosis through interactions with the core components2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 47, p. 46772-46781Article in journal (Refereed)
    Abstract [en]

    Sorting nexin 9 (SNX9) belongs to a family of proteins, the sorting nexins, that are characterized by the presence of a subclass of the phosphoinositide-binding phox domain. SNX9 has in its amino terminus a Src homology 3 domain and a region with predicted low complexity followed by a carboxyl-terminal part containing the phox domain. We previously found that SNX9 is one of the major proteins in hematopoietic cells that binds to the alpha and beta2-appendages of adaptor protein complex 2 (AP-2), a protein with a critical role in the formation of clathrin-coated vesicles at the plasma membrane. In the present study we show that clathrin and dynamin-2, two other essential molecules in the endocytic process, also interact with SNX9. We found that both AP-2 and clathrin bind to the low complexity region in SNX9 in a cooperative manner, whereas dynamin-2 binds to the Src homology 3 domain. In the cytosol, SNX9 is present in a 14.5 S complex containing dynamin-2 and an unidentified 41-kDa protein. In HeLa cells, SNX9 co-localized with both AP-2 and dynamin-2 at the plasma membrane or on vesicular structures derived from it but not with the early endosomal marker EEA1 or with AP-1. The results suggest that SNX9 may be recruited together with dynamin-2 and become co-assembled with AP-2 and clathrin at the plasma membrane. Overexpression in both K562 and HeLa cells of truncated forms of SNX9 interfered with the uptake of transferrin, consistent with a role of SNX9 in endocytosis.

  • 9.
    Lundmark, Richard
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The beta-appendages of the four adaptor-protein (AP) complexes: structure and binding properties, and identification of sorting nexin 9 as an accessory protein to AP-22002In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 362, no 3, p. 597-607Article in journal (Refereed)
    Abstract [en]

    Adaptor protein (AP) complexes are essential components for the formation of coated vesicles and the recognition of cargo proteins for intracellular transport. Each AP complex exposes two appendage domains with that function to bind regulatory accessory proteins in the cytosol. Secondary structure predictions, sequence alignments and CD spectroscopy were used to relate the beta-appendages of all human AP complexes to the previously published crystal structure of AP-2. The results suggested that the beta-appendages of AP-1, AP-2 and AP-3 have similar structures, consisting of two subdomains, whereas that of AP-4 lacks the inner subdomain. Pull-down and overlay assays showed partial overlap in the binding specificities of the beta-appendages of AP-1 and AP-2, whereas the corresponding domain of AP-3 displayed a unique binding pattern. That AP-4 may have a truncated, non-functional domain was indicated by its apparent inability to bind any proteins from cytosol. Of several novel beta-appendage-binding proteins detected, one that had affinity exclusively for AP-2 was identified as sorting nexin 9 (SNX9). SNX9, which contains a phox and an Src homology 3 domain, was found in large complexes and was at least partially associated with AP-2 in the cytosol. SNX9 may function to assist AP-2 in its role at the plasma membrane.

  • 10.
    Lundmark, Richard
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven R
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Driving membrane curvature in clathrin-dependent and clathrin-independent endocytosis.2010In: Seminars in Cell and Developmental Biology, ISSN 1084-9521, E-ISSN 1096-3634, Vol. 21, no 4, p. 363-70Article in journal (Refereed)
    Abstract [en]

    Cellular activity depends to a large extent on membrane bilayer dynamics. Many processes, such as organelle biogenesis and vesicular transport, rely on alterations in membrane structure and shape. It is now widely accepted that intracellular membrane curvature generation and remodelling is mediated and regulated by protein action, and the mechanisms behind the processes are currently being revealed. Here, we will briefly discuss the key principles of membrane deformation and focus on different endocytic events that use various kinds of proteins to shape the plasma membrane into transport carriers. The entry routes are adopted to make sure that a vast variety of molecules on the cell surface can be regulated by endocytosis. The principles for membrane sculpting of endocytic carriers can be viewed either from a perspective of rigid coat budding or of flexible opportunistic budding. We will discuss these principles and their implications, focusing on clathrin-dependent and -independent carrier formation and the proteins involved in the respective pathways.

  • 11.
    Lundmark, Richard
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven R
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Expression and properties of sorting nexin 9 in dynamin-mediated endocytosis2005In: Methods in Enzymology, ISSN 0076-6879, E-ISSN 1557-7988, Vol. 404, p. 545-556Article in journal (Refereed)
    Abstract [en]

    Sorting nexin 9 (SNX9) is identified as an important regulator of dynamin function in clathrin-mediated endocytosis. SNX9 recruits dynamin to the plasma membrane and promotes its GTPase activity, resulting in membrane constriction and ultimate transport vesicle scission. This chapter describes procedures to express recombinant SNX9, to biochemically characterize the cytosolic complex between SNX9 and dynamin, and to identify additional interacting partners of SNX9. Assays are presented to investigate the requirements for SNX9-dependent membrane recruitment of dynamin in vitro and in vivo.

  • 12.
    Lundmark, Richard
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven R
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Regulated membrane recruitment of dynamin-2 mediated by sorting nexin 9.2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 41, p. 42694-42702Article in journal (Refereed)
    Abstract [en]

    The endocytic proteins sorting nexin 9 (SNX9) and dynamin-2 (Dyn2) assemble in the cytosol as a resting complex, together with a 41-kDa protein. We show here that the complex can be activated for membrane binding of SNX9 and Dyn2 by incubation of cytosol in the presence of ATP. SNX9 was essential for Dyn2 recruitment, whereas the reverse was not the case. RNA interference experiments confirmed that SNX9 functions as a mediator of Dyn2 recruitment to membranes in cells. The 41-kDa component was identified as the glycolytic enzyme aldolase. Aldolase bound with high affinity to a tryptophan-containing acidic sequence in SNX9 located close to its Phox homology domain, thereby blocking the membrane binding activity of SNX9. Phosphorylation of SNX9 released aldolase from the native cytosolic complex and rendered SNX9 competent for membrane binding. The results suggest that SNX9-dependent recruitment of Dyn2 to the membrane is regulated by an interaction between SNX9 and aldolase.

  • 13.
    Lundmark, Richard
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven R
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    SNX9 - a prelude to vesicle release2009In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 122, no 1, p. 5-11Article in journal (Refereed)
    Abstract [en]

    The sorting nexin SNX9 has, in the past few years, been singled out as an important protein that participates in fundamental cellular activities. SNX9 binds strongly to dynamin and is partly responsible for the recruitment of this GTPase to sites of endocytosis. SNX9 also has a high capacity for modulation of the membrane and might therefore participate in the formation of the narrow neck of endocytic vesicles before scission occurs. Once assembled on the membrane, SNX9 stimulates the GTPase activity of dynamin to facilitate the scission reaction. It has also become clear that SNX9 has the ability to activate the actin regulator N-WASP in a membrane-dependent manner to coordinate actin polymerization with vesicle release. In this Commentary, we summarize several aspects of SNX9 structure and function in the context of membrane remodeling, discuss its interplay with various interaction partners and present a model of how SNX9 might work in endocytosis.

  • 14.
    Lundmark, Richard
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Doherty, Gary J
    Howes, Mark T
    Cortese, Katia
    Vallis, Yvonne
    Parton, Robert G
    McMahon, Harvey T
    The GTPase-activating protein GRAF1 regulates the CLIC/GEEC endocytic pathway2008In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 18, no 22, p. 1802-1808Article in journal (Refereed)
    Abstract [en]

    Clathrin-independent endocytosis is an umbrella term for a variety of endocytic pathways that internalize numerous cargoes independently of the canonical coat protein Clathrin [1, 2]. Electron-microscopy studies have defined the pleiomorphic CLathrin-Independent Carriers (CLICs) and GPI-Enriched Endocytic Compartments (GEECs) as related major players in such uptake [3, 4]. This CLIC/GEEC pathway relies upon cellular signaling and activation through small G proteins, but mechanistic insight into the biogenesis of its tubular and tubulovesicular carriers is lacking. Here we show that the Rho-GAP-domain-containing protein GRAF1 marks, and is indispensable for, a major Clathrin-independent endocytic pathway. This pathway is characterized by its ability to internalize bacterial exotoxins, GPI-linked proteins, and extracellular fluid. We show that GRAF1 localizes to PtdIns(4,5)P2-enriched, tubular, and punctate lipid structures via N-terminal BAR and PH domains. These membrane carriers are relatively devoid of caveolin1 and flotillin1 but are associated with activity of the small G protein Cdc42. This study provides the first specific noncargo marker for CLIC/GEEC endocytic membranes and demonstrates how GRAF1 can coordinate small G protein signaling and membrane remodeling to facilitate internalization of CLIC/GEEC pathway cargoes.

  • 15.
    Lundmark, Richard
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Doherty, Gary J
    Vallis, Yvonne
    Peter, Brian J
    McMahon, Harvey T
    Arf family GTP loading is activated by, and generates, positive membrane curvature.2008In: The Biochemical journal, ISSN 1470-8728, Vol. 414, no 2, p. 189-94Article in journal (Refereed)
    Abstract [en]

    Small G-proteins belonging to the Arf (ADP-ribosylation factor) family serve as regulatory proteins for numerous cellular processes through GTP-dependent recruitment of effector molecules. In the present study we demonstrate that proteins in this family regulate, and are regulated by, membrane curvature. Arf1 and Arf6 were shown to load GTP in a membrane-curvature-dependent manner and stabilize, or further facilitate, changes in membrane curvature through the insertion of an amphipathic helix.

  • 16. Marchès, O
    et al.
    Batchelor, M
    Shaw, RK
    Patel, A
    Cummings, N
    Nagai, T
    Sasakawa, C
    Carlsson, Sven R
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Cougoule, C
    Caron, E
    Knutton, S
    Connerton, I
    Frankel, G
    EspF of enteropathogenic Escherichia coli binds sorting nexin 92006In: Journal of Bacteriology, Vol. 188, no 8, p. 3110-3115Article in journal (Refereed)
  • 17.
    Mohan, Jagan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Morén, Björn
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Holst, Mikkel
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Cavin3 interacts with cavin1 and caveolin1 to increase surface dynamics of caveolae2015In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 128, no 5, p. 979-991Article in journal (Refereed)
    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.

  • 18.
    Morén, Björn
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Shah, Claudio
    Howes, Mark T
    Schieber, Nicole L
    McMahon, Harvey T
    Parton, Robert G
    Daumke, Oliver
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    EHD2 regulates caveolar dynamics via ATP-driven targeting and oligomerization2012In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 23, no 7, p. 1316-1329Article in journal (Refereed)
    Abstract [en]

    Eps15 homology domain-containing 2 (EHD2) belongs to the EHD-containing protein family of dynamin-related ATPases involved in membrane remodeling in the endosomal system. EHD2 dimers oligomerize into rings on highly curved membranes, resulting in stimulation of the intrinsic ATPase activity. In this paper, we report that EHD2 is specifically and stably associated with caveolae at the plasma membrane and not involved in clathrin-mediated endocytosis or endosomal recycling, as previously suggested. EHD2 interacts with pacsin2 and cavin1, and ordered membrane assembly of EHD2 is dependent on cavin1 and caveolar integrity. While the EHD of EHD2 is dispensable for targeting, we identified a loop in the nucleotide-binding domain that, together with ATP binding, is required for caveolar localization. EHD2 was not essential for the formation or shaping of caveolae, but high levels of EHD2 caused distortion and loss of endogenous caveolae. Assembly of EHD2 stabilized and constrained caveolae to the plasma membrane to control turnover, and depletion of EHD2, resulting in endocytic and more dynamic and short-lived caveolae. Thus, following the identification of caveolin and cavins, EHD2 constitutes a third structural component of caveolae involved in controlling the stability and turnover of this organelle.

  • 19.
    Olofsson, Annelie
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nygård Skalman, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Obi, Ikenna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Uptake of Helicobacter pylori vesicles is facilitated by clathrin-dependent and clathrin-independent endocytic pathways2014In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 5, no 3, p. e00979-14-Article in journal (Refereed)
    Abstract [en]

    UNLABELLED: Bacteria shed a diverse set of outer membrane vesicles that function as transport vehicles to deliver effector molecules and virulence factors to host cells. Helicobacter pylori is a gastric pathogen that infects half of the world's population, and in some individuals the infection progresses into peptic ulcer disease or gastric cancer. Here we report that intact vesicles from H. pylori are internalized by clathrin-dependent endocytosis and further dynamin-dependent processes, as well as in a cholesterol-sensitive manner. We analyzed the uptake of H. pylori vesicles by gastric epithelial cells using a method that we refer to as quantification of internalized substances (qIS). The qIS assay is based on a near-infrared dye with a cleavable linker that enables the specific quantification of internalized substances after exposure to reducing conditions. Both chemical inhibition and RNA interference in combination with the qIS assay showed that H. pylori vesicles enter gastric epithelial cells via both clathrin-mediated endocytosis and additional endocytic processes that are dependent on dynamin. Confocal microscopy revealed that H. pylori vesicles colocalized with clathrin and dynamin II and with markers of subsequent endosomal and lysosomal trafficking. Interestingly, however, knockdown of components required for caveolae had no significant effect on internalization and knockdown of components required for clathrin-independent carrier (CLIC) endocytosis increased internalization of H. pylori vesicles. Furthermore, uptake of vesicles by both clathrin-dependent and -independent pathways was sensitive to depletion, but not sequestering, of cholesterol in the host cell membrane suggesting that membrane fluidity influences the efficiency of H. pylori vesicle uptake.

    IMPORTANCE: Bacterial vesicles act as long-distance tools to deliver toxins and effector molecules to host cells. Vesicles can cause a variety of host cell responses via cell surface-induced cell signaling or internalization. Vesicles of diverse bacterial species enter host cells via different endocytic pathways or via membrane fusion. With the combination of a fluorescence-based quantification assay that quantifies internalized vesicles in a large number of cells and either chemical inhibition or RNA interference, we show that clathrin-mediated endocytosis is the major pathway for uptake of Helicobacter pylori vesicles and that lipid microdomains of the host cell membrane affect uptake of vesicles via clathrin-independent pathways. Our results provide important insights about membrane fluidity and its important role in the complex process that directs the H. pylori vesicle to a specific endocytic pathway. Understanding the mechanisms that operate in vesicle-host interactions is important to fully recognize the impact of vesicles in pathogenesis.

  • 20. Pylypenko, Olena
    et al.
    Ignatev, Alexander
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rasmuson, Erika
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven R
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rak, Alexey
    A combinatorial approach to crystallization of PX-BAR unit of the human Sorting Nexin 9.2008In: Journal of Structural Biology, ISSN 1047-8477, Vol. 162, no 2, p. 356-360Article in journal (Refereed)
  • 21. Pylypenko, Olena
    et al.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rasmuson, Erika
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven R
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rak, Alexey
    The PX-BAR membrane-remodeling unit of sorting nexin 9.2007In: EMBO Journal, ISSN 1460-2075, Vol. 26, no 22, p. 4788-800Article in journal (Refereed)
  • 22.
    Rompikuntal, Pramod Kumar
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Åhlund, Monika
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lindmark, Barbro
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Johnson, Tanya L
    Department of Microbiology and Immunology, University of Michigan Medical School..
    Sandkvist, Maria
    Department of Microbiology and Immunology, University of Michigan Medical School..
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Outer membrane vesicle-mediated export of PrtV protease from Vibrio choleraeManuscript (preprint) (Other academic)
    Abstract [en]

    Background: Gram-negative bacteria release large amounts of outer membrane vesicles (OMVs) during normal growth. OMVs from pathogenic bacteria are known to carry different biologically active toxins and enzymes into the surrounding environment. We hypothesized that OMVs may therefore be able to mediate the transport of bacterial products into host cells. We present here an analysis of the V. cholerae OMV-associated virulence factor PrtV. 

    Methodology/Principal Findings: We observed that PrtV, a M6 family, zinc-binding metalloprotease, is secreted from V. cholerae wild type strain C6706 into the culture supernatant in association with OMVs. The association of PrtV with OMVs was determined by immunoblotting and electron microscopy using immunogold labeling. In addition, we observed that the PKD-domain(s) of PrtV has a role in the protein’s association with OMVs. We also demonstrated that OMV-associated PrtV was biologically active because HCT8 cells treated with OMVs from the wild type V. cholerae strain C6706 exhibited altered morphology, whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Moreover, our data suggest that OMV-associated PrtV might be transported into target eukaryotic cells by a vesicle fusion mechanism in association with lipid raft microdomains in the plasma membrane.

    Conclusion/Significance: Our findings suggest that OMVs can deliver biologically active PrtV into target host cells.

  • 23. Shah, Claudio
    et al.
    Hegde, Balachandra G
    Morén, Björn
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Behrmann, Elmar
    Mielke, Thorsten
    Moenke, Gregor
    Spahn, Christian MT
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Daumke, Oliver
    Langen, Ralf
    Structural insights into membrane interaction and caveolar targeting of dynamin-like EHD22014In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 22, no 3, p. 409-420Article in journal (Refereed)
    Abstract [en]

    The dynamin-related Eps15-homology domain-containing protein 2 (EHD2) is a membrane-remodeling ATPase that regulates the dynamics of caveolae. Here, we established an electron paramagnetic resonance (EPR) approach to characterize structural features of membrane-bound EHD2. We show that residues at the tip of the helical domain can insert into the membrane and may create membrane curvature by a wedging mechanism. Using EPR and X-ray crystallography, we found that the N terminus is folded into a hydrophobic pocket of the GTPase domain in solution and can be released into the membrane. Cryoelectron microscopy demonstrated that the N terminus is not essential for oligomerization of EHD2 into a membrane-anchored scaffold. Instead, we found a function of the N terminus in regulating targeting and stable association of EHD2 to caveolae. Our data uncover an unexpected, membrane-induced regulatory switch in EHD2 and demonstrate the versatility of EPR to study structure and function of dynamin superfamily proteins.

1 - 23 of 23
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